Applied Biosystems Sequencing Chemistry Guide

Applied Biosystems Sequencing Chemistry Guide
Automated DNA
Sequencing
Chemistry Guide
© Copyright 2000, Applied Biosystems
For Research Use Only. Not for use in diagnostic procedures.
ABI PRISM and its design, Applied Biosystems, and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and
certain other countries.
ABI, BigDye, CATALYST, POP, POP-4, POP-6, and Primer Express are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain
other countries.
AmpliTaq, AmpliTaq Gold, and GeneAmp are registered trademarks of Roche Molecular Systems, Inc.
Centricon is a registered trademark of W. R. Grace and Co.
Centri-Sep is a trademark of Princeton Separations, Inc.
Long Ranger is a trademark of The FMC Corporation.
Macintosh and Power Macintosh are registered trademarks of Apple Computer, Inc.
pGEM is a registered trademark of Promega Corporation.
Contents
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
New DNA Sequencing Chemistry Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
Introduction to Automated DNA Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
ABI PRISM Sequencing Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5
Applied Biosystems DNA Sequencing Instruments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Data Collection and Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-12
2 ABI PRISM DNA Sequencing Chemistries . . . . . . . . . . . . . . . . . . 2-1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Dye Terminator Cycle Sequencing Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
Dye Primer Cycle Sequencing Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8
Dye Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12
Chemistry/Instrument/Filter Set Compatibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13
Dye/Base Relationships for Sequencing Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-14
Choosing a Sequencing Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-15
3 Performing DNA Sequencing Reactions . . . . . . . . . . . . . . . . . . . 3-1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
DNA Template Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
Sequencing PCR Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10
DNA Template Quality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15
DNA Template Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17
Primer Design and Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18
Reagent and Equipment Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20
Preparing Cycle Sequencing Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21
Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-27
Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-33
Removing Unincorporated Dye Terminators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-34
Preparing Dye Primer Reaction Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . 3-46
Preparing and Loading Samples for Gel Electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-50
Preparing and Loading Samples for Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . 3-53
4 Optimizing Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2
i
Avoiding Problems with Sequencing Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4
5 Optimizing Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . 5-1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Capillary Electrophoresis Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing Electrokinetic Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing Electrophoresis Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run Parameters for Specific Sequencing Chemistries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5-1
5-2
5-4
5-7
5-8
6 Optimizing Software Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1
Choosing a Run Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2
Choosing a Dye Set/Primer (Mobility) File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3
Choosing the Correct Basecaller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6
Creating an Instrument (Matrix) File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-7
Setting the Data Analysis Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15
7 Data Evaluation and Troubleshooting . . . . . . . . . . . . . . . . . . . . 7-1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1
Data Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2
Practical Examples of Data Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-10
Troubleshooting Sequencing Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16
Troubleshooting DNA Sequence Composition Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30
Troubleshooting Sequencing Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-39
Troubleshooting Gel Electrophoresis on the ABI 373 and ABI PRISM 377 . . . . . . . . . . . . . . 7-44
Troubleshooting Capillary Electrophoresis on the ABI PRISM 310. . . . . . . . . . . . . . . . . . . . . 7-55
Troubleshooting Software Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-62
A Gel Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1
Protocol and Run Conditions for 19:1 Polyacrylamide Gels . . . . . . . . . . . . . . . . . . . . . . . . . . A-2
Protocol and Run Conditions for 29:1 Polyacrylamide Gels. . . . . . . . . . . . . . . . . . . . . . . . . . . A-6
Protocol and Run Conditions for Long Ranger and PAGE-PLUS Gels . . . . . . . . . . . . . . . . . A-10
Preparing APS, TBE Buffer, and Deionized Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-15
ii
B IUB Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1
C References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1
D Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .D-1
To Reach Us on the Web. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hours for Telephone Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
To Reach Us by Telephone or Fax in North America. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Documents on Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
To Reach Us by E-Mail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Regional Offices Sales and Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D-1
D-1
D-1
D-2
D-3
D-3
E Part Numbers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1
ABI PRISM DNA Sequencing Kits and Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1
ABI PRISM 310 Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-5
ABI PRISM 377 DNA Sequencer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-8
ABI 373 DNA Sequencer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-9
Documentation and Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-10
Index
iii
Introduction
1
1
New DNA Sequencing Chemistry Guide
Purpose Since the original DNA Sequencing Chemistry Guide was published in early 1995,
Applied Biosystems has released two new instrument platforms, five new sequencing
chemistries, and a new sequencing enzyme.
To accommodate this new information, we have written the Automated DNA
Sequencing Chemistry Guide. This updated guide provides the following:
♦
An introduction to automated DNA sequencing
♦
Descriptions of Applied Biosystems sequencing instruments, chemistries, and
software
♦
Detailed protocols for preparing DNA templates, performing cycle sequencing,
and preparing the extension products for electrophoresis
♦
Guidelines for optimizing electrophoresis and interpreting and troubleshooting
sequencing data
Introduction 1-1
Introduction to Automated DNA Sequencing
Sanger Dideoxy DNA polymerases copy single-stranded DNA templates, by adding nucleotides to a
Sequencing growing chain (extension product). Chain elongation occurs at the 3´ end of a primer,
an oligonucleotide that anneals to the template. The deoxynucleotide added to the
extension product is selected by base-pair matching to the template.
The extension product grows by the formation of a phosphodiester bridge between the
3´-hydroxyl group at the growing end of the primer and the 5´-phosphate group of the
incoming deoxynucleotide (Watson et al., 1987). The growth is in the 5´Æ3´ direction
(Figure 1-1).
DNA polymerases can also incorporate analogues of nucleotide bases. The dideoxy
method of DNA sequencing developed by Sanger et al. (1977) takes advantage of this
ability by using 2´,3´-dideoxynucleotides as substrates. When a dideoxynucleotide is
incorporated at the 3´ end of the growing chain, chain elongation is terminated
selectively at A, C, G, or T because the chain lacks a 3´-hydroxyl group (Figure 1-1).
Extension product
Template
3´ hydroxyl group
no 3´ hydroxyl group
Figure 1-1 DNA strand synthesis by formation of phosphodiester bonds. The chain is
terminated by the use of dideoxycytidine triphosphate (ddC) in place of deoxycytidine
triphosphate (dCTP). The inset shows a schematic representation of the process.
1-2 Introduction
Fluorescent In the Applied Biosystems strategy for automated fluorescent sequencing, fluorescent
Sequencing dye labels are incorporated into DNA extension products using 5´-dye labeled primers
(dye primers) or 3´-dye labeled dideoxynucleotide triphosphates (dye terminators).
The most appropriate labeling method to use depends on your sequencing objectives,
the performance characteristics of each method, and on personal preference.
Applied Biosystems DNA sequencers detect fluorescence from four different dyes that
are used to identify the A, C, G, and T extension reactions. Each dye emits light at a
different wavelength when excited by an argon ion laser. All four colors and therefore
all four bases can be detected and distinguished in a single gel lane or capillary
injection (Figure 1-2).
Figure 1-2 Four-color/one-lane fluorescent sequencing vs. one-color/four-lane method such
as radioactive sequencing
Introduction 1-3
Cycle Sequencing Cycle sequencing is a simple method in which successive rounds of denaturation,
annealing, and extension in a thermal cycler result in linear amplification of extension
products (Figure 1-3). The products are then loaded onto a gel or injected into a
capillary. All current ABI PRISM DNA sequencing kits use cycle sequencing protocols.
See Chapter 3 for information on cycle sequencing protocols.
Figure 1-3 Cycle sequencing
Advantages of Cycle ♦
Sequencing ♦
1-4 Introduction
Protocols are robust and easy to perform.
Cycle sequencing requires much less template DNA than single-temperature
extension methods.
♦
Cycle sequencing is more convenient than traditional single-temperature labeling
methods that require a chemical denaturation step for double-stranded templates.
♦
High temperatures reduce secondary structure, allowing for more complete
extension.
♦
High temperatures reduce secondary primer-to-template annealing.
♦
The same protocol is used for double- and single-stranded DNA.
♦
The protocols work well for direct sequencing of PCR products (see page 3-14).
♦
Difficult templates, such as bacterial artificial chromosomes (BACs), can be
sequenced.
ABI PRISM Sequencing Chemistries
AmpliTaq DNA AmpliTaq® DNA Polymerase, FS is the sequencing enzyme used in ABI PRISM cycle
Polymerase, FS sequencing kits. It is a mutant form of Thermus aquaticus (Taq) DNA polymerase and
contains a point mutation in the active site, replacing phenylalanine with tyrosine at
residue 667 (F667Y). This mutation results in less discrimination against
dideoxynucleotides, and leads to a much more even peak intensity pattern (Tabor and
Richardson, 1995).
AmpliTaq DNA Polymerase, FS also contains a point mutation in the amino terminal
domain, replacing glycine with aspartate at residue 46 (G46D), which removes almost
all of the 5´Æ3´ nuclease activity. This eliminates artifacts that arise from the
exonuclease activity.
The enzyme has been formulated with a thermally stable inorganic pyrophosphatase
that cleaves the inorganic pyrophosphate (PPi) byproduct of the extension reaction
and prevents its accumulation in the sequencing reaction.
In the presence of high concentrations of PPi the polymerization reaction can be
reversed (Kornberg and Baker, 1992), a reaction called pyrophosphorolysis. In this
reaction, a nucleoside monophosphate is removed from the extension product with the
addition of PPi to form the nucleoside triphsphate.
In a sequencing reaction, if a dideoxynucleotide is frequently removed at a particular
position and replaced by a deoxynucleotide, eventually there is little or no chain
termination at that location. This results in a weak or missing peak in the sequence
data (Tabor and Richardson, 1990).
Dye-Labeled With dye terminator labeling, each of the four dideoxy terminators (ddNTPs) is tagged
Terminators with a different fluorescent dye. The growing chain is simultaneously terminated and
labeled with the dye that corresponds to that base (Figure 1-4).
Figure 1-4 One cycle of dye terminator cycle sequencing
Features of Dye-labeled Terminator Reactions
♦ An unlabeled primer can be used.
♦
Dye terminator reactions are performed in a single tube. They require fewer
pipetting steps than dye primer reactions.
♦
Four-color dye labeled reactions are loaded in a single gel lane or capillary
injection.
♦
False stops, i.e., fragments that are not terminated by a dideoxynucleotide (see
page 7-30), go undetected because no dye is attached.
See Chapter 2 for information on ABI PRISM™ DNA sequencing kits.
Introduction 1-5
Dye-Labeled With dye primer labeling, primers are tagged with four different fluorescent dyes.
Primers Labeled products are generated in four separate base-specific reactions. The
products from these four reactions are then combined and loaded into a single gel
lane or capillary injection (Figure 1-5).
Figure 1-5 One cycle of dye primer cycle sequencing
Features of Dye-labeled Primer Reactions
♦ Dye primer chemistries generally produce more even signal intensities than dye
terminator chemistries.
♦
Labeled primers are available for common priming sites. Custom primers can also
be labeled.
♦
Four-color dye-labeled reactions are loaded onto a single lane or capillary
injection.
See Chapter 2 for information on ABI PRISM™ DNA sequencing kits.
1-6 Introduction
Applied Biosystems DNA Sequencing Instruments
ABI 373 The ABI™ 373 DNA Sequencer is an automated instrument for analyzing fluorescently
DNA Sequencer labeled DNA fragments by gel electrophoresis. You can use three sizes of gel plates
for sequencing applications: 24-cm, 34-cm and 48-cm well-to-read lengths (see
Table 1-1 on page 1-10). The longer the well-to-read length, the better the resolution
of the gel.
Sequencing reaction products labeled with four different fluorescent dyes are loaded
into each lane of a 0.3-mm or 0.4-mm vertical slab gel made of polymerized
acrylamide or acrylamide derivatives. You can run up to 36 lanes simultaneously on a
single gel.
The dye-labeled DNA fragments migrate through the acrylamide gel and separate
according to size. At the lower portion of the gel they pass through a region where a
laser beam scans continuously across the gel. The laser excites the fluorescent dyes
attached to the fragments, and they emit light at a specific wavelength for each dye.
The fluorescence intensity is detected by a photomultiplier tube (PMT) and recorded
as a function of time. A moving stage contains the optical equipment (filter wheel and
photomultiplier tube). The PMT detects the fluorescence emission and converts it into
a digital signal. Each time the stage traverses across the gel (a scan) a different
bandpass filter is positioned in front of the PMT to detect each of the four dyes.
A single scan of the gel with one filter takes 1.5 seconds and measures signal in 194
channels. A complete scan with four filters takes 6 seconds and equals one data point.
The data is then transmitted to the Macintosh® computer and stored for processing.
The Sequencing Analysis software (see page 1-16) interprets the result, calling the
bases from the fluorescence intensity at each data point.
Refer to the 373 DNA Sequencing System User’s Manual (P/N 902376) for more
information.
XL Upgrade
The ABI 373 DNA Sequencer with XL Upgrade increases the number of samples that
can be analyzed simultaneously. This increased throughput is made possible by
reengineering the instrument to collect data from 388 channels instead of 194. With
the XL Upgrade, the operation of the ABI 373 DNA Sequencer is controlled from the
Power Macintosh® computer supplied with the upgrade.
After the initial calibration by the Field Service Engineer, the instrument automatically
increases the PMT voltage to compensate for the smaller amount of signal generated
per lane when running 48- or 64-lane gels.
The XL Upgrade also includes new combs and spacers. For sequencing applications,
48-well and 64-well shark’ s tooth combs are available. You can still use 24-well or
36-well combs if desired.
Note
These combs are not interchangeable with combs for the ABI PRISM® 377 DNA
Sequencer.
Refer to the 373 DNA Sequencer With XL Upgrade User’s Manual (P/N 904258) for
more information.
Introduction 1-7
Filter Sets The ABI 373 and ABI 373 with XL Upgrade DNA Sequencers use filters mounted on a
filter wheel to separate light of different wavelengths. The instruments record the light
intensity in four regions, collectively called Filter Set A, centered at the following
wavelengths:
♦
Four-filter wheel: 540 nm, 560 nm, 580 nm, 610 nm
♦
Five-filter wheel: 531 nm, 560 nm, 580 nm, and 610 nm
Note
The five-filter wheel instruments also have Filter Set B (531 nm, 545 nm, 560nm, and
580 nm), but it is not used with existing Applied Biosystems sequencing chemistries. Filter Set
B was used for the T7 (Sequenase) terminator chemistries, which have been discontinued.
BigDye Filter Wheel
To use the new dRhodamine terminator, BigDye™ terminator, and BigDye™ primer
sequencing chemistries (see Chapter 2) on the ABI 373 and ABI 373 with XL Upgrade
DNA Sequencers, the ABI PRISM™ BigDye™ Filter Wheel has been developed.
Its Filter Set A is as follows: 540 nm, 570 nm, 595 nm, and 625 nm.
Note
The BigDye Filter Wheel also has Filter Set B (540 nm, 555 nm, 570, and 595 nm), but
it is not used with existing Applied Biosystems sequencing chemistries.
Refer to the Using the ABI 373 BigDye Filter Wheel User Bulletin (P/N 4304367) for
more information.
ABI PRISM 377 The ABI PRISM 377 DNA Sequencer is a medium- to high-throughput, automated
DNA Sequencer instrument for analyzing fluorescently labeled DNA fragments by gel electrophoresis.
You can use two sizes of gel plates for sequencing applications: 36-cm and 48-cm
well-to-read lengths. The 48-cm well-to-read plates are used to obtain longer read
lengths.
Sequencing reaction products labeled with four different fluorescent dyes are loaded
into each lane of a 0.2-mm vertical slab gel made of polymerized acrylamide or
acrylamide derivatives. You can run up to 36 lanes simultaneously on one gel.
The dye-labeled DNA fragments migrate through the acrylamide gel and separate
according to size. At the lower portion of the gel they pass through a region where a
laser beam scans continuously across the gel. The laser excites the fluorescent dyes
attached to the fragments, and they emit light at a specific wavelength for each dye.
The light is collected in 194 channels during each scan and separated according to
wavelength by a spectrograph onto a cooled, charge-coupled device (CCD) camera,
so all four types of fluorescent emissions can be detected with one pass of the laser.
The data collection software collects the light intensities from the CCD at particular
wavelength bands (virtual filters) and stores them on a Power Macintosh computer as
digital signals for processing. The Sequencing Analysis software (see page 1-16)
interprets the result, calling the bases from the fluorescence intensity at each data
point.
Refer to the ABI PRISM 377 DNA Sequencer User’s Manual (P/N 903433) for more
information.
1-8 Introduction
377-18
The ABI PRISM 377-18 DNA Sequencer is a lower-cost, lower-throughput version of
the ABI PRISM 377 DNA Sequencer. It can run up to 18 lanes on a single gel.
XL Upgrade
The ABI PRISM 377 DNA Sequencer with XL Upgrade increases the number of
samples that can be analyzed simultaneously. This increased throughput is made
possible by reengineering the instrument to collect data from 388 channels instead of
194 during each scan.
The XL Upgrade also includes new combs. For sequencing applications, 48-well and
64-well shark’ s tooth combs are available. You can still use 36-well or other lower lane
density combs if desired.
Refer to the ABI PRISM 377 DNA Sequencer XL Upgrade User’s Manual (P/N 904412)
for more information.
96-Lane Upgrade
The ABI PRISM 377 DNA Sequencer with 96-Lane Upgrade increases the number of
samples that can be run on each gel. The increased throughput is made possible by
reengineering the instrument to collect data from 480 channels instead of 388 for the
ABI PRISM 377 DNA Sequencer with XL Upgrade or 194 for the ABI PRISM 377 DNA
Sequencer.
The 96-lane upgrade includes new combs and new notched front glass plates. You
can still use lower lane density combs, but only with the original notched front glass
plates that were provided with the instrument.
The new notched front glass plate has a bevel in the loading region that increases the
thickness of the gel in this region from 0.2 mm to 0.4 mm. In addition, the scan region
has been increased from 6 inches to 7.5 inches. This makes sample loading easier
than for a 64-lane gel.
Refer to the ABI PRISM 377 DNA Sequencer 96-Lane Upgrade User’s Manual
(P/N 4305423) for more information.
Introduction 1-9
Gel Electrophoresis Instruments
Table 1-1 Applied Biosystems Gel Electrophoresis Instruments
Instrument
ABI 370
Well-to-Read
Length (cm)
24
Number of
Lanes
Maximum
Throughput
(bases/hr)a
Detection System
PMT, 4-filter wheel
16
800
24
1200
24, 36
1800
24, 36, 48, 64
3200
24, 36 or
24, 36, 48, 64
1800 or
3200
24, 36
7200
18
3600
ABI PRISM 377 with
XL Upgrade
24, 36, 48, 64
12,800
ABI PRISM 377 with
96-Lane Upgrade
24, 36, 48, 64,
96
19,200
ABI 373
ABI 373 Leon Model
6, 12, 24, 34
ABI 373 Stretch Model
6, 12, 24, 34, 48
ABI 373 with XL
Upgrade
24 or
6, 12, 24, 34 or
6, 12, 24, 34, 48
ABI 373 with BigDye
Filter Wheelb
ABI PRISM 377
ABI PRISM 377-18
12, 36, 48
Computer
HP Vectra
Macintosh
PMT, 5-filter wheel
Power
Macintosh
PMT, new 5-filter
wheel
Macintosh or
Power
Macintosh
CCD camera,
spectrograph
Power
Macintosh
a. Maximum throughput = maximum number of lanes ¥ maximum electrophoresis speed (50 bph for ABI 370 and ABI 373 models, 200 bph
for ABI PRISM 377 models)
b. Allows use of dRhodamine-based chemistries on any ABI 373 or ABI 373 with XL Upgrade instrument with a 5-filter wheel. See page 1-8
for ABI 373 filter sets.
ABI PRISM 310 The ABI PRISM® 310 Genetic Analyzer is an automated instrument for analyzing
Genetic Analyzer fluorescently labeled DNA fragments by capillary electrophoresis.
The sequencing reaction sample tubes are placed in an autosampler tray that holds
either 48 or 96 samples. The autosampler successively brings each sample into
contact with the cathode electrode and one end of a glass capillary filled with a
separation polymer. An anode electrode at the other end of the capillary is immersed
in buffer.
The sample enters the capillary as current flows from the cathode to the anode. The
short period of electrophoresis conducted while the capillary and cathode are
immersed in the sample is called electrokinetic injection. The sample forms a tight
band in the capillary during this injection. The end of the capillary near the cathode is
then placed in buffer. Current is applied again to continue electrophoresis.
When the DNA fragments reach a detector window in the capillary, a laser excites the
fluorescent dye labels. Emitted fluorescence from the dyes is collected once per
second by a cooled, charge-coupled device (CCD) camera at particular wavelength
bands (virtual filters) and stored as digital signals on a Power Macintosh computer for
processing. The Sequencing Analysis software (see page 1-16) interprets the result,
calling the bases from the fluorescence intensity at each data point.
Refer to the ABI PRISM 310 Genetic Analyzer User’s Manual (P/N 903565) for more
information.
1-10 Introduction
Virtual Filter Sets ABI PRISM 310 and ABI PRISM 377 (All Models)1
These instruments use virtual filter sets to detect light intensity in four non-overlapping
regions on a CCD camera. Each region corresponds to a wavelength range that
contains or is close to the emission maximum of an ABI PRISM dye.
The process is similar to using a physical filter to separate light of different
wavelengths. However, the filter sets are called “virtual filters” because the
instruments use no physical filtering hardware to perform the separation.2
The exact positions of the CCD regions and the dye combinations appropriate to
these positions depend upon the virtual filter set used. For example, with Virtual Filter
Set E the instrument records the light intensity in four regions, or “windows,” centered
at 540 nm, 570 nm, 595 nm, and 625 nm. The window positions in each virtual filter
set have been optimized to provide the maximum possible separation among the
centers of detection for the different dyes while maintaining good signal strength.
The Data Collection Software color-codes the intensity displays from the four
light-collection regions. These appear as the blue, green, black (yellow on gel images),
and red peaks in the raw data.
The Sequencing Analysis Software uses the same four colors to color-code analyzed
data from all dye/virtual filter set combinations. The display colors represent the
relative, not the actual, detection wavelengths. For consistency, the software always
displays analyzed data with A as green, C as blue, G as black, and T as red in the
electropherogram view.
Table 1-2 shows the wavelengths of the “windows” in the virtual filter sets used in cycle
sequencing applications.
Table 1-2 Wavelength Ranges of Virtual Filter Sets
Virtual
Filter Set
Color
Wavelength Range of
Virtual Filter (nm)
A
blue
530–541
green
554–564
E
yellow/black
581–591
red
610–620
blue
535–545
green
565–575
yellow/black
590–600
red
620–630
1.
Includes the ABI PRISM 377, ABI PRISM 377-18, ABI PRISM 377 with XL Upgrade, and the
ABI PRISM 377 with 96-Lane Upgrade instruments.
2.
The ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencer have a long-pass filter to
prevent light from the instrument’s argon ion laser from interfering with the detection of the dye signals.
Introduction 1-11
Data Collection and Analysis Settings
Overview This section is intended to provide an introduction to the data collection and analysis
settings, which are dealt with in more detail in Chapter 6.
Many users sequence DNA using more than one chemistry. Take care when entering
data collection and analysis settings in the software. If your data is analyzed with the
wrong software settings, the resulting electropherograms will show overlapping peaks
and gaps between peaks rather than the evenly spaced peaks characteristic of
correctly analyzed data.
Run Modules ABI 373 with XL Upgrade
A run module file contains all the parameters required for a particular function or
application. The parameters include the following:
♦
Electrophoresis power
♦
Current and voltage settings
♦
Laser settings
♦
Scanner settings
♦
PMT settings
There are three types of run module files. Not all of the parameters listed above are in
each module file.
♦
Plate Check
This module is for checking the cleanliness and alignment of the gel plates. Laser,
scanning, and PMT settings are associated with it.
♦
Pre Run
This module is for prerunning sequencing gels. Laser, scanning, electrophoresis,
and PMT settings are associated with it.
♦
Seq Run
This module is for running sequencing gels. Laser, scanning, electrophoresis, and
PMT settings are associated with it.
IMPORTANT
When you select a run module, the filter set is chosen automatically. You must
edit the run module to change the filter set used to collect the data. Refer to the 373 DNA
Sequencer With XL Upgrade User’s Manual (P/N 904258) for more information.
Note
The ABI 373 DNA Sequencer does not use run modules. Run parameters are set on
the instrument’s keypad. Refer to the 373 DNA Sequencing System User’s Manual
(P/N 902376) for information on setting run parameters.
ABI PRISM 310 and ABI PRISM 377 (All Models)
A run module file contains all the parameters required for a particular function or
application. The parameters include the following:
1-12 Introduction
♦
Electrophoresis voltage
♦
Current and power settings
♦
Laser settings
♦
Scanner settings (ABI PRISM 377 DNA Sequencer only)
♦
Virtual filters and CCD gain and offset
♦
Run temperature settings
♦
Injection time and voltage (ABI PRISM 310 Genetic Analyzer)
There are three types of module files. Not all of the parameters listed above are in
each module file.
♦
Plate check
These modules are for checking the cleanliness and alignment of the gel plates.
Laser, scanning, virtual filter, and CCD conditions are associated with these types
of files.
♦
Prerun
These modules are for prerunning the gel or polymer. Laser, scanning, virtual
filter, and electrophoresis, CCD, and gel temperature conditions are associated
with these types of files.
Note
Plate check and prerun modules are not used with the ABI PRISM 310 Genetic
Analyzer.
♦
Run
These modules are for running the gel or polymer. Laser, scanning, virtual filter,
CCD, and electrophoresis parameters and gel temperature are associated with
these types of files.
IMPORTANT
When you select a run module, the virtual filter set is chosen automatically.
You must be careful to select the correct run module for your sequencing chemistry.
The available run modules are listed in Table 6-1 on page 6-2.
Dye Set/Primer Files Mobility Correction
The different dyes affect the electrophoretic mobility of cycle sequencing extension
products. The relative mobility of the dye-labeled fragments is specific to each
sequencing chemistry (see page 6-4 for more information). Under the same set of
conditions, the mobilities are very reproducible.
The analysis software is able to compensate for these mobility differences by applying
mobility shifts to the data so that evenly spaced peaks are presented in the analyzed
data. The files that contain the mobility shift information are called dye set/primer files.
Dye set/primer files also tell the Sequencing Analysis software (see page 1-16) the
following:
♦
Which matrix file in the instrument file (see page 1-14) to use to analyze the data
♦
Dye/base relationships for converting raw data colors to base calls (see
page 2-14)
The dye set/primer files available are listed in Table 6-2 on page 6-5.
Introduction 1-13
Instrument Files Multicomponent Analysis
Multicomponent analysis is the process that separates the four different fluorescent
dye colors into distinct spectral components. Although each of these dyes emits its
maximum fluorescence at a different wavelength, there is some overlap in the
emission spectra between the four dyes (Figure 1-6). The goal of multicomponent
analysis is to isolate the signal from each dye so that there is as little noise in the data
as possible.
Figure 1-6 Spectral overlap of the dRhodamine dyes in the four virtual filters (vertical gray
bars) of Filter Set E
The precise spectral overlap between the four dyes is measured by running DNA
fragments labeled with each of the dyes in separate lanes of a gel or in separate
injections on a capillary. These dye-labeled DNA fragments are called matrix
standards.
The Data Utility software (see page 6-7) then analyzes the data from each of the four
matrix standard samples and creates an instrument file. The instrument file contains
three matrix files, which have tables of numbers with four columns and four rows
(Figure 1-7 on page 1-15). These numbers are normalized fluorescence intensities
and represent a mathematical description of the spectral overlap that is observed
between the four dyes.
The rows in the tables represent the virtual filters and the columns represent the dyes.
The top lefthand value, 1.000, represents the normalized fluorescence of the blue dye
in the blue filter. It follows that all matrix tables should have values of 1.000 on the
diagonal from top left to bottom right.
The other values in the table should all be less than 1. These values represent the
amount of spectral overlap observed for each dye in each virtual filter. For example,
the values in the third row reflect quantitatively the amount of each dye detected in the
third (“yellow”) virtual filter.
1-14 Introduction
Figure 1-7 Instrument file created in the Data Utility software, indicating the values obtained
with the dRhodamine matrix standards for Filter Set E on a particular ABI PRISM 377 instrument
Note that the numbers decrease moving away from the diagonal in any direction. For
example, in the first column the amount of blue fluorescence seen through the red
filter (fourth row) should be less than that seen in the yellow filter (third row), which
should be less than that seen in the green filter (second row).
These values will vary between different instruments and between filter sets on a
single instrument. An instrument file must be made for each filter set used on each
instrument.
The instrument file is created for a specific filter set or virtual filter set when the
instrument is installed. Whenever a new filter set is used, a new instrument file must
be created for that filter set. Refer to your instrument user’s manual or the protocol for
the sequencing chemistry you are using for instructions on creating instrument files.
The appropriate instrument file can be applied to data on subsequent capillary runs or
gels on the same instrument, as long as the same filter set is used. This is because
the spectral overlap between the four dyes is very reproducible.
Multicomponent analysis of sequencing data is performed automatically by the
Sequencing Analysis software (see below), which applies a mathematical matrix
calculation, using the values in the instrument file, to all sample data.
See page 6-7 for instructions for creating instrument files.
Introduction 1-15
What Is In a Matrix File
The matrix files in an instrument file are used for specific types of chemistry, and
provide information to the Sequencing Analysis software to allow it to correct for
spectral overlap.
Matrix files also contain the following:
♦
Baselining algorithm for the chemistry being used
♦
Information that the Sequencing Analysis software uses to determine Peak 1
Locations and Start Points for data analysis
Sequencing Analysis The DNA Sequencing Analysis Software analyzes the raw data collected by the Data
Software Collection software:
♦
Tracks gel files (if using the ABI 373 or ABI PRISM 377 DNA Sequencer)
♦
Extracts sample information from gel files (if using the ABI 373 or ABI PRISM 377
DNA Sequencer)
♦
Performs multicomponent analysis
♦
Applies mobility corrections
♦
Normalizes the base spacing
♦
Baselines data
♦
Determines analysis starting points
♦
Calls bases
See Chapter 7 for information on interpreting and troubleshooting sequencing data.
Refer to the ABI PRISM DNA Sequencing Analysis Software User’s Manual for specific
information about the Sequencing Analysis software.
1-16 Introduction
ABI PRISM DNA
Sequencing Chemistries 2
2
Overview
In This Chapter This chapter describes the Applied Biosystems cycle sequencing chemistries, the
dyes used in them, and how to choose a sequencing chemistry.
Topic
See page
Dye Terminator Cycle Sequencing Kits
2-2
Dye Primer Cycle Sequencing Kits
2-8
Dye Spectra
2-12
Chemistry/Instrument/Filter Set Compatibilities
2-13
Dye/Base Relationships for Sequencing Chemistries
2-14
Choosing a Sequencing Chemistry
2-15
ABI PRISM DNA Sequencing Chemistries 2-1
Dye Terminator Cycle Sequencing Kits
Rhodamine Dye The rhodamine dye terminators have the following dye labels. The structures of the
Terminators rhodamine dye terminators are shown in Figure 2-1.
Terminator
Dye Label
A
R6G
C
ROX
G
R110
T
TAMRA
Figure 2-1 Rhodamine dye terminators
Rhodamine Dye The ABI PRISM™ Dye Terminator Cycle Sequencing Kits combine AmpliTaq® DNA
Terminator Kits Polymerase, FS, rhodamine dye terminators, and all the required components for the
sequencing reaction.
Note
Throughout this manual, these kits will be referred to as “rhodamine dye terminators.”
The concentrations of the dye-labeled dideoxynucleotides and deoxynucleotides in
the dNTP mix have been optimized to give a balanced distribution of signal above 700
bases. The dNTP mix includes dITP in place of dGTP to minimize band compressions.
In the Ready Reaction format, the dye terminators, deoxynucleoside triphosphates,
AmpliTaq DNA Polymerase, FS, rTth pyrophosphatase, magnesium chloride, and
buffer are premixed into a single tube of Ready Reaction Mix and are ready to use.
These reagents are suitable for performing fluorescence-based cycle sequencing
reactions on single-stranded or double-stranded DNA templates, or on polymerase
chain reaction (PCR) fragments.
2-2 ABI PRISM DNA Sequencing Chemistries
In the Core Kit format, the reagents are supplied in individual tubes to maximize kit
flexibility. For convenience when sequencing large quantities of templates, the
reagents can be premixed and stored.
The cycle sequencing protocols are optimized for GeneAmp® PCR Instrument
Systems thermal cyclers, the CATALYST™ 800 Molecular Biology LabStation, and the
ABI PRISM® 877 Integrated Thermal Cycler. For more information, refer to the
ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit Protocol
(P/N 402078) or the ABI PRISM Dye Terminator Cycle Sequencing Core Kit Protocol
(P/N 402116).
dRhodamine Applied Biosystems has designed new dichlororhodamine (dRhodamine) dye
Terminators terminators to give more even peak heights than the rhodamine dye terminators
(Rosenblum et al., 1997). The new dyes have narrower emission spectra, giving less
spectral overlap and therefore less noise (Figure 2-7 on page 2-12).
The new dRhodamine dye terminators have the following dye labels. The dye
terminator structures are shown in Figure 2-2.
Terminator
Dye Label
A
dichloro[R6G]
C
dichloro[TAMRA]
G
dichloro[R110]
T
dichloro[ROX]
Figure 2-2 dRhodamine terminators
ABI PRISM DNA Sequencing Chemistries 2-3
Three of the four dRhodamine terminators use the new ethylene oxide (EO) linker to
attach the dye to the dideoxynucleotide. This improves the incorporation of the
dye-labeled terminators by the AmpliTaq DNA Polymerase, FS enzyme.
Data collected in Applied Biosystems laboratories shows more uniform signal
intensities with the new dyes and a reduction of the weak G after A pattern that is a
problem with the rhodamine dye terminators.
With less noise, better signal uniformity, and more even peak heights, the new
dRhodamine dye terminators can give better sequencing results than the rhodamine
dye terminators (Figure 2-3).
Figure 2-3 Sequence data obtained from a plasmid with dRhodamine terminators. Reactions
were run on an ABI PRISM® 377 DNA Sequencer with a 48-cm well-to-read gel.
dRhodamine The ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kits
Terminator Ready combine AmpliTaq DNA Polymerase, FS and the new dRhodamine dye terminators. In
Reaction Kits the Ready Reaction format, the dye terminators, deoxynucleoside triphosphates,
AmpliTaq DNA Polymerase, FS, rTth pyrophosphatase, magnesium chloride, and
buffer are premixed into a single tube of Ready Reaction Mix and are ready to use.
The dNTP mix includes dITP in place of dGTP to minimize band compressions.
These reagents are suitable for performing fluorescence-based cycle sequencing
reactions on single-stranded or double-stranded DNA templates, or on polymerase
chain reaction (PCR) fragments. The cycle sequencing protocols are optimized for
GeneAmp PCR Instrument Systems thermal cyclers, the CATALYST 800 Molecular
Biology LabStation, and the ABI PRISM 877 Integrated Thermal Cycler.
For more information, refer to the ABI PRISM dRhodamine Terminator Cycle
Sequencing Ready Reaction Kit Protocol (P/N 403041).
2-4 ABI PRISM DNA Sequencing Chemistries
Instrument Platforms
The ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kits are
for use with the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencer
(all models).1
These kits can also be used with ABI™ 373 DNA Sequencers2 on which the new
ABI PRISM BigDye Filter Wheel has been installed. Refer to the ABI PRISM BigDye
Filter Wheel User Bulletin (P/N 4304367) for more information.
IMPORTANT
This kit is not designed for use with ABI 373 DNA Sequencers and ABI 373
DNA Sequencers with XL Upgrade that do not have the ABI PRISM BigDye Filter Wheel.
BigDye Terminators Applied Biosystems has developed a set of dye terminators labeled with novel,
high-sensitivity dyes (Rosenblum et al., 1997). The new dye structures contain a
fluorescein donor dye, e.g., 6-carboxyfluorescein (6-FAM), linked to one of four
dichlororhodamine (dRhodamine) acceptor dyes. The excitation maximum of each dye
label is that of the fluorescein donor, and the emission spectrum is that of the
dRhodamine acceptor (Figure 2-7 on page 2-12).
The donor dye is optimized to absorb the excitation energy of the argon ion laser in
the Applied Biosystems DNA sequencing instruments. The linker affords extremely
efficient energy transfer (quantum efficiency nearly 1.0, i.e., 100%) between the donor
and acceptor dyes. The BigDye™ terminators are 2–3 times brighter than the
rhodamine dye terminators when incorporated into cycle sequencing products.
The BigDye terminators are labeled with the following dRhodamine acceptor dyes:
Terminator
Acceptor Dye
A
dichloro[R6G]
Note
C
dichloro[ROX]
G
dichloro[R110]
T
dichloro[TAMRA]
The individual dRhodamine dye structures are shown in Figure 2-2 on page 2-3.
The BigDye terminators also have narrower emission spectra than the rhodamine dye
terminators, giving less spectral overlap and therefore less noise (Figure 2-7 on
page 2-12). The brighter signal and decreased noise provide an overall 4–5X gain in
signal-to-noise ratio (Figure 2-4 on page 2-6).
♦
The nucleotide/dideoxynucleotide mixes have been optimized to give longer, more
accurate reads above 700 bases.
♦
Large templates can be sequenced more readily. One such application is BAC
end sequencing.
♦
Reactions using half the amount of Ready Reaction Premix can be run on some
templates, such as PCR products and plasmids (see page 3-22).
1.
Includes the ABI PRISM 377, ABI PRISM 377-18, ABI PRISM 377 with XL Upgrade, and the
ABI PRISM 377 with 96-Lane Upgrade instruments.
2.
Includes the ABI 373 and ABI 373 with XL Upgrade instruments.
ABI PRISM DNA Sequencing Chemistries 2-5
Figure 2-4 Sequence data obtained from a plasmid with BigDye terminators. Reactions were
run on an ABI PRISM 377 DNA Sequencer with a 5.25% PAGE-PLUS, 48-cm well-to-read gel.
BigDye Terminator The ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits combine
Ready Reaction Kits AmpliTaq DNA Polymerase, FS, the new BigDye terminators, and all the required
components for the sequencing reaction.
In the Ready Reaction format, the dye terminators, deoxynucleoside triphosphates,
AmpliTaq DNA Polymerase, FS, rTth pyrophosphatase, magnesium chloride, and
buffer are premixed into a single tube of Ready Reaction Mix and are ready to use.
These reagents are suitable for performing fluorescence-based cycle sequencing
reactions on single-stranded or double-stranded DNA templates, on polymerase chain
reaction (PCR) fragments, and on large templates, e.g., BAC clones.
The dNTP mix includes dITP in place of dGTP to minimize band compressions.The
dNTP mix also uses dUTP in place of dTTP. dUTP improves the incorporation of the T
terminator and results in a better T pattern.
The cycle sequencing protocols are optimized for GeneAmp PCR Instrument Systems
thermal cyclers, the CATALYST 800 Molecular Biology LabStation, and the
ABI PRISM 877 Integrated Thermal Cycler.
For more information, refer to the ABI PRISM BigDye Terminator Cycle Sequencing
Ready Reaction Kit Protocol (P/N 4303237).
2-6 ABI PRISM DNA Sequencing Chemistries
Instrument Platforms
The ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits are for use
with the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencer (all
models).1
These kits can also be used with ABI 373 DNA Sequencers2 on which the new
ABI PRISM BigDye Filter Wheel has been installed. Refer to the ABI PRISM BigDye
Filter Wheel User Bulletin (P/N 4304367) for more information.
IMPORTANT
This kit is not designed for use with ABI 373 DNA Sequencers and ABI 373
DNA Sequencers with XL Upgrade that do not have the ABI PRISM BigDye Filter Wheel.
1.
Includes the ABI PRISM 377, ABI PRISM 377-18, ABI PRISM 377 with XL Upgrade, and the
ABI PRISM 377 with 96-Lane Upgrade instruments.
2.
Includes the ABI 373 and ABI 373 with XL Upgrade instruments.
ABI PRISM DNA Sequencing Chemistries 2-7
Dye Primer Cycle Sequencing Kits
Fluorescein/ The fluorescein/rhodamine dye primers (see note below) have the following dye
Rhodamine Dye labels:
Primers
Primer
Dye Label
A
JOE
C
5-FAM
G
TAMRA
T
ROX
The structures of the fluorescein/rhodamine dye primers are shown in Figure 2-5.
Note that 5-FAM and JOE are fluorescein dyes and TAMRA and ROX are rhodamine
dyes. The emission spectra of the dyes are shown in Figure 2-8 on page 2-12.
Note
Throughout this manual, this chemistry will be referred to as “fluorescein/rhodamine
dye primer” to distinguish it from BigDye primer chemistry.
Figure 2-5 Fluorescein/rhodamine dye primers
Fluorescein/ The ABI PRISM Dye Primer Cycle Sequencing Kits include AmpliTaq DNA
Rhodamine Dye Polymerase, FS, dye-labeled primers and all the required components for the
Primer Kits sequencing reaction.
The deoxy- and dideoxynucleotide ratios in the dNTP mix have been optimized to give
a balanced distribution of signal between base 10 and base 700+ after the primer. The
dNTP mix includes 7-deaza-dGTP in place of dGTP to minimize band compressions.
In the Ready Reaction format, the dye-labeled primers, deoxynucleoside
triphosphates, dideoxynucleoside triphosphates, AmpliTaq DNA Polymerase, FS, rTth
2-8 ABI PRISM DNA Sequencing Chemistries
pyrophosphatase, magnesium chloride, and buffer are premixed into A, C, G, and T
Ready Reaction cocktails to eliminate time-consuming reagent preparation. These
reagents are suitable for performing fluorescence-based cycle sequencing reactions
on single-stranded or double-stranded DNA templates, or on polymerase chain
reaction (PCR) fragments.
In the Core Kit format, the reagents are supplied in individual tubes to maximize kit
flexibility. For convenience when sequencing large quantities of templates, the
reagents can be premixed and stored for later use.
The cycle sequencing protocols are optimized for GeneAmp PCR Instrument Systems
thermal cyclers, the CATALYST 800 Molecular Biology LabStation, and the
ABI PRISM 877 Integrated Thermal Cycler.
Note
We do not recommend using fluorescein/rhodamine dye primers with the POP-6™
polymer on the ABI PRISM 310 Genetic Analyzer.
For more information, refer to the ABI PRISM Dye Primer Cycle Sequencing Ready
Reaction Kit Protocol (P/N 402113) or the ABI PRISM Dye Primer Cycle Sequencing
Core Kit Protocol (P/N 402114).
BigDye Primers Applied Biosystems has developed a set of dye primers labeled with novel,
high-sensitivity dyes (Lee et al., 1997). The new dye structures contain a fluorescein
donor dye, e.g., 6-carboxyfluorescein (6-FAM), linked to one of four dichlororhodamine
(dRhodamine) acceptor dyes. The excitation maximum of each dye label is that of the
fluorescein donor, and the emission spectrum is that of the dRhodamine acceptor
(Figure 2-7 on page 2-12).
The donor dye is optimized to absorb the excitation energy of the argon ion laser in
the Applied Biosystems DNA sequencing instruments. The linker affords extremely
efficient energy transfer (quantum efficiency nearly 1.0, i.e., 100%) between the donor
and acceptor dyes. Hence, the BigDye™ primers are 2–3 times brighter than the
fluorescein/rhodamine dye primers when incorporated into cycle sequencing
products.
The BigDye primers are labeled with the following dRhodamine acceptor dyes:
Primer
Acceptor Dye
A
dichloro[R6G]
C
dichloro[R110]
G
dichloro[TAMRA]
T
dichloro[ROX]
Note
The individual dRhodamine dye structures are shown in Figure 2-2 on page 2-3. The
BigDye primers use the same dyes as the BigDye terminators.
The BigDye primers also have narrower emission spectra than the fluorescein/
rhodamine dye primers, giving less spectral overlap and therefore less noise
(Figure 2-7 on page 2-12). The brighter signal and decreased noise provide an overall
4–5X gain in signal-to-noise ratio, giving added flexibility in sequencing applications:
♦
The A, C, G, and T reactions are carried out in a 1:1:1:1 ratio.
♦
The nucleotide/dideoxynucleotide mixes have been optimized to give longer, more
accurate reads above 700 bases.
ABI PRISM DNA Sequencing Chemistries 2-9
♦
Large templates can be sequenced more readily. One such application is BAC
end sequencing.
♦
Reactions using half the amount of Ready Reaction Premix can be run on some
templates, such as PCR products and plasmids (see page 3-25).
♦
In some cases, reactions can be loaded onto the sequencing instrument without
precipitation (see page 3-49).
Figure 2-6 shows BigDye primer sequencing data.
Figure 2-6 Sequence data obtained from a plasmid with BigDye primers. Reactions were run
on an ABI PRISM 377 DNA Sequencer with a 5.25% PAGE-PLUS, 48-cm well-to-read gel.
BigDye Primer The ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kits combine
Ready Reaction Kits AmpliTaq DNA Polymerase, FS, the new BigDye primers, and all the required
components for the sequencing reaction.
The kits contain nucleotide mixes that have been specifically optimized for AmpliTaq
DNA Polymerase, FS. The deoxy- and dideoxynucleotide ratios in the nucleotide
mixes have been formulated to give a good signal balance above 700 bases. These
formulations also contain 7-deaza-dGTP in place of dGTP to minimize band
compressions.
In the Ready Reaction format, the dye-labeled primers, deoxynucleoside
triphosphates, dideoxynucleoside triphosphates, AmpliTaq DNA Polymerase, FS, rTth
pyrophosphatase, magnesium chloride, and buffer are premixed into A, C, G, and T
Ready Reaction cocktails to eliminate time-consuming reagent preparation. These
reagents are suitable for performing fluorescence-based cycle sequencing reactions
2-10 ABI PRISM DNA Sequencing Chemistries
on single-stranded or double-stranded DNA templates, on polymerase chain reaction
(PCR) fragments, and on large templates, e.g., the ends of BAC clones.
The cycle sequencing protocols are optimized for GeneAmp PCR Instrument Systems
thermal cyclers, the CATALYST 800 Molecular Biology LabStation, and the
ABI PRISM 877 Integrated Thermal Cycler.
For more information, refer to the ABI PRISM BigDye Primer Cycle Sequencing Ready
Reaction Kit Protocol (P/N 403057).
Instrument Platforms
The ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kits are for use with
the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencer (all
models).1
These kits can also be used with ABI 373 DNA Sequencers2 on which the new
ABI PRISM BigDye Filter Wheel has been installed. Refer to the ABI PRISM BigDye
Filter Wheel User Bulletin (P/N 4304367) for more information.
IMPORTANT
This kit is not designed for use with ABI 373 DNA Sequencers and ABI 373
DNA Sequencers with XL Upgrade that do not have the ABI PRISM BigDye Filter Wheel.
1.
Includes the ABI PRISM 377, ABI PRISM 377-18, ABI PRISM 377 with XL Upgrade, and the
ABI PRISM 377 with 96-Lane Upgrade instruments.
2.
Includes the ABI 373 and ABI 373 with XL Upgrade instruments.
ABI PRISM DNA Sequencing Chemistries 2-11
Dye Spectra
Rhodamine and The normalized emission spectra of the rhodamine and dRhodamine dyes are shown
dRhodamine Dyes in Figure 2-7. The dRhodamine dyes are used in the ABI PRISM dRhodamine
Terminator, BigDye Primer, and BigDye Terminator Cycle Sequencing Ready Reaction
Kits.
Figure 2-7 Emission spectra of rhodamine and dRhodamine dyes. Note the narrower
emission spectra of the dRhodamine dyes.
Fluorescein/ The normalized emission spectra of the fluorescein and rhodamine dyes used in the
Rhodamine Dyes ABI PRISM Dye Primer Cycle Sequencing Kits are shown in Figure 2-8.
Figure 2-8 Emission spectra of the dyes used in ABI PRISM Dye Primer Cycle Sequencing
Kits
2-12 ABI PRISM DNA Sequencing Chemistries
Chemistry/Instrument/Filter Set Compatibilities
Chemistry and Table 2-1 shows which chemistries can be used on which instruments.
Instrument
Table 2-1 Chemistry/Instrument Compatibilities
Compatibilities
Sequencing Chemistry
Rhodamine
Dye
Terminator
dRhodamine
Terminator
BigDye
Terminator
Fluorescein/
Rhodamine
Dye Primer
BigDye
Primer
ABI 373a
yes
no
no
yes
no
373a
with
ABI
BigDye
Filter Wheel
no
yes
yes
no
yes
ABI PRISM® 310
and
ABI PRISM 377b
yes
yes
yes
yes
yes
Instrument
a. Includes the ABI 373 and ABI 373 with XL Upgrade instruments.
b. All models.
Filter Sets Table 2-2 shows the filter sets and virtual filter sets that are used with the Applied
Biosystems cycle sequencing chemistries.
Table 2-2 Sequencing Chemistries and Filter Sets
Filter Set
Chemistry
Rhodamine
Dye Terminator
ABI
with
BigDye Filter Wheel
ABI PRISM 310 and
ABI PRISM 377b
A
Cannot use these
chemistries with this
instrument
configuration
A
Cannot use these
chemistries with this
instrument
configuration
A
E
ABI 373a
Fluorescein/
Rhodamine
Dye Primer
dRhodamine
Terminator
BigDye™
Terminator
Virtual Filter Set
373a
BigDye™
Primer
a. Includes the ABI 373 and ABI 373 with XL Upgrade instruments.
b. All models.
ABI PRISM DNA Sequencing Chemistries 2-13
Dye/Base Relationships for Sequencing Chemistries
Overview During the development of a new sequencing chemistry, alternative dye/base
relationships are investigated to see which produces the most uniform signal in the
analyzed data. For this reason, different sequencing chemistries may have different
dye/base relationships.
The Sequencing Analysis software compensates for this when the correct dye set/
primer (mobility) file is used (see page 6-3). The software always displays A as green,
C as blue, G as black, and T as red in the electropherogram view of analyzed data.
Dye/Base Relationships
Table 2-3 dRhodamine Terminator Dye/Base Relationships
Color of Raw Data
on Gel Imageb
Color of
Analyzed Data on
Electropherogramsc
Terminator
Dye
Color of
Unanalyzed Data
on Electropherograma
A
dR6G
green
green
green
C
dTAMRA
black
yellow
blue
G
dR110
blue
blue
black
T
dROX
red
red
red
Table 2-4 Rhodamine Dye Terminator and BigDye Terminator Dye/Base Relationships
Dye
Terminator
Rhodamine
Dye
Terminator
BigDye
Terminator
Color of
Unanalyzed Data
on Electropherograma
Color of Raw Data
on Gel Imageb
Color of
Analyzed Data on
Electropherogramsc
A
R6G
dR6G
green
green
green
C
ROX
dROX
red
red
blue
G
R110
dR110
blue
blue
black
T
TAMRA
dTAMRA
black
yellow
red
Table 2-5 Fluorescein/Rhodamine Dye Primer and BigDye Primer Dye/Base Relationships
Dye
Base
Fluorescein/
Color of
Rhodamine
Unanalyzed Data
Dye Primer BigDye Primer on Electropherograma
Color of Raw Data
on Gel Imageb
Color of
Analyzed Data on
Electropherogramsc
A
JOE
dR6G
green
green
green
C
5-FAM
dR110
blue
blue
blue
G
TAMRA
dTAMRA
black
yellow
black
T
ROX
dROX
red
red
red
a. ABI PRISM 310 Genetic Analyzer.
b. ABI 373 and ABI PRISM 377 DNA Sequencers. See Table 2-1 on page 2-13 for chemistry/instrument compatibilities.
c. All instruments. See Table 2-1 on page 2-13 for chemistry/instrument compatibilities.
2-14 ABI PRISM DNA Sequencing Chemistries
Choosing a Sequencing Chemistry
Overview Although all of the sequencing chemistries are relatively versatile, some are better
choices than others for specific types of templates. No single chemistry works with
every template. While you can choose a single kit for most work, a second chemistry
or modifications to the standard protocol of the main sequencing chemistry may be
necessary. See “Troubleshooting DNA Sequence Composition Problems” on
page 7-30 for more information.
ABI PRISM 310,
ABI 373 with BigDye
Filter Wheel, and
ABI PRISM 377
We generally recommend the BigDye terminators because of their optimal
signal-to-noise characteristics, ease of use, and versatility. Table 2-6 shows the
chemistry recommendations for various applications.
Table 2-6 ABI PRISM 310, ABI 373 with BigDye Filter Wheel, and ABI PRISM 377
Chemistry Recommendations
dRhodamine
Terminator
BigDye
Terminator
BigDye
Primer
De novo sequencing—high throughput
S
Ra
R
De novo sequencing—mid-to-low throughput
S
R
S
Comparative sequencing
(germline mutations 50:50 heterozygotes)
S
R
R
Comparative sequencing
(somatic mutations 30:70 heterozygotes)
N
S
R
Comparative sequencing
(somatic mutations 10:90 heterozygotes)
N
N
S
Gene walking (custom primers)
S
R
N
Shotgun sequencing (universal primers, M13)
S
R
R
DNA Sequencing Application
Deletion clone sequencing (universal primers)
S
R
R
Gap closure (custom primers)
S
R
N
GC-rich >65%
S
R
S
AT-rich >65%
R
R
R
GT-rich regions
R
N
R
Homopolymer A or T >25 bpb
R
N
R
DNA Sequence Context
Template
Plasmid (<15 kb)
R
R
R
M13
R
R
R
BAC, cosmid, lambda, large PCR product
S
R
S
Bacterial genomic DNA
N
R
N
PCR amplicon
R
R
R
PCR amplicon (heterozygous 50:50)
S
R
R
PCR amplicon (heterozygous 30:70)
N
S
R
PCR amplicon (heterozygous 10:90)
N
N
S
a. R = recommended, S = satisfactory, N = not recommended
b. All cycle sequencing chemistries can have difficulties with homopolymers >40 bp.
ABI PRISM DNA Sequencing Chemistries 2-15
The dRhodamine terminators are useful for templates with long homopolymer
(>25 bases) stretches or templates with GT-rich motifs. However, the dRhodamine
terminators produce weaker signals than the BigDye chemistries. More of the sample
must be loaded to ensure adequate signal is available. This is especially important for
running 48-, 64- and 96-lane gels on the ABI PRISM 377 DNA Sequencer, where less
signal is detected because the lanes are narrower. To compensate for the decreased
signal strength with dRhodamine terminators, increase the CCD gain from 2 to 4.
ABI 373 DNA Table 2-7 provides a list of sequencing applications and suggests kits that best suit
Sequencer them.
Table 2-7 ABI 373 Chemistry Recommendations
Rhodamine
Dye Terminator
Fluorescein/
Rhodamine
Dye Primer
Ra
R
DNA Sequencing Application
De novo sequencing—high throughput
De novo sequencing—mid-to-low throughput
R
S
Comparative sequencing
(germline mutations 50:50 heterozygotes)
N
R
Comparative sequencing
(somatic mutations 30:70 heterozygotes)
N
R
Comparative sequencing
(somatic mutations 10:90 heterozygotes)
N
S
Gene walking (custom primers)
R
N
Shotgun sequencing (universal primers, M13)
R
R
Deletion clone sequencing (universal primers)
R
R
Gap closure (custom primers)
R
N
DNA Sequence Context
GC-rich >65%
R
S
AT-rich >65%
R
R
S
R
S
R
Plasmid (<15 kb)
R
R
M13
R
R
BAC, cosmid, lambda, large PCR product
S
S
GT-rich regions
Homopolymer A or T >25
bpb
Template
Bacterial genomic DNA
N
N
PCR amplicon
R
R
PCR amplicon (heterozygous 50:50)
N
R
PCR amplicon (heterozygous 30:70)
N
R
PCR amplicon (heterozygous 10:90)
N
R
a. R = recommended, S = satisfactory, N = not recommended
b. All cycle sequencing chemistries can have difficulties with homopolymers >40 bp.
2-16 ABI PRISM DNA Sequencing Chemistries
Performing DNA
Sequencing Reactions
3
3
Overview
Factors That Affect With careful template preparation and sequencing techniques, you can obtain reliable
Data Quality sequence data for both dye primer and dye terminator chemistries. This section
describes the factors affecting data quality, how they can be controlled during sample
preparation and the sequencing reactions, and how some fundamental errors can be
avoided and corrected:
Topic
See page
DNA Template Preparation
3-2
Sequencing PCR Templates
3-10
DNA Template Quality
3-15
DNA Template Quantity
3-17
Primer Design and Quantitation
3-18
Reagent and Equipment Considerations
3-20
Preparing Cycle Sequencing Reactions
3-21
Cycle Sequencing
3-27
Preparing Extension Products for Electrophoresis
3-33
Preparing and Loading Samples for Gel Electrophoresis
3-50
Preparing and Loading Samples for Capillary Electrophoresis
3-53
Performing DNA Sequencing Reactions 3-1
DNA Template Preparation
Overview The DNA purification method used can affect the quality of the template. Some
recommendations for purifying DNA templates are given below.
Prepare adequate template to check purity (see “Determining DNA Quality” on
page 3-16), to quantitate the DNA accurately (see “Quantitating DNA” on page 3-17),
and to perform the sequencing reactions. The recommended quantities for
sequencing reactions are shown in Table 3-1 on page 3-17.
Single-stranded You can use the following methods to prepare single-stranded templates such as M13:
DNA Templates ♦ QIAGEN (http://www.qiagen.com) QIAprep Spin M13 Kit (P/N 27704,
50 reactions)
♦
High-throughput (ThermoMAX procedure, see below)
♦
PEG precipitation followed by phenol extraction (see below)
ThermoMAX Procedure
Cells infected with recombinant M13 phage are grown in liquid medium. The growth
medium is clarified by centrifugation and PEG precipitation. The phage particles are
resuspended in buffer and then heated to release the single-stranded DNA.
Reagents and equipment required:
♦
2X TY medium, pH 7.2–7.4
Step
1
Action
Combine the following:
♦
Bactotryptone, 16.0 g
♦
Yeast extract, 5.0 g
♦
NaCl, 5.0 g
Make up to 1 L in autoclaved water.
2
♦
Adjust the pH to 7.2–7.4 with NaOH.
PEG solution (20% PEG, 2.5 M NaCl)
Make up fresh as needed from equal volumes of 40% PEG (in deionized water)
and 5 M NaCl stocks.
♦
TTE buffer (0.25% v/v Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
Step
1
2
Action
Combine the following:
♦
Tris-HCl (pH 8.0), 1 M, 500 µL
♦
Na2EDTA, 0.5 M, 10 µL
♦
Triton X-100, 250 µL
Make up to 50 mL in deionized water.
♦
96-cap sealer (Beckman)
♦
Adhesive-backed aluminum foil tape (3M Scotch Tape 425-3)1
3-2 Performing DNA Sequencing Reactions
♦
Centrifuge with 96-tube tray adapter
♦
Sterile 1.2-mL culture tubes, 96/box
♦
Sterile toothpicks
To grow M13-infected cells:
Step
Action
1
Inoculate 250 mL of 2X TY with 1 mL of JM101.
2
Transfer 0.8 mL of the JM101 culture to each of 96 1.2-mL mini-tubes (one box).
3
Wearing clean gloves, pick M13 plaques using sterile toothpicks. Drop each
toothpick into a culture tube. Remove toothpicks after all 96 have been picked.
4
Cover the rack of tubes with the provided cover. Shake the tubes at 37 °C for
18–19 hours.
To precipitate M13 phage particles with PEG:
Step
Action
1
Spin the cultures in a centrifuge at 3250 rpm for 15 minutes to pellet the cells.
2
While the cells are pelleting, add 120 µL of PEG solution to each tube in a second
96-tube box.
3
Carefully transfer 0.6 mL of M13 supernatant from each tube from the first box to
the corresponding tube in the second box.
4
Cover the tubes with a Beckman 96-cap sealer, and invert several times to mix.
5
Leave the tubes at room temperature for 30 minutes, then chill at 2–6 °C for
30 minutes.
6
Spin the cultures in a centrifuge at 3250 rpm for 15 minutes to pellet the M13
particles.
7
When centrifugation is finished, check for pellets by discarding the supernatant
from a single row. Be sure to place the row back in place correctly.
If the pellets are present then discard the supernatant from the rest of the samples.
8
Place the inverted tubes on a paper towel for a few minutes to drain.
9
With the tubes still inverted, place them on a dry paper towel in the centrifuge
carrier. Spin the inverted tubes in a centrifuge at 300 rpm for 3–5 minutes to remove
all traces of PEG.
After spinning, check to see that the pellets have stayed at the bottoms of the tubes.
To extract the M13 DNA:
Step
Action
1
Add 20 µL of TTE buffer to each tube.
2
Seal the tubes with aluminum foil tape and vortex vigorously.
Note
3
1.
Ensure that the pellet is well suspended.
Heat the tubes at 80 °C for 10 minutes in a water bath.
Contact 3M in the USA at (800) 364-3577 for your local 3M representative. Use of other tapes may result
in leakage or contamination of the sample.
Performing DNA Sequencing Reactions 3-3
To extract the M13 DNA:
Step
(continued)
Action
4
While the samples are heating, add 70 µL of deionized water to each well of a fresh
96-well plate.
5
When heating is complete, spin the samples briefly in a centrifuge to return the
liquid to the bottoms of the tubes. Transfer the samples to the 96-well plate
containing the deionized water.
6
Cover the samples with foil tape and place in a non-frost-free, –15 to –25 °C
freezer.
For more information, refer to the Washington University School of Medicine Genome
Sequencing Center Genome Sequencing Manual (http://genome.wustl.edu/gsc/
manual/protocols/M13_ThermoMAX_prep.html).
Preparing DNA from M13 Phage by PEG Precipitation and Phenol Extraction
Cells infected with recombinant M13 phage are grown in liquid medium. After
clarification of the growth medium by centrifugation and PEG precipitation,
single-stranded DNA is extracted from the phage particles in the supernatant.
Reagents and equipment required:
♦
2X TY medium, pH 7.2–7.4
Step
1
Action
Combine the following:
♦
Bactotryptone, 16.0 g
♦
Yeast extract, 5.0 g
♦
NaCl, 5.0 g
Make up to 1 L in autoclaved water.
2
♦
Adjust the pH to 7.2–7.4 with NaOH.
Chloroform
! WARNING ! CHEMICAL HAZARD. Chloroform is extremely toxic and a potential
human carcinogen. This chemical is highly corrosive to skin and eyes. Always work in a
fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate
protective eyewear, clothing, and gloves.
♦
Ethanol, 95%
♦
Ethanol, 70%
♦
PEG solution (20% PEG, 2.5 M NaCl)
Make up fresh as needed from equal volumes of 40% PEG (in deionized water)
and 5 M NaCl stocks.
♦
Sodium acetate (NaOAc), 3 M, pH 5.2
♦
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
♦
TE0.1 buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0)
♦
Tris buffer (10 mM, pH 8.0)
♦
Tris-saturated phenol, pH >7.6
3-4 Performing DNA Sequencing Reactions
! WARNING ! CHEMICAL HAZARD. Phenol is a highly toxic, combustible, and
vesicant chemical that causes burns and is readily absorbed through the skin. It is
extremely destructive to mucous membranes, eyes, and skin. Inhalation and ingestion
can cause CNS, liver, pancreas, and spleen damage, and can be fatal. Always work in a
fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate
protective eyewear, clothing, and gloves.
♦
1.5-mL microcentrifuge tubes
♦
Sterile 10-mL tubes (centrifugable at 10,000 ¥ g)
♦
Sterile cotton-tipped swabs
♦
Sterile Pasteur pipettes
To grow M13-infected cells:
Step
Action
1
Inoculate 10 mL of 2X TY medium with a single colony of E. coli grown on glucose
minimal agar.
2
Shake overnight at 37 °C.
3
Dilute an aliquot of the culture 1:100 in 2X TY medium and dispense 5-mL aliquots
into sterile 10-mL tubes.
4
Using the tip of a Pasteur pipette, add one agar plug from the recombinant plaque
to each 10-mL tube.
5
Shake the 10-mL tubes at 37 °C for 6–7 hours.
Note
Incubation longer than 6–7 hours may complicate purification.
To clarify the culture by centrifugation and PEG precipitation:
Step
1
Action
Spin the culture in a centrifuge at 2500 ¥ g for 10 minutes. Transfer the supernatant
containing the M13 particles to a sterile tube.
Note
2
The supernatant can be stored for up to 1 month at 2–6 °C.
Spin the supernatant in a centrifuge for 10 minutes at 5000 ¥ g. Transfer the
resulting supernatant to a fresh 10-mL tube that will withstand centrifugation at
10,000 ¥ g.
Note
This second centrifugation is necessary even if supernatants have not
been stored.
Note
The supernatant from the second centrifugation can be stored after
addition of 50 µL chloroform/mL of supernatant. The chloroform kills any remaining
bacteria and destroys any enzymes without damaging the DNA.
3
Add 1 mL PEG solution to aggregate the M13 particles. Mix the culture and PEG
solution well and allow the mixture to stand at 2–6 °C for 30 minutes.
4
Centrifuge the PEG/phage suspension for 10 minutes at 10,000 ¥ g to sediment the
M13 particles.
5
Aspirate and discard the supernatant. Invert the tube to drain excess liquid, and
carefully remove any remaining traces of supernatant with cotton-tipped swabs.
IMPORTANT
Removal of all supernatant is critical. PEG and salt inhibit
sequencing reactions.
The PEG/phage pellet should be visible at this stage of preparation.
Performing DNA Sequencing Reactions 3-5
To extract DNA from the phage particles:
Step
Action
1
Resuspend the pellet in 400 µL TE buffer. Transfer the suspension to
microcentrifuge tubes.
2
Extract the suspension twice with Tris-saturated phenol:
a.
Add 400 µL of Tris-saturated phenol.
b.
Vortex to mix.
c.
Centrifuge the extraction mix for 1 minute in a microcentrifuge to separate the
organic and aqueous phases.
d.
Remove the upper aqueous phase, and transfer it to a fresh microcentrifuge
tube.
IMPORTANT
Take care not to disturb the aqueous/organic interface, which
contains lipid and denatured protein that can inhibit the DNA polymerase used for
cycle sequencing reactions.
3
Extract the aqueous phase twice with chloroform to remove phenol, which can
affect sequencing data:
a.
Add 400 µL of chloroform.
b.
Vortex each extraction until all cloudiness disappears.
c.
Spin the emulsion 30 seconds in a microcentrifuge to separate the phases.
d.
Transfer the upper aqueous phase to a fresh microcentrifuge tube.
Remove any remaining chloroform by vacuum centrifugation.
4
Precipitate the DNA:
a.
Add 40 µL of 3 M sodium acetate and 1 mL of 95% ethanol.
b.
Invert the tube to mix.
c.
Chill the mixture for at least 20 minutes at –70 °C or overnight at –15 to –25 °C.
5
Spin the mixture in a microcentrifuge for 15 minutes at maximum speed. Aspirate all
of the supernatant and discard.
6
Wash the pellet with 70% ethanol:
a.
7
Add 250 µL of 70% ethanol to the pellet.
b.
Spin in a microcentrifuge at maximum speed for 5 minutes.
c.
Carefully draw off the ethanol and dry the pellet in a vacuum centrifuge for no
more than 5 minutes.
Dissolve the DNA pellet in 20 µL of deionized water or TE0.1 buffer.
Assess the quality of the DNA spectrophotometrically or by agarose gel
electrophoresis (see “Determining DNA Quality” on page 3-16). Quantitate the DNA
spectrophotometrically (see “Quantitating DNA” on page 3-17).
8
Store the DNA at –15 to –25 °C until needed for sequencing reactions.
Plasmid DNA When purifying recombinant plasmids in bacteria, plate out the transformants to
Templates obtain isolated colonies. Select a single colony and restreak out on a plate. Select an
isolated colony from that plate to obtain plasmids with the desired insert.
The optimal procedure for preparing a particular plasmid depends on the particular
bacterial strain and the yield of each construct. Good sequencing data has been
obtained from the following methods:
♦
ABI PRISM™ Plasmid Miniprep Kit (P/N 402790 or 402791)
3-6 Performing DNA Sequencing Reactions
♦
Cesium chloride (CsCl) banding
♦
Modified alkaline lysis/PEG precipitation method (see below)
♦
PureGene DNA Isolation Kit (Gentra Systems, Inc., P/N D-5500A)
♦
QIAGEN Plasmid Kits (http://www.qiagen.com):
–
Mini (P/N 12123, 25 reactions; 12125, 100 reactions)
–
Midi (P/N 12143, 25 reactions; 12144, 50 reactions; 12145, 100 reactions)
–
Maxi (P/N 12162, 10 reactions; 12163, 25 reactions; 12165, 100 reactions)
Modified Alkaline Lysis/PEG Method
Reagents required:
♦
Chloroform
! WARNING ! CHEMICAL HAZARD. Chloroform is extremely toxic and a potential
human carcinogen. This chemical is highly corrosive to skin and eyes. Always work in a
fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate
protective eyewear, clothing, and gloves.
♦
Deionized water
♦
Ethanol, 70%
♦
GET buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0)
♦
Isopropanol, 100% (anhydrous)
♦
PEG 8000, 13% (sterilized by autoclaving, rather than by filtration)
! WARNING ! CHEMICAL HAZARD. Polyethylene glycol 8000 (PEG) can be harmful
if inhaled, ingested, or absorbed through the skin. It is irritating to the eyes, skin, and
mucous membranes. Always work in a fume hood. Obtain a copy of the MSDS from the
manufacturer. Wear appropriate protective eyewear, clothing, and gloves.
♦
Potassium acetate, 3 M, pH 4.8
♦
RNase A (DNase-free), 10 mg/mL
♦
Sodium chloride (NaCl), 4 M
♦
Sodium hydroxide (NaOH), 0.2 N, with 1% SDS (freshly made)
! WARNING ! CHEMICAL HAZARD. Sodium hydroxide (NaOH) can cause severe
burns to the skin, eyes, and respiratory tract. Always work in a fume hood. Obtain a copy
of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and
gloves.
! WARNING ! CHEMICAL HAZARD. Sodium dodecyl sulfate (SDS) is a toxic
chemical that is harmful to the lungs and internal organs if swallowed. Contact with the
eyes can cause serious damage. Always work in a fume hood. Obtain a copy of the
MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves.
Performing DNA Sequencing Reactions 3-7
Note
To minimize shearing of contaminating chromosomal DNA, do not use a vortexer
during this procedure.
To prepare plasmid DNA by alkaline lysis/PEG treatment:
Step
1
Action
Pellet 1.5-mL aliquots of culture for 1 minute in a microcentrifuge at maximum
speed.
Note
A total culture volume of 4.5 mL can be spun down per tube without
changing volumes in the procedure. This allows you to achieve a threefold increase
in yield while eliminating the need for extra tubes and additional handling.
2
Remove the supernatant by aspiration.
3
Resuspend the bacterial pellet in 200 µL of GET buffer by pipetting up and down.
4
Add 300 µL of freshly prepared 0.2 N NaOH/1% SDS. Mix the contents of the tube
by inversion. Incubate on ice for 5 minutes.
5
Neutralize the solution by adding 300 µL of 3.0 M potassium acetate, pH 4.8. Mix by
inverting the tube. Incubate on ice for 5 minutes.
6
Remove cellular debris by spinning in a microcentrifuge at maximum speed for 10
minutes at room temperature. Transfer the supernatant to a clean tube.
7
Add RNase A (DNase-free) to a final concentration of 20 µg/mL. Incubate the tube
at 37 °C for 20 minutes.
8
Extract the supernatant twice with chloroform:
a.
Add 400 µL of chloroform.
b.
Mix the layers by inversion for 30 seconds.
c.
Centrifuge the tube for 1 minute to separate the phases.
d.
Transfer the upper aqueous phase to a clean tube.
9
Add an equal volume of 100% isopropanol. Mix the contents of the tube by
inversion.
10
Spin the tube in a microcentrifuge at maximum speed for 10 minutes at room
temperature.
11
Remove the isopropanol completely by aspiration.
12
Wash the DNA pellet with 500 µL of 70% ethanol. Dry under vacuum for 3 minutes.
13
Dissolve the pellet in 32 µL of deionized water.
14
Add 8.0 µL of 4 M NaCl, then 40 µL of autoclaved 13% PEG 8000.
15
Mix thoroughly, then leave the sample on ice for 20 minutes.
16
Pellet the plasmid DNA by spinning in a microcentrifuge at maximum speed for
15 minutes at 2–6 °C.
17
Carefully remove the supernatant. Rinse the pellet with 500 µL of 70% ethanol.
18
Resuspend the pellet in 20 µL of deionized water. Store at –15 to –25 °C.
3-8 Performing DNA Sequencing Reactions
BAC DNA Templates With larger DNA targets such as bacterial artificial chromosomes (BACs), the quality
of DNA template is important to the success of the sequencing reaction. Two methods
have given good sequencing results:
♦
Alkaline lysis, with extra phenol extraction and isopropanol precipitation if very
clean DNA is desired (Marra et al., 1996)
♦
Cesium chloride (CsCl) banding
Commercial kits are also available for BAC DNA preparation:
♦
♦
ProPrep BAC (LigoChem, http://www.ligochem.com)
–
Individual reactions: P/N PLK-100, 100 reactions; PLK-1000, 1000 reactions
–
96-well plates: P/N PLF-1000, 1 plate; PLF-1000, 10 plates; PLF-2500,
25 plates)
QIAGEN-tip 100 (QIAGEN: P/N 10043, 25 reactions; 10045, 100 reactions) and
QIAGEN-tip 500 (QIAGEN: P/N 10063, 25 reactions; 10065, 100 reactions)
For other BAC DNA preparation protocols, refer to the following Web sites:
♦
Centre National de Séquençage (CNS, or Génoscope):
http://www.cns.fr/externe/arabidopsis/protoBAC.html
♦
The Institute for Genome Research (TIGR):
http://www.tigr.org/softlab/TPFBACmultiprep.052397.html
♦
University of Oklahoma Advanced Center for Genome Technology (ACGT):
http://www.genome.ou.edu/DblAcetateProcV3.html
♦
Washington University School of Medicine Genome Sequencing Center:
http://genome.wustl.edu/gsc/manual/protocols/BAC.html
Performing DNA Sequencing Reactions 3-9
Sequencing PCR Templates
Overview This section provides information about preparing and sequencing PCR products, but
it is not meant to be a detailed guide to PCR amplification. General information on
PCR amplification can be found in the Guide to PCR Enzymes (Stock No. 700905)
and in the product inserts included with GeneAmp® PCR reagents. For PCR
amplification, use GeneAmp® PCR Instrument Systems and GeneAmp PCR Core
Reagents.
Although PCR fragments can be difficult to denature with traditional
single-temperature sequencing methods, cycle sequencing provides several chances
to denature and extend the template, which ensures adequate signal in the
sequencing reaction.
Visualize PCR products by agarose gel electrophoresis (see “Determining DNA
Quality” on page 3-16).
For more detailed information about PCR sequencing, refer to Comparative PCR
Sequencing, A Guide to Sequencing-Based Mutation Detection (Stock No.
770901-001). This booklet can also be obtained from the Applied Biosystems WWW
site (www.appliedbiosystems.com/techsupport).
PCR Strategies Single Amplification
In the simplest PCR sequencing case, the target DNA is amplified with a single set of
primers and then sequenced using the same primers. For many samples, this works
well. For the samples that do not work well with this method, optimization of the PCR
amplification may be required (see page 3-12). Optimizing the PCR minimizes the
presence of non-specific product bands and ensures adequate yield.
A single PCR amplification is also compatible with the use of a sequencing primer that
binds internally (semi-nested or nested) to one or both of the PCR primers. This can
be helpful if primer-dimer (primer oligomerization) artifacts are a problem (see
Figure 7-31 on page 7-24).
Nested and Semi-Nested PCR
If you encounter difficulty with more complex samples, such as bacterial genomic
DNA, use a nested or semi-nested PCR. These techniques are useful when the target
is present in small quantity. They offer more specificity, which provides superior
sequencing data with reduced background signal.
Both nested and semi-nested PCR require two amplifications. The first amplification is
identical for nested and semi-nested, but the second amplification differs as described
in the following paragraphs.
♦
Amplify with one set of PCR primers, which converts a complex sample (such as
bacterial genomic DNA) into a non-complex sample consisting of the first PCR
product and some side products.
♦
Nested PCR—Amplify 1% or less of the first PCR reaction product using a second
set of PCR primers that hybridize at positions internal to the first set.
♦
Semi-nested PCR—Only one primer of the second set of PCR primers is internal.
The other primer is one of the original PCR primers.
3-10 Performing DNA Sequencing Reactions
Universal-Tailed PCR Primers
A PCR primer can be synthesized with a universal sequencing primer binding site
added to the 5´ end (see Appendix E for universal primer sequences). This allows any
PCR product to be sequenced with universal primers.
Universal-tailed PCR primers enable the use of commercially available dye-labeled
sequencing primers. This technique is also useful with dye terminator chemistries,
because universal sequencing primers have good annealing characteristics. However,
the longer PCR primers add to the overall cost of the reactions.
Using universal-tailed primers sometimes results in primer oligomerization. As these
products have priming sites present, they can result in noisy data for the first 20–100
bases (see page 7-24). Redesigning the PCR primer, optimizing the PCR
amplification further, and employing Hot Start methods can help overcome this
situation.
Contaminants That Excess Primers and dNTPs
Affect PCR After PCR amplification, the resulting PCR product is in solution along with PCR
Sequencing primers, dNTPs, enzyme, and buffer components. The method used to prepare the
PCR product for sequencing depends on the amounts of these components that are
carried over and on the chemistry used for sequencing.
Excess PCR primers carried over from the amplification reaction compete with the
sequencing primer for binding sites and reagents in the sequencing reaction. This
carryover of PCR primers presents more of a problem in dye terminator chemistries
because the dye label is incorporated into the extension product after the primer
anneals to the template. If more than one primer is present, multiple dye-labeled
sequence ladders are generated, resulting in noisy data.
Excess dNTPs from the amplification reaction can affect the balance of the
sequencing reaction, resulting in decreased termination in shorter extension
fragments.
Nonspecific PCR Products
Nonspecific PCR products include primer-dimer artifacts and secondary PCR
products. The presence of any significant quantity of either in a PCR product can
result in poor quality sequencing data. Nonspecific PCR products behave as
templates in the sequencing reaction and produce extension products, which results
in noisy data.
These products often can be visualized on an agarose gel before sequencing. If they
are present, the PCR amplification should be optimized and repeated before
sequencing. Use of a nested or semi-nested sequencing primer can also allow good
sequence data to be obtained. Alternatively, the PCR product of interest can be
purified by agarose gel electrophoresis.
Performing DNA Sequencing Reactions 3-11
Minimizing There are several ways to minimize contaminants in a PCR amplification:
Contaminants ♦ PCR optimization (Innis and Gelfand, 1990):
–
Amount of starting DNA
–
Careful primer design
–
Primer concentration
–
Enzyme concentration
–
Magnesium ion (Mg2+) concentration
–
Nucleotide concentration
–
Buffer composition
–
Number of cycles
–
pH
♦
Manual Hot Start method
♦
AmpliTaq Gold® DNA Polymerase as an automatic Hot Start
♦
Limiting dNTPs and primers
All of these methods increase the specificity of the PCR amplification and decrease
the amount of contaminants that can interfere with a sequencing reaction.
Preparing PCR Purification
Products for There are several methods for purifying PCR products:
Sequencing
♦
Column purification
♦
Ethanol precipitation
♦
Gel purification
IMPORTANT
If more than one PCR product is present, neither column purification nor
ethanol precipitation will isolate the desired product. Use gel purification to isolate the desired
product or reoptimize the PCR to obtain a single product.
Commercially available products for PCR product purification are listed below:
♦
Centricon®-100 columns (P/N N930-2119)
These columns contain an ultrafiltration membrane that separates primers and
dNTPs from larger PCR products. However, they may not work as well for short
PCR products (<125 bases).
To purify PCR fragments by ultrafiltration:
Step
Action
1
Assemble the Centricon-100 column according to the manufacturer’s
recommendations.
2
Load 2 mL deionized water onto the column.
3
Add the entire sample to the column.
4
Spin the column at 3000 ¥ g in a fixed-angle centrifuge for 10 minutes.
Note
The manufacturer recommends a maximum speed of 1000 ¥ g, but
3000 ¥ g has worked well in Applied Biosystems. If you are following the
manufacturer’s guidelines, increase the time to compensate.
3-12 Performing DNA Sequencing Reactions
To purify PCR fragments by ultrafiltration:
Step
♦
(continued)
Action
5
Remove the waste receptacle and attach the collection vial.
6
Invert the column and spin it at 270 ¥ g for 2 minutes to collect the sample. This
should yield approximately 40–60 µL of sample.
7
Add deionized water to bring the purified PCR fragments to the original volume.
QIAquick PCR Purification Kits (QIAGEN: P/N 28104, 50 reactions; 28106,
250 reactions)
These kits work well for PCR products ranging from 100 bp–10 kbp.
♦
QIAquick Gel Extraction Kits (QIAGEN: P/N 28704, 50 reactions; 28706,
250 reactions)
These kits are used to purify PCR fragments from agarose gels. The kits work well
for DNA ranging from 70 bp–10 kbp. Fragments larger than this should be
extracted with the QIAEX II Gel Extraction Kits (QIAGEN: P/N 20021,
150 reactions; 20051, 500 reactions).
Refer to the manufacturer’s instructions for the procedures.
The recommended DNA quantities for sequencing reactions are shown in Table 3-1
on page 3-17.
Shrimp Alkaline Phosphatase and Exonuclease I Treatment
An alternative to one of the more stringent purification methods listed above is
treatment of PCR products with shrimp alkaline phosphatase (SAP) and
exonuclease I (Exo I) before sequencing. The SAP/Exo I procedure degrades
nucleotides and single-stranded DNA (primers) remaining after PCR (Werle et al.,
1994). This procedure is particularly useful in cases where limiting concentrations of
primers and nucleotides cannot be used for direct PCR sequencing (see page 3-14).
IMPORTANT
This method only works when a single PCR product is obtained.
To degrade PCR primers and nucleotides using SAP/Exo I:
Step
1
Action
For each sample, combine the following:
♦
SAP(1 Unit/µL), 2 µL
♦
Exo I (10 Units/µL), 0.2 µL
♦
Deionized water, 6.0 µL
Note
In general this procedure works well using 0.5 units of each enzyme per
microliter of PCR products used. The procedure seems to work equally well with or
without the use of SAP buffer, so this has been excluded in this protocol.
2
Add 4.0 µL of PCR product to the above mix.
3
Incubate at 37 °C for 1 hour.
4
Incubate at 72 °C for 15 minutes to inactivate the enzymes.
The PCR product may need dilution before sequencing. Determine the dilution ratio
empirically (start with 1:2 and 1:10 dilutions with deionized water).
Performing DNA Sequencing Reactions 3-13
The recommended DNA quantities for sequencing reactions are shown in Table 3-1
on page 3-17.
Direct PCR PCR protocols that limit amounts of primers and dNTPs allow the product of the
Sequencing reaction to be used for sequencing with no purification. This is usually carried out by
setting up the PCR amplification with 5–10 pmol of primers and 20–40 µM dNTPs, so
that most of the primers and dNTPs are exhausted during amplification.
If the yield of the desired PCR product is high and the product is specific, i.e., it
produces a single band when analyzed by agarose gel electrophoresis, the sample
can be diluted before sequencing and will give good results. The dilution ratio depends
on the concentration of your PCR product and needs to be determined empirically
(start with 1:2 and 1:10 dilutions with deionized water).
When you limit concentrations of primers and dNTPs and dilute the PCR products, the
PCR parameters have to be robust. Direct PCR sequencing is most useful in
applications where the same target is being amplified and sequenced repeatedly and
PCR conditions have been optimized.
Direct PCR sequencing is usually done with dye primer chemistries. With dye
terminator chemistries, it is much more critical that the PCR primers be consumed.
Excess PCR primers will be extended and labeled by the cycle sequencing reaction,
resulting in noisy data.
Direct PCR sequencing does not work for XL PCR because limiting amounts of
primers and dNTPs cannot be used. The PCR product should be purified (see
page 3-12) or the excess primers and nucleotides should be degraded by SAP/Exo I
treatment (see page 3-13).
3-14 Performing DNA Sequencing Reactions
DNA Template Quality
Using Control DNA Include a control DNA template as one of the templates in a set of sequencing
reactions. The results from the control can help determine whether failed reactions are
the result of poor template quality or sequencing reaction failure.
We recommend M13mp18 as a single-stranded control and pGEM®-3Zf(+) as a
double-stranded control. All Applied Biosystems DNA sequencing kits provide pGEM
control DNA. All dye terminator cycle sequencing kits include a –21 M13 control
primer.
Sequencing Standards
The Cycle Sequencing Standards provide an additional control to help in
troubleshooting electrophoresis runs. These standards contain lyophilized sequencing
reactions that only require resuspension and denaturation before use. There are four
standards available:
♦
Dye Primer Cycle Sequencing Standard (P/N 401920)
♦
Dye Terminator Cycle Sequencing Standard (P/N 402830)
♦
dRhodamine Terminator Cycle Sequencing Standard (P/N 4303120)
♦
BigDye™ Terminator Cycle Sequencing Standard (P/N 4304154)
Poor Template Poor template quality is the most common cause of sequencing problems. Always
Quality follow recommended procedures to prepare templates (see “DNA Template
Preparation” on page 3-2).
The following are characteristics of poor quality templates:
♦
Noisy data or peaks under peaks (see page 7-11)
♦
No usable sequence data (see Figure 7-9 on page 7-10)
♦
Weak signal (see Figure 7-10 on page 7-11)
Contamination Potential contaminants include:
♦
Proteins
♦
RNA
♦
Chromosomal DNA
♦
Excess PCR primers, dNTPs, enzyme, and buffer components (from a PCR
amplification used to generate the sequencing template)
♦
Residual salts
♦
Residual organic chemicals, e.g., phenol, chloroform, and ethanol
♦
Residual detergents
Performing DNA Sequencing Reactions 3-15
Host Strain The host strain used for template preparation can impact template quality. One host
Variability strain may produce better sequencing results for a specific template than another.
If you plan to use a commercial template preparation kit, contact the vendor for
information about host strains that work well with that kit.
A good source of information relating to host strain effects can be found in the
QIAGEN Guide to Template Preparation and DNA Sequencing (2nd edition). Contact
your local QIAGEN office (http://www.qiagen.com/qiagenww.html) to obtain a copy of
this guide.
Determining DNA The following methods can be used to examine DNA quality:
Quality ♦ Agarose gel electrophoresis
Purified DNA should run as a single band on an agarose gel.
Note
♦
Uncut plasmid DNA can run as three bands: supercoiled, nicked, and linear.
Spectrophotometry
The A260/A280 ratio should be 1.7–1.9. Smaller ratios usually indicate
contamination by protein or organic chemicals.
Agarose gels reveal the presence of contaminating DNAs and RNAs, but not proteins.
Spectrophotometry can indicate the presence of protein contamination, but not DNA
and RNA contamination. These methods should be used together to get the most
information about your DNA template before sequencing.
Note
RNA contamination up to 1 µg can be tolerated, but it will affect DNA quantitation
greatly.
Neither of these methods shows the presence of contaminating salts that can cause
noisy data. If you suspect that your DNA is contaminated with salt, remove the salt
before sequencing. The most efficient method for salt removal is ultrafiltration with a
Centricon-100 column (see page 3-12). Spin columns and ethanol precipitation can
also be used (see page 3-33).
Cleaning Up Dirty A “dirty” template preparation sometimes can be cleaned up with one of the following
Templates methods:
♦
Purify the DNA by ultrafiltration. Use Centricon-100 Micro-Concentrator columns
(see “Preparing PCR Products for Sequencing” on page 3-12).
♦
Purify by extraction:
Step
Action
1
Extract the DNA twice with 1 volume of chloroform or chloroform:isoamyl alcohol
(24:1 v/v).
2
Add 0.16 volumes of 5M NaCl and 1 total volume of 13% PEG.
3
Incubate on ice for 20 minutes, then centrifuge at maximum speed in a
microcentrifuge at 2–6 °C for 20 minutes.
4
Rinse the pellet twice with 70% ethanol.
5
Dry the pellet in a vacuum centrifuge for 3–5 minutes or to dryness.
3-16 Performing DNA Sequencing Reactions
DNA Template Quantity
Quantitating DNA DNA template quantitation is critical for successful sequencing reactions. The most
common way to determine DNA quantity is to measure the absorbance (optical
density or O.D.) of a sample at 260 nm in a spectrophotometer.
One O.D. unit is the amount of a substance dissolved in 1.0 mL that gives an
absorbance reading of 1.00 in a spectrophotometer with a 1-cm path length. The
wavelength is assumed to be 260 nm unless stated otherwise. A260 values can be
converted into ng/µL using Beer´s Law:
Absorbance (260 nm)
= sum of extinction coefficient contributions ¥ cuvette pathlength ¥
concentration
The following formulas are derived from Beer’s Law (Ausubel et al., 1998):
♦
One A260 unit of single-stranded DNA contains 33 ng/µL.
♦
One A260 unit of double-stranded DNA contains 50 ng/µL.
Note
Absorbance measurements of highly concentrated (O.D.>1.0) or very dilute
(O.D.<0.05) DNA samples can be inaccurate.
DNA can also be quantitated by fluorometric analysis employing either Hoechst dye
#33258 (Hoefer, Inc., 1993) or Picogreen (Molecular Probes, Inc., 1995).
Amount to Use in The amount of DNA template used in a sequencing reaction can affect the quality of
Sequencing the data. Too much template makes data appear top heavy with strong peaks at the
Reactions beginning of the run that fade rapidly. Too little template or primer reduces the signal
strength and peak height. In the worst case, the noise level increases so that bases
cannot be called. Table 3-1 shows the recommended quantities for each sequencing
chemistry.
Table 3-1 Recommended Ranges of DNA Template Quantity for Each Chemistry
Cycle Sequencing Chemistry
Rhodamine
Dye Terminator
dRhodamine
Terminator
BigDye
Terminator
Fluorescein/
Rhodamine
Dye Primer
BigDye Primer
100–200 bp
1–3 ng
1–3 ng
1–3 ng
2–5 ng
2–5 ng
200–500 bp
3–10 ng
3–10 ng
3–10 ng
5–10 ng
5–10 ng
500–1000 bp
5–20 ng
5–20 ng
5–20 ng
10–20 ng
10–20 ng
1000–2000 bp
10–40 ng
10–40 ng
10–40 ng
20–50 ng
20–50 ng
>2000 bp
40–100 ng
40–100 ng
40–100 ng
50–150 ng
50–150 ng
Template
PCR product:
single-stranded
100–250 ng
50–100 ng
50–100 ng
150–300 ng
150–400 ng
double-stranded
200–500 ng
200–500 ng
200–500 ng
300–600 ng
200–800 ng
cosmid, BAC
0.5–2.0 µg
not
recommended
0.5–1.0 µg
0.5–2.0 µg
0.5–1.0 µg
bacterial genomic
DNA
not recommended
2–3 µg
not recommended
Performing DNA Sequencing Reactions 3-17
Primer Design and Quantitation
Overview The choice of primer sequence, method of primer synthesis, and approach to primer
purification can have a significant effect on the quality of the sequencing data obtained
in dye terminator cycle sequencing reactions. Dye primer cycle sequencing kits
include dye-labeled primers that are already optimized and quantitated.
Some of the recommendations given here are based on information that is general
knowledge, while others are based on practical experience gained by scientists at
Applied Biosystems.
Primer Design The following recommendations are provided to help optimize primer selection:
♦
Use Primer Express™ software (P/N 402089) for primer design.
Primer Express software is useful in identifying potential secondary structure
problems, calculating melting temperature (Tm) more accurately, and determining
if a secondary hybridization site exists on the target DNA.
♦
Primers should be at least 18 bases long to ensure good hybridization.
♦
Avoid runs of an identical nucleotide, especially runs of four or more Gs.
♦
Keep the G-C content in the range 30–80%, preferably 50–55%.
♦
For cycle sequencing, primers with Tm>45 °C produce better results than primers
with lower Tm using our recommended thermal cycling parameters.
♦
For primers with a G-C content less than 50%, it may be necessary to extend the
primer sequence beyond 18 bases to keep the Tm>45 °C.
♦
Use of primers longer than 18 bases also minimizes the chance of having a
secondary hybridization site on the target DNA.
♦
Avoid primers that can hybridize to form dimers.
♦
Avoid palindromes because they can form secondary structures.
♦
The primer should be as pure as possible, preferably purified by HPLC.
Estimating Melting The following formula can be used for a rough estimate of melting temperature:
Temperature
Tm = (number of A + T residues) ¥ 2 °C + (number of G + C residues) ¥ 4 °C
3-18 Performing DNA Sequencing Reactions
Primer Quantitation The following formula, which is derived from Beer’s Law, converts A260 readings into
pmol/µL concentrations:
C (pmol/µL or µM) = (A 260 ¥ 100)/(1.54nA + 0.75nC + 1.17nG + 0.92nT)
where:
C = concentration
nx = number of residues of base x in the oligonucleotide
Oligonucleotide Molecular weight of a DNA oligonucleotide (sodium salt, pH≥7):
Molecular Weights
MW = (NA x 335.2) + (NC x 311.2) + (NG x 351.2)+ (NT x 326.2) + P
where:
NX = number of residues of base x in the oligonucleotide
P = –101.0 for dephosphorylated oligonucleotides, 40.0 for phosphorylated
oligonucleotides
Primer Problems
Table 3-2 Primer Problems and Possible Causes
and Possible Causes
Problems
Possible Causes
Poor priming resulting in
weak or no signal
Melting temperature is too low due to low GC content and/or
short primer length
Secondary structure of the primer, particularly at the 3´ end
Secondary structure of the template in the region of
hybridization
Incorrect primer concentration
Priming site not present
Adequate signal
strength with noisy data
Secondary hybridization site, which results in many extra
peaks
Impure primer. You may see a shadow sequence of N–1.
Custom You can obtain custom primers from the Applied Biosystems Custom Oligonucleotide
Oligonucleotides Synthesis Service:
♦
Phone: (800) 345-5224
♦
E-mail: [email protected]
♦
Online: www.appliedbiosystems.com/techsupport
Performing DNA Sequencing Reactions 3-19
Reagent and Equipment Considerations
Reagent Handling The freshest reagents are likely to perform the best. The following methods are
and recommended for guaranteeing reagent freshness.
Reaction Storage ♦ Store reagents at –15 to –25 °C when not in use, and thaw completely at room
temperature or in an ice bath (do not heat) before use.
Note
Do not use a frost-free freezer. The automatic cycling of the temperature for defrosting
can damage reagents, particularly enzymes.
♦
Avoid excess (more than ten) freeze-thaw cycles. Aliquot reagents in smaller
amounts if necessary.
♦
Shield reagents and sequencing reactions from light. Fluorescent dyes are
susceptible to bleaching.
♦
If you would like to store sequencing reactions for future use, purify and dry them.
Store the reactions at –15 to –25 °C.
Reaction Tubes The type of tube required depends on the type of thermal cycler used. For the DNA
Thermal Cycler (TC1) and DNA Thermal Cycler 480, use 0.5-mL GeneAmp®
Thin-Walled PCR tubes. For the GeneAmp PCR Systems 9700, 9600, and 2400, use
0.2-mL MicroAmp® PCR tubes. If using the CATALYST™ 800 Molecular Biology
LabStation or ABI PRISM® 877 Integrated Thermal Cycler, refer to your instrument
user’s manual for reaction tube selection.
Thermal Cyclers The type and performance of the thermal cycler used to prepare sequencing reactions
can affect the quality of the reactions. Ensure that the thermal cycler is calibrated
regularly by the manufacturer and that ramping rates are 1 °C/second.
3-20 Performing DNA Sequencing Reactions
Preparing Cycle Sequencing Reactions
Rhodamine Dye The procedure given here is for the ABI PRISM Dye Terminator Cycle Sequencing
Terminators Ready Reaction Kits. Refer to the ABI PRISM Dye Terminator Cycle Sequencing Core
Kit Protocol (P/N 402116) for information on preparing reactions with the core kits.
Step
1
Action
For each reaction, add the following reagents to a separate tube:
Reagent
Quantity
Terminator Ready Reaction Mix
8.0 µL
Template
–
single-stranded DNA
100–250 ng
double-stranded DNA
200–500 ng
PCR product
1–100 ng
(depending on size, see
Table 3-1 on page 3-17)
Primer
3.2 pmol
Deionized water
q.s.
Total Volume
20 µL
2
Mix well and spin briefly.
3
If using the DNA Thermal Cycler (TC1) or DNA Thermal Cycler 480:
Overlay the reaction mixture with 40 µL of light mineral oil.
The cycle sequencing procedures for rhodamine dye terminators start on page 3-27.
dRhodamine
Terminators
Step
1
Action
For each reaction, add the following reagents to a separate tube:
Reagent
Quantity
Terminator Ready Reaction Mix
8.0 µL
Template
–
single-stranded DNA
50–100 ng
double-stranded DNA
200–500 ng
PCR product
1–100 ng
(depending on size, see
Table 3-1 on page 3-17)
Primer
3.2 pmol
Deionized water
q.s.
Total Volume
20 µL
2
Mix well and spin briefly.
3
If using the DNA Thermal Cycler (TC1) or DNA Thermal Cycler 480:
Overlay the reaction mixture with 40 µL of light mineral oil.
The cycle sequencing procedures for dRhodamine terminators start on page 3-27.
Performing DNA Sequencing Reactions 3-21
BigDye Terminators The flexibility of the BigDye terminators allows three options for cycle sequencing:
Reaction Type
Template
Cycle
1X
♦
PCR product
standard
♦
plasmid
♦
M13
♦
PCR product
♦
plasmid
♦
M13
♦
Large DNA templates
♦
bacterial genomic DNA
0.5X
High-sensitivity (2X)
standard
modified
The cycle sequencing procedures for BigDye terminators start on page 3-27.
1X Reactions
Step
1
Action
For each reaction, add the following reagents to a separate tube:
Reagent
Terminator Ready Reaction Mix
Template
Quantity
8.0 µL
–
single-stranded DNA
50–100 ng
double-stranded DNA
200–500 ng
PCR product DNA
Primer
Deionized water
Total Volume
1–100 ng
(depending on size, see
Table 3-1 on page 3-17)
3.2 pmol
q.s.
20 µL
2
Mix well and spin briefly.
3
If using the DNA Thermal Cycler (TC1) or DNA Thermal Cycler 480:
Overlay the reaction mixture with 40 µL of light mineral oil.
3-22 Performing DNA Sequencing Reactions
0.5X Reactions
Dilute 5X Sequencing Buffer (400 mM Tris-HCl, 10 mM MgCl2, pH 9.0—P/N 4305605,
600 reactions; 4305603, 5400 reactions) with an equal volume of deionized water to
2.5X for use in this procedure.
Step
1
Action
For each reaction, add the following reagents to a separate tube:
Reagent
Quantity
Terminator Ready Reaction Mix
4.0 µL
2.5X Sequencing Buffer
4.0 µL
Template
–
single-stranded DNA
50–100 ng
double-stranded DNA
200–500 ng
PCR product DNA
1–100 ng
(depending on size, see
Table 3-1 on page 3-17)
Primer
3.2 pmol
Deionized water
q.s.
Total Volume
20 µL
2
Mix well and spin briefly.
3
If using the DNA Thermal Cycler (TC1) or DNA Thermal Cycler 480:
Overlay the reaction mixture with 40 µL of light mineral oil.
High-Sensitivity (2X) Reactions for BACs, PACs, YACs, and Cosmids
Step
1
Action
For each reaction, add the following reagents to a separate tube:
Reagent
Quantity
Terminator Ready Reaction Mix
16 µL
DNA Template
0.5–1.0 µg
Primer
5–10 pmol
Deionized water
q.s.
Total Volume
2
40 µL
Mix well and spin briefly.
Note
These high-sensitivity reactions have been optimized on the GeneAmp PCR System
9600 or 9700 (in 9600 emulation mode). The protocols would need to be reoptimized for use on
other thermal cyclers.
The cycle sequencing procedure is on page 3-28.
Performing DNA Sequencing Reactions 3-23
High-Sensitivity (2X) Reactions for Bacterial Genomic DNA
Step
1
Action
For each reaction, add the following reagents to a separate tube:
Reagent
Quantity
Terminator Ready Reaction Mix
16 µL
DNA Templatea
2–3 µg
Primer
6–13 pmol
Deionized water
q.s.
Total Volume
2
40 µL
Mix well and spin briefly.
a. Shearing the DNA by passing it seven times through a 21-gauge, 1-inch long needle can improve signals.
Note
These high-sensitivity reactions have been optimized on the GeneAmp PCR System
9600 or 9700 (in 9600 emulation mode). The protocols would need to be reoptimized for use on
other thermal cyclers.
The cycle sequencing procedure is on page 3-29.
Fluorescein/ The procedure given here is for the ABI PRISM Dye Primer Cycle Sequencing Ready
Rhodamine Reaction Kits. Refer to the ABI PRISM Dye Primer Cycle Sequencing Core Kit Protocol
Dye Primers (P/N 402114) for information on preparing reactions with the core kits.
Step
1
Action
Aliquot the following reagents into four PCR tubes:
Reagent
2
A (mL)
C (mL)
G (mL)
T (mL)
Ready Reaction Premix
4
4
8
8
DNA Template (see Table 3-1
on page 3-17 for quantity)
1
1
2
2
Total Volume
5
5
10
10
If using the DNA Thermal Cycler (TC1) or DNA Thermal Cycler 480:
a.
Add 20 µL of light mineral oil.
b.
Spin to layer the oil over the aqueous reaction.
The cycle sequencing procedures for fluorescein/rhodamine dye primers start on
page 3-29.
3-24 Performing DNA Sequencing Reactions
BigDye Primers The flexibility of the BigDye™ primers allows three options for cycle sequencing:
Reaction Type
Template
Cycle
1X
♦
PCR product
standard
♦
plasmid
♦
M13
♦
PCR product
♦
plasmid
♦
M13
♦
Large DNA template containing –21 M13
and/or M13 Reverse priming site
0.5X
High-sensitivity (2X)
IMPORTANT
standard
modified
Prepare separate tubes for each of the four reactions (A, C, G, and T).
The cycle sequencing procedures for BigDye primers start on page 3-29.
1X Reactions
Step
1
Action
Aliquot the following reagents into four PCR tubes:
Reagent
2
A (mL)
C (mL)
G (mL)
T (mL)
Ready Reaction Premix
4
4
4
4
DNA Template (see Table 3-1
on page 3-17 for quantity)
1
1
1
1
Total Volume
5
5
5
5
If using the DNA Thermal Cycler (TC1) or DNA Thermal Cycler 480:
a.
Add 20 µL of light mineral oil.
b.
Spin to layer the oil over the aqueous reaction.
Performing DNA Sequencing Reactions 3-25
0.5X Reactions
Dilute 5X Sequencing Buffer (400 mM Tris-HCl, 10 mM MgCl2, pH 9.0—P/N 4305605,
600 reactions; 4305603, 5400 reactions) with four parts deionized water to 1X for use
in this procedure.
Step
Action
1
Dilute each Ready Reaction Premix (A, C, G, T) 1:1 with 1X Sequencing Buffer in a
separate tube (e.g., 2 µL of A Mix and 2 µL of 1X Sequencing Buffer).
2
Aliquot the following reagents into four PCR tubes for each DNA template:
Reagent
3
A (mL)
C (mL)
G (mL)
T (mL)
Diluted Ready Reaction
Premix
4
4
4
4
DNA Template (see
Table 3-1 on page 3-17
for quantity)
1
1
1
1
Total Volume
5
5
5
5
If using the DNA Thermal Cycler (TC1) or DNA Thermal Cycler 480:
a.
Add 20 µL of light mineral oil.
b.
Spin to layer the oil over the aqueous reaction.
High-Sensitivity (2X) Reactions
Aliquot the following reagents into four PCR tubes:
Reagent
A (µL)
C (µL)
G (µL)
T (µL)
Ready Reaction Premix
8
8
8
8
DNA Template (see Table 3-1 on
page 3-17 for quantity)
2
2
2
2
Total Volume
10
10
10
10
Note
These high-sensitivity reactions have been optimized on the GeneAmp PCR System
9600 or 9700 (in 9600 emulation mode). The protocols would need to be reoptimized for use on
other thermal cyclers.
The cycle sequencing procedure is on page 3-30.
3-26 Performing DNA Sequencing Reactions
Cycle Sequencing
Overview These protocols have been optimized for all Applied Biosystems thermal cyclers,
including the DNA Thermal Cycler (TC1), the DNA Thermal Cycler 480, the
CATALYST 800 Molecular Biology LabStation, the ABI PRISM 877 Integrated Thermal
Cycler, and the GeneAmp PCR Systems 9600, 2400, and 9700 in 9600 emulation
mode. The protocols contained in this section should work for all seven instruments.
If you use a thermal cycler not manufactured by Applied Biosystems, you may need to
optimize thermal cycling conditions. Ramping time is very important. If the thermal
ramping time is too fast (>1 °C/sec), poor (noisy) data may result.
Dye Terminator These protocols, except for sequencing BAC DNA and other large templates, are used
Chemistries for the rhodamine dye terminator, dRhodamine terminator, and BigDye terminator
chemistries.
These conditions work for a variety of templates and primers. However, if necessary,
these parameters can be changed to suit particular situations, including the following:
♦
For short PCR products, you can use reduced numbers of cycles (e.g., 20 cycles
for a 300-bp or smaller fragment).
♦
If the Tm of a primer is >60 °C, the annealing step can be eliminated.
♦
If the Tm of a primer is <50 °C, increase the annealing time to 30 seconds or
decrease the annealing temperature to 48 °C.
♦
For templates with high GC content (>70%), heat the tubes at 98 °C for 5 minutes
before cycling to help denature the template.
GeneAmp 9700 (in 9600 Emulation Mode), 9600, or 2400
Step
Action
1
Place the tubes in a thermal cycler and set the volume to 20 µL.
2
Repeat the following for 25 cycles:
♦
Rapid thermal rampa to 96 °C
♦
96 °C for 10 sec.
♦
Rapid thermal ramp to 50 °C
♦
50 °C for 5 sec.
♦
Rapid thermal ramp to 60 °C
♦
60 °C for 4 min.
3
Rapid thermal ramp to 4 °C and hold until ready to purify.
4
Spin down the contents of the tubes in a microcentrifuge.
a. Rapid thermal ramp is 1 °C/sec.
Performing DNA Sequencing Reactions 3-27
DNA Thermal Cycler (TC1) or DNA Thermal Cycler 480
Step
Action
1
Place the tubes in a thermal cycler and set the volume to 20 µL.
2
Repeat the following for 25 cycles:
♦
Rapid thermal rampa to 96 °C
♦
96 °C for 30 sec.
♦
Rapid thermal ramp to 50 °C
♦
50 °C for 15 sec.
♦
Rapid thermal ramp to 60 °C
♦
60 °C for 4 min.
3
Rapid thermal ramp to 4 °C and hold until ready to purify.
4
Spin down the contents of the tubes in a microcentrifuge.
a. Rapid thermal ramp is 1 °C/sec.
BACs, PACs, YACs, and Cosmids on the GeneAmp 9700 (in 9600 Emulation Mode), 9600,
or 2400
Note
Step
This protocol is for use only with the BigDye terminator kits.
Action
1
Place the tubes in a thermal cycler and set the volume to 40 µL.
2
Heat the tubes at 95 °C for 5 minutes.
3
Repeat the following for 30 cycles:a
♦
Rapid thermal rampb to 95 °C
♦
95 °C for 30 sec.
♦
Rapid thermal ramp to 50–55 °C (depending on template)
♦
50–55 °C for 10 sec.
♦
Rapid thermal ramp to 60 °C
♦
60 °C for 4 min.
4
Rapid thermal ramp to 4 °C and hold until ready to purify.
5
Spin down the contents of the tubes in a microcentrifuge.
a. Some laboratories have found that increasing the number of cycles gives better results.
b. Rapid thermal ramp is 1 °C/sec.
3-28 Performing DNA Sequencing Reactions
Bacterial Genomic DNA on the GeneAmp 9700 (in 9600 Emulation Mode), 9600, or 2400
Note
Step
This protocol is for use only with the BigDye terminator kits.
Action
1
Place the tubes in a thermal cycler and set the volume to 40 µL.
2
Heat the tubes at 95 °C for 5 minutes.
3
Repeat the following for 45 cycles:
♦
Rapid thermal rampa to 95 °C
♦
95 °C for 30 sec.
♦
Rapid thermal ramp to 50–55 °C (depending on template)
♦
55 °C for 20 sec.
♦
Rapid thermal ramp to 60 °C
♦
60 °C for 4 min.
4
Rapid thermal ramp to 4 °C and hold until ready to purify.
5
Spin down the contents of the tubes in a microcentrifuge.
a. Rapid thermal ramp is 1 °C/sec.
Dye Primer These protocols, except for BAC DNA sequencing, are used for the fluorescein/
Chemistries rhodamine dye primer and BigDye primer chemistries.
GeneAmp 9700 (in 9600 Emulation Mode), 9600, or 2400
Step
Action
1
Place the tubes in a thermal cycler and set the volume to 5 µL.
2
Repeat the following for 15 cycles:
3
4
♦
Rapid thermal rampa to 96 °C
♦
96 °C for 10 sec.
♦
Rapid thermal ramp to 55 °C
♦
55 °C for 5 sec.
♦
Rapid thermal ramp to 70 °C
♦
70 °C for 1 min.
Repeat the following for 15 cycles:
♦
Rapid thermal ramp to 96 °C
♦
96 °C for 10 sec.
♦
Rapid thermal ramp to 70 °C
♦
70 °C for 1 min.
Rapid thermal ramp to 4 °C and hold until ready to pool and precipitate.
a. Rapid thermal ramp is 1 °C/sec.
Performing DNA Sequencing Reactions 3-29
TC1 or DNA Thermal Cycler 480
Step
Action
1
Place the tubes in a thermal cycler preheated to 95 °C.
2
Repeat the following for 15 cycles:
3
4
♦
Rapid thermal rampa to 95 °C
♦
95 °C for 30 sec.
♦
Rapid thermal ramp to 55 °C
♦
55 °C for 30 sec.
♦
Rapid thermal ramp to 70 °C
♦
70 °C for 1 min.
Repeat the following for 15 cycles:
♦
Rapid thermal ramp to 95 °C
♦
95 °C for 30 sec.
♦
Rapid thermal ramp to 70 °C
♦
70 °C for 1 min.
Rapid thermal ramp to 4 °C and hold until ready to pool and precipitate.
a. Rapid thermal ramp is 1 °C/sec.
BAC DNA on the GeneAmp 9700 (in 9600 Emulation Mode) or 9600
Note
Step
1
2
3
4
This protocol is for use only with the BigDye primer kits.
Action
Place the tubes in a thermal cycler, set the volume at 10 µL, and begin thermal
cycling with the following parameters:
♦
Rapid thermal rampa to 95 °C
♦
95 °C for 5 min.
Repeat the following for 20 cycles:
♦
Rapid thermal ramp to 95 °C
♦
95 °C for 30 sec.
♦
Rapid thermal ramp to 50 °C
♦
50 °C for 15 sec.
♦
Rapid thermal ramp to 70 °C
♦
70 °C for 1 min.
Repeat the following for 15 cycles:
♦
Rapid thermal ramp to 95 °C
♦
95 °C for 30 sec.
♦
Rapid thermal ramp to 70 °C
♦
70 °C for 1 min.
Rapid thermal ramp to 4 °C and hold until ready to pool and precipitate.
a. Rapid thermal ramp is 1 °C/sec.
3-30 Performing DNA Sequencing Reactions
Cycle Sequencing on Templates that have been prepared as described in this chapter should be suitable for
the CATALYST 800 use on the CATALYST 800 Molecular Biology LabStation using LabStation 3.0
protocols. Follow the protocols in the Turbo Appendix of the CATALYST 800 Molecular
Biology LabStation User’s Manual (P/N 903939) to set up your reactions.
Dye Terminator Sequencing Options
Terminator Sequencing has two options:
♦
Using a reaction premix containing the sequencing primer or premixing template
with primer in the sample tube
♦
Combining reaction cocktail (lacking primers), water, and primer from one tube
and template from another tube
This eliminates the requirement for premixing samples and primers.
Dye Primer Sequencing Options
Predefined temperature profiles are provided for the following:
♦
Double-Stranded Forward (Universal) Primer
♦
Double-Stranded Reverse (Universal) Primer
♦
Single-Stranded Forward Primer
♦
Quick Cycle
These are chosen during the pre-run dialogue, and can be edited to make custom
profiles.
IMPORTANT
worksurface.
Load only the reagents that you plan to use. Do not store kit reagents on the
Performing DNA Sequencing Reactions 3-31
Cycle Sequencing on Dye Terminator Sequencing Options
the ABI PRISM 877 Predefined temperature profiles are provided for the following on the ABI PRISM 877
ITC Integrated Thermal Cycler:
♦
Terminator Sequencing uses a reaction premix containing the sequencing primer,
or else requires premixing template with primer in the sample tube.
♦
Terminator Automix Sequencing combines reaction cocktail (lacking primers),
water, primer from one tube, and template from another tube. This eliminates the
requirement for premixing of samples and primers.
The profile is chosen on the Chemistry page of the Sequencing Notebook and can be
edited to make custom profiles. Refer to Chapter 4, “Using the ABI PRISM 877
Software,” in the ABI PRISM 877 Integrated Thermal Cycler User’s Manual
(P/N 904414).
Dye Primer Sequencing Options
Predefined temperature profiles are provided for the following on the ABI PRISM 877
Integrated Thermal Cycler:
♦
Double-Stranded Forward (Universal) Primer
♦
Double-Stranded Reverse (Universal) Primer
♦
Single-Stranded Forward Primer
♦
Quick Cycle
These are chosen on the Chemistry page of the Sequencing Notebook, and can be
edited to make custom profiles. Refer to Chapter 4, “Using the ABI PRISM 877
Software,” in the ABI PRISM 877 Integrated Thermal Cycler User’s Manual for
instructions on editing temperature profiles.
IMPORTANT
worksurface.
3-32 Performing DNA Sequencing Reactions
Load only the reagents that you plan to use. Do not store kit reagents on the
Preparing Extension Products for Electrophoresis
Overview Preparation of extension products for electrophoresis will vary depending on the cycle
sequencing chemistry used.
Dye Terminator Chemistries
Unincorporated dye terminators must be removed before the samples can be
analyzed by electrophoresis. Excess dye terminators in sequencing reactions obscure
data in the early part of the sequence and can interfere with basecalling.
Several protocols for each sequencing chemistry are presented to offer a choice of
reagents and process. We recommend performing controlled reactions with each
method to determine the one that works best for you.
♦
Precipitation methods are cheaper and faster, but if performed poorly can leave
unincorporated dye-labeled terminators that can obscure data at the beginning of
the sequence.
Refer to the Precipitation Methods to Remove Residual Dye Terminators from
Sequencing Reactions User Bulletin (P/N 4304655). This document can be
obtained from the Applied Biosystems WWW site
(www.appliedbiosystems.com/techsupport).
♦
The spin column and 96-well plate procedures remove all excess terminators if
performed correctly, but are more costly than precipitation methods.
Dye Primer Chemistries
The standard procedure is ethanol precipitation, which concentrates the sample. An
Express Load option is also available for BigDye primers (see page 3-49).
Table 3-3 Recommended Methods for Preparing Extension Products for
Electrophoresis
Chemistry
Recommended Methods
See Page
Rhodamine
Dye Terminator and
dRhodamine
Terminator
Spin Column Purification
3-34
96-Well Plate Purification Protocol
3-35
BigDye Terminator
Ethanol/Sodium Acetate Precipitation
3-41
Ethanol/MgCl2 Precipitation
3-43
Shrimp Alkaline Phosphatase Digestion
3-45
Spin Column Purification
3-34
96-Well Plate Purification Protocol
3-35
Isopropanol Precipitation
3-36
Ethanol Precipitation for BigDye Terminators
3-38
Ethanol/Sodium Acetate Precipitation
3-41
Fluorescein/
Rhodamine
Dye Primer
Ethanol Precipitation for Fluorescein/Rhodamine
Dye Primers
3-46
BigDye Primer
Ethanol Precipitation for BigDye Primers
3-47
Express Load Option for BigDye Primers Run on
36-Lane Gels
3-49
Performing DNA Sequencing Reactions 3-33
Removing Unincorporated Dye Terminators
Spin Column We recommend Centri-Sep™ spin columns from Princeton Separations (P/N CS-901).
Purification IMPORTANT
For the BigDye terminators, hydrate the column for 2 hours.
Tips for optimizing spin column purification:
♦
Use one column for each sample.
♦
Do not process more columns than you can handle conveniently at one time.
♦
Load the sample in the center of the column bed. Make sure that the sample does
not touch the sides of the column and that the pipet tip does not touch the gel
surface.
If samples are not properly loaded, peaks from unincorporated dye terminators
can result (see Figure 7-36 on page 7-29).
♦
Spin the column at 325–730 ¥ g for best results. Use the following formula to
calculate the best speed for your centrifuge:
g = 11.18 ¥ r ¥ (rpm/1000)2
where:
g = relative centrifugal force
rpm = revolutions per minute
r = radius of the rotor in cm
♦
Do not spin for more than 2 minutes.
♦
Perform the entire procedure without interruption to ensure optimal results. Do not
allow the column to dry out.
To perform spin column purification:
Step
Action
1
Gently tap the column to cause the gel material to settle to the bottom of the
column.
2
Remove the upper end cap and add 0.8 mL of deionized water.
3
Replace the upper end cap and vortex or invert the column a few times to mix the
water and gel material.
4
Allow the gel to hydrate at room temperature for 30 minutes (at least 2 hours for
BigDye terminators).
Note
Hydrated columns can be stored for a few days at 2–6 °C. Longer storage
in water is not recommended. Allow columns that have been stored at 2–6 °C to
warm to room temperature before use.
5
Remove any air bubbles by inverting or tapping the column and allowing the gel to
settle.
6
Remove the upper end cap first, then remove the bottom cap. Allow the column to
drain completely by gravity.
Note
If flow does not begin immediately, apply gentle pressure to the column
with a pipette bulb.
7
Insert the column into the wash tube provided.
8
Spin the column in a microcentrifuge at 730 ¥ g for 2 minutes to remove the
interstitial fluid.
3-34 Performing DNA Sequencing Reactions
To perform spin column purification:
Step
(continued)
Action
9
Remove the column from the wash tube and insert it into a sample collection tube
(e.g., a 1.5-mL microcentrifuge tube).
10
Remove the extension reaction mixture from its tube and load it carefully onto the
center of the gel material.
Note
If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling,
remove the reactions from the tubes as shown in step 1 on page 3-37.
11
Spin the column in a microcentrifuge at 730 ¥ g for 2 minutes.
Note
If using a centrifuge with a fixed-angle rotor, place the column in the same
orientation as it was in for the first spin. This is important because the surface of the
gel will be at an angle in the column after the first spin.
12
Discard the column. The sample is in the sample collection tube.
13
Dry the sample in a vacuum centrifuge for 10–15 minutes, or until dry. Do not
over-dry.
96-Well Plate For large-scale procedures, you can use the following commercially available 96-well
Purification Protocol plates:
♦
96-Well Gel Filtration Block (Edge Biosystems, P/N 21520, 192 reactions; 91751,
960 reactions)
♦
Multiscreen 96-Well Filter Plates (Millipore, P/N MADYEKIT1)
Refer to the manufacturer’s instructions for the procedures.
Performing DNA Sequencing Reactions 3-35
Isopropanol Note
Precipitation
These procedures are for use only with the BigDye terminators.
Precipitation in 96-Well MicroAmp Trays
Reagents and equipment required:
♦
Variable speed table-top centrifuge with microtiter plate tray, capable of reaching
at least 1400 ¥ g
♦
Strip caps or adhesive-backed aluminum foil tape (3M Scotch Tape 425-3)1
♦
75% Isopropanol (2-propanol) or 100% isopropanol (anhydrous) at room
temperature
Note
This procedure does not use salt.
To precipitate extension products in MicroAmp Trays:
Step
Action
1
Remove the MicroAmp Tray from the thermal cycler. Remove the caps from each
tube.
2
Add one of the following:
♦
80 µL of 75% isopropanol
or
♦
20 µL of deionized water and 60 µL of 100% isopropanol
The final isopropanol concentration should be 60 ± 5%.
3
Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 425-3
adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any
leakage.
4
Invert the tray a few times to mix.
5
Leave the tray at room temperature for 15 minutes to precipitate the extension
products.
Note
Precipitation times <15 minutes will result in the loss of very short
extension products. Precipitation times >24 hours will increase the precipitation of
unincorporated dye terminators.
6
Place the tray in a table-top centrifuge with tube-tray adaptor and spin it at the
maximum speed, which must be ≥1400 ¥ g but <3000 ¥ g:
♦
1400–2000 ¥ g: 45 minutes
♦
2000–3000 ¥ g: 30 minutes
Note
A MicroAmp tube in a MicroAmp Tray can withstand 3000 ¥ g for
30 minutes.
IMPORTANT
Proceed to the next step immediately. If not possible, then spin
the tubes for 2 minutes more immediately before performing the next step.
1.
7
Without disturbing the precipitates, remove the adhesive tape and discard the
supernatant by inverting the tray onto a paper towel folded to the size of the tray.
8
Place the inverted tray with the towel into the table-top centrifuge and spin at
700 ¥ g for 1 minute.
Contact 3M in the USA at (800) 364-3577 for your local 3M representative. Use of other tapes may result
in leakage or contamination of the sample.
3-36 Performing DNA Sequencing Reactions
To precipitate extension products in MicroAmp Trays:
Step
9
(continued)
Action
Remove the tray and discard the paper towel.
Note
Pellets may or may not be visible. Vacuum drying of the samples is not
necessary.
Precipitation in Microcentrifuge Tubes
Reagents and equipment required for this method:
♦
1.5-mL microcentrifuge tubes
♦
Benchtop microcentrifuge, capable of reaching at least 14000 ¥ g
♦
Vacuum centrifuge
♦
75% Isopropanol (2-propanol) or 100% isopropanol (anhydrous) at room
temperature
Note
This procedure does not use salt.
To precipitate extension products in microcentrifuge tubes:
Step
1
Action
Pipet the entire contents of each extension reaction into a 1.5-mL microcentrifuge
tube.
To remove reactions run on the
TC1 or DNA Thermal Cycler 480:
Place the pipette tip into the bottom
of the reaction and carefully remove
the reaction from the oil.
Oil
Reaction
IMPORTANT
2
Transfer as little oil as possible.
Add one of the following:
♦
80 µL of 75% isopropanol
or
♦
20 µL of deionized water and 60 µL of 100% isopropanol
The final isopropanol concentration should be 60 ± 5%.
3
Close the tubes and vortex briefly.
4
Leave the tubes at room temperature for 15 minutes to precipitate the extension
products.
Note
Precipitation times <15 minutes will result in the loss of very short
extension products. Precipitation times >24 hours will increase the precipitation of
unincorporated dye terminators.
5
Place the tubes in a microcentrifuge and mark their orientations. Spin the tubes for
20 minutes at maximum speed.
IMPORTANT
Proceed to the next step immediately. If not possible, then spin
the tubes for 2 minutes more immediately before performing the next step.
Performing DNA Sequencing Reactions 3-37
To precipitate extension products in microcentrifuge tubes:
Step
6
(continued)
Action
Carefully aspirate the supernatants with a separate pipette tip for each sample and
discard. Pellets may or may not be visible.
IMPORTANT
The supernatants must be removed completely, as
unincorporated dye terminators are dissolved in them. The more residual
supernatant left in the tubes, the more unincorporated dye terminators will remain
in the samples.
7
Add 250 µL of 75% isopropanol to the tubes and vortex them briefly.
8
Place the tubes in the microcentrifuge in the same orientation as in step 5 and spin
for 5 minutes at maximum speed.
9
Aspirate the supernatants carefully as in step 6.
10
Dry the samples in a vacuum centrifuge for 10–15 minutes or to dryness.
(Alternatively, place the tubes with the lids open in a heat block or thermal cycler at
90 °C for 1 minute.)
Ethanol Note These procedures are for use with BigDye terminators only.
Precipitation for
With ethanol precipitation, traces of unincorporated terminators may be seen at the
BigDye Terminators
beginning of the sequence data (up to base 40), but this is usually minimal. Some loss
in the recovery of the smallest fragments may also be observed.
IMPORTANT
Where 95% ethanol is recommended in precipitation protocols, purchase
non-denatured ethanol at this concentration rather than absolute (100%) ethanol. Absolute
ethanol absorbs water from the atmosphere, gradually decreasing its concentration. This can
lead to inaccurate final concentrations of ethanol, which can affect some protocols.
Precipitation in 96-Well MicroAmp Trays
Reagents and equipment required:
♦
Variable speed table-top centrifuge with microtiter plate tray, capable of reaching
at least 1400 ¥ g
♦
Strip caps or adhesive-backed aluminum foil tape (3M Scotch Tape 425-3)1
♦
95% Ethanol (ACS reagent grade), non-denatured
Note
This procedure does not use salt.
To precipitate extension products in MicroAmp Trays:
Step
Action
1
Remove the MicroAmp Tray from the thermal cycler. Remove the caps from each
tube.
2
Add the following:
♦
16 µL of deionized water
♦
64 µL of non-denatured 95% ethanol
The final ethanol concentration should be 60 ± 3%.
1.
Contact 3M in the USA at (800) 364-3577 for your local 3M representative. Use of other tapes may result
in leakage or contamination of the sample.
3-38 Performing DNA Sequencing Reactions
To precipitate extension products in MicroAmp Trays:
Step
(continued)
Action
3
Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 425-3
adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any
leakage.
4
Invert the tray a few times to mix.
5
Leave the tray at room temperature for 15 minutes to precipitate the extension
products.
Note
Precipitation times <15 minutes will result in the loss of very short
extension products. Precipitation times >24 hours will increase the precipitation of
unincorporated dye terminators.
6
Place the tray in a table-top centrifuge with tube-tray adaptor and spin it at the
maximum speed, which must be ≥1400 ¥ g but <3000 ¥ g:
♦
1400–2000 ¥ g: 45 minutes
♦
2000–3000 ¥ g: 30 minutes
Note
A MicroAmp tube in a MicroAmp Tray can withstand 3000 ¥ g for
30 minutes.
IMPORTANT
Proceed to the next step immediately. If not possible, then spin
the tubes for 2 minutes more immediately before performing the next step.
7
Without disturbing the precipitates, remove the adhesive tape and discard the
supernatant by inverting the tray onto a paper towel folded to the size of the tray.
8
Place the inverted tray with the towel into the table-top centrifuge and spin at
700 ¥ g for 1 minute.
9
Remove the tray and discard the paper towel.
Note
Pellets may or may not be visible. Vacuum drying of the samples is not
necessary.
Precipitation in Microcentrifuge Tubes
Reagents and equipment required for this method:
♦
1.5-mL microcentrifuge tubes
♦
Benchtop microcentrifuge, capable of reaching at least 14000 ¥ g
♦
Vacuum centrifuge
♦
95% Ethanol (ACS reagent grade), non-denatured
Note
This procedure does not use salt.
To precipitate extension products in microcentrifuge tubes:
Step
1
Action
Pipet the entire contents of each extension reaction into a 1.5-mL microcentrifuge
tube.
Note
If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling,
remove the reactions from the tubes as shown in step 1 on page 3-37.
2
Add the following:
♦
16 µL of deionized water
♦
64 µL of non-denatured 95% ethanol
The final ethanol concentration should be 60 ± 3%.
Performing DNA Sequencing Reactions 3-39
To precipitate extension products in microcentrifuge tubes:
Step
(continued)
Action
3
Close the tubes and vortex briefly.
4
Leave the tubes at room temperature for 15 minutes to precipitate the extension
products.
Note
Precipitation times <15 minutes will result in the loss of very short
extension products. Precipitation times >24 hours will increase the precipitation of
unincorporated dye terminators.
5
Place the tubes in a microcentrifuge and mark their orientations. Spin the tubes for
20 minutes at maximum speed.
IMPORTANT
Proceed to the next step immediately. If not possible, then spin
the tubes for 2 minutes more immediately before performing the next step.
6
Carefully aspirate the supernatants with a separate pipette tip for each sample and
discard. Pellets may or may not be visible.
IMPORTANT
The supernatants must be removed completely, as
unincorporated dye terminators are dissolved in them. The more residual
supernatant left in the tubes, the more unincorporated dye terminators will remain
in the samples.
7
Add 250 µL of 70% ethanol to the tubes and vortex them briefly.
8
Place the tubes in the microcentrifuge in the same orientation as in step 5 and spin
for 10 minutes at maximum speed.
9
Aspirate the supernatants carefully as in step 6.
10
Dry the samples in a vacuum centrifuge for 10–15 minutes or to dryness.
(Alternatively, place the tubes with the lids open in a heat block or thermal cycler at
90 °C for 1 minute.)
3-40 Performing DNA Sequencing Reactions
Ethanol/Sodium IMPORTANT
Use non-denatured 95% ethanol rather than absolute (100%) ethanol.
Absolute
ethanol
absorbs
water from the atmosphere, gradually decreasing its concentration.
Acetate Precipitation
This can lead to inaccurate final concentrations of ethanol, which can affect some protocols.
Precipitation in 96-Well MicroAmp Trays
Reagents and equipment required:
♦
Variable speed table-top centrifuge with microtiter plate tray, capable of reaching
at least 1400 ¥ g
♦
Strip caps or adhesive-backed aluminum foil tape (3M Scotch Tape 425-3)1
♦
Sodium acetate (NaOAc), 3 M, pH 4.6 (P/N 400320)
♦
95% Ethanol (ACS reagent grade), non-denatured
To precipitate extension products in MicroAmp Trays:
Step
Action
1
Remove the MicroAmp Tray from the thermal cycler. Remove the caps from each
tube.
2
Add the following:
♦
2.0 µL of 3 M sodium acetate (NaOAc), pH 4.6
♦
50 µL of 95% ethanol (EtOH)
The final ethanol concentration should be 65%.
3
Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 425-3
adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any
leakage.
4
Invert the tray a few times to mix.
5
Leave the tray at room temperature for 15 minutes to precipitate the extension
products.
Note
Precipitation times <15 minutes will result in the loss of very short
extension products. Precipitation times >24 hours will increase the precipitation of
unincorporated dye terminators.
6
Place the tray in a table-top centrifuge with tube-tray adaptor and spin it at the
maximum speed, which must be ≥1400 ¥ g but <3000 ¥ g:
♦
1400–2000 ¥ g: 45 minutes
♦
2000–3000 ¥ g: 30 minutes
Note
A MicroAmp tube in a MicroAmp Tray can withstand 3000 ¥ g for
30 minutes.
IMPORTANT
Proceed to the next step immediately. If not possible, then spin
the tubes for 2 minutes more immediately before performing the next step.
1.
7
Without disturbing the precipitates, remove the adhesive tape and discard the
supernatant by inverting the tray onto a paper towel folded to the size of the tray.
8
Place the inverted tray with the towel into the table-top centrifuge and spin at
700 ¥ g for 1 minute.
9
Add 150 µL of 70% ethanol to each pellet.
Contact 3M in the USA at (800) 364-3577 for your local 3M representative. Use of other tapes may result
in leakage or contamination of the sample.
Performing DNA Sequencing Reactions 3-41
To precipitate extension products in MicroAmp Trays:
Step
(continued)
Action
10
Cap or seal the tubes, then invert the tray a few times to mix.
11
Spin the tray for 10 minutes at maximum speed.
12
Repeat steps 7 and 8.
13
Remove the tray and discard the paper towel.
Note
Pellets may or may not be visible. Vacuum drying of the samples is not
necessary.
Precipitation in Microcentrifuge Tubes
Reagents and equipment required:
♦
1.5-mL microcentrifuge tubes
♦
Benchtop microcentrifuge, capable of reaching at least 14000 ¥ g
♦
Vacuum centrifuge
♦
Sodium acetate (NaOAc), 3 M, pH 4.6 (P/N 400320)
♦
95% Ethanol (ACS reagent grade), non-denatured
Step
1
Action
For each sequencing reaction, prepare a 1.5-mL microcentrifuge tube containing
the following:
♦
2.0 µL of 3 M sodium acetate (NaOAc), pH 4.6
♦
50 µL of 95% ethanol (EtOH)
Note
If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling,
remove the reactions from the tubes as shown in step 1 on page 3-37.
2
Pipet the entire contents of each extension reaction into a tube of sodium acetate/
ethanol mixture. Mix thoroughly.
3
Vortex the tubes and leave at room temperature for 15 minutes to precipitate the
extension products.
Precipitation times <15 minutes will result in the loss of very short extension
products. Precipitation times >24 hours will increase the precipitation of
unincorporated dye terminators.
4
Spin the tubes in a microcentrifuge for 20 minutes at maximum speed.
5
Carefully aspirate the supernatant with a pipette tip and discard.
IMPORTANT
The supernatants must be removed completely, as
unincorporated dye terminators are dissolved in them. The more residual
supernatant left in the tubes, the more unincorporated dye terminators will remain
in the samples.
6
Rinse the pellet with 250 µL of 70% ethanol.
7
Vortex briefly.
8
Spin for 5 minutes in a microcentrifuge at maximum speed. Again, carefully aspirate
the supernatant and discard.
9
Dry the pellet in a vacuum centrifuge for 10–15 minutes, or until dry. Do not
over-dry. (Alternatively, place the tubes with the lids open in a heat block or thermal
cycler at 90 °C for 1 minute.)
3-42 Performing DNA Sequencing Reactions
Ethanol/MgCl2 Note These procedures are for use only with the rhodamine dye terminator and
Precipitation dRhodamine terminator chemistries.
These procedures require 70% ethanol (EtOH) containing 0.5 mM MgCl2. This
reagent can be prepared in situ or as a stock solution.
IMPORTANT
Use non-denatured 95% ethanol rather than absolute (100%) ethanol to
prepare 70% ethanol solutions. Absolute ethanol absorbs water from the atmosphere, gradually
decreasing its concentration. This can lead to inaccurate final concentrations of ethanol, which
can affect some protocols.
To prepare 70% EtOH/0.5 mM MgCl2 stock solution:
Step
1
2
Action
Combine the following in a 1.5-mL microcentrifuge tube:
♦
1 mL 70% EtOH
♦
1 µL 0.5 M MgCl2
Vortex briefly to mix.
Precipitation in 96-Well MicroAmp Trays
Equipment required:
♦
Variable speed table-top centrifuge with microtiter plate tray, capable of reaching
at least 1400 ¥ g
♦
Strip caps or adhesive-backed aluminum foil tape (3M Scotch Tape 425-3)1
To precipitate extension products in MicroAmp Trays:
Step
Action
1
Remove the MicroAmp Tray from the thermal cycler. Remove the caps from each
tube.
2
Add 74 µL of 70% EtOH/0.5 mM MgCl2 to each tube. (Alternatively, add 20 µL of
2 mM MgCl2 and then 55 µL of 95% ethanol.)
3
Seal the tubes by applying a piece of 3M Scotch Tape 425-3 adhesive-backed
aluminum foil tape. Press the foil onto the tubes to prevent any leakage.
4
Invert the tray a few times to mix.
5
Leave the tray at room temperature for 15 minutes to precipitate the extension
products.
Note
Precipitation times <15 minutes will result in the loss of very short
extension products. Precipitation times >24 hours will increase the precipitation of
unincorporated dye terminators.
1.
Contact 3M in the USA at (800) 364-3577 for your local 3M representative. Use of other tapes may result
in leakage or contamination of the sample.
Performing DNA Sequencing Reactions 3-43
To precipitate extension products in MicroAmp Trays:
Step
6
(continued)
Action
Place the tray in a table-top centrifuge with tube-tray adaptor and spin it at the
maximum speed, which must be ≥1400 ¥ g but <3000 ¥ g:
♦
1400–2000 ¥ g: 45 minutes
♦
2000–3000 ¥ g: 30 minutes
Note
A MicroAmp tube in a MicroAmp Tray can withstand 3000 ¥ g for
30 minutes.
IMPORTANT
Proceed to the next step immediately. If not possible, then spin
the tubes for 2 minutes more immediately before performing the next step.
7
Without disturbing the precipitates, remove the adhesive tape and discard the
supernatant by inverting the tray onto a paper towel folded to the size of the tray.
8
Place the inverted tray with the towel into the table-top centrifuge and spin at
700 ¥ g for 1 minute.
9
Remove the tray and discard the paper towel.
Note
Pellets may or may not be visible. Vacuum drying of the samples is not
necessary.
Precipitation in Microcentrifuge Tubes
Reagents and equipment required:
♦
1.5-mL microcentrifuge tubes
♦
Benchtop microcentrifuge, capable of reaching at least 14000 ¥ g
♦
Vacuum centrifuge
To precipitate extension products in microcentrifuge tubes:
Step
1
Action
Pipet the entire contents of each extension reaction into a 1.5-mL microcentrifuge
tube.
Note
If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling,
remove the reactions from the tubes as shown in step 1 on page 3-37
2
Add 74 µL of 70% EtOH/0.5 mM MgCl2 to each tube. (Alternatively, add 20 µL of
2 mM MgCl2 and then 55 µL of 95% ethanol.)
3
Close the tubes and vortex briefly.
4
Leave the tubes at room temperature for 15 minutes to precipitate the extension
products.
Note
Precipitation times <15 minutes will result in the loss of very short
extension products. Precipitation times >24 hours will increase the precipitation of
unincorporated dye terminators.
5
Place the tubes in a microcentrifuge and mark their orientations. Spin the tubes for
20 minutes at maximum speed.
IMPORTANT
Proceed to the next step immediately. If not possible, then spin
the tubes for 2 minutes more immediately before performing the next step.
3-44 Performing DNA Sequencing Reactions
To precipitate extension products in microcentrifuge tubes:
Step
6
(continued)
Action
Carefully aspirate the supernatant with a separate pipette tip for each sample and
discard. Pellets may or may not be visible.
IMPORTANT
The supernatant must be removed completely, as unincorporated
dye terminators are dissolved in it. The more residual supernatant left in the tubes,
the more unincorporated dye terminators will remain in the samples.
7
Visually inspect the sample tubes for residual supernatant. If there is any residual
supernatant:
a.
Place the tubes in the microcentrifuge in the same orientation as in step 5 and
spin for 10 seconds.
b.
Aspirate the supernatant carefully as in step 6.
8
Rinse the pellet with 250 µL of 70% ethanol.
9
Vortex briefly.
10
Spin for 5 minutes in a microcentrifuge at maximum speed. Again, carefully aspirate
the supernatant and discard.
11
Dry the samples in a vacuum centrifuge for 10–15 minutes or to dryness.
(Alternatively, place the tubes with the lids open in a heat block or thermal cycler at
90 °C for 1 minute.)
Shrimp Alkaline Note This procedure is for use only with the rhodamine dye terminator and dRhodamine
Phosphatase terminator chemistries. It can be used for more efficient removal of unincorporated dye
Digestion terminators, but adds additional time and expense.
Step
Action
1
At the end of thermal cycling, add 2 µL of SAP (1 U/µL) and 18 µL of 1X SAP buffer
to each tube. Seal each tube and incubate at 37 °C for 30 minutes.
2
For precipitation in 96-Well MicroAmp Trays:
a.
Add 150 µL of 70% EtOH/0.5 mM MgCl2 to each tube. (Alternatively, add 40 µL
of 2 mM MgCl2 and then 110 µL of 95% ethanol.)
b.
Proceed to step 3 of “Precipitation in 96-Well MicroAmp Trays” on page 3-43.
For precipitation in microcentrifuge tubes:
a.
Transfer the contents of each tube to a 1.5-mL microcentrifuge tube.
b.
Add 150 µL of 70% EtOH/0.5 mM MgCl2 to each tube. (Alternatively, add 40 µL
of 2 mM MgCl2 and then 110 µL of 95% ethanol.)
c.
Proceed to step 3 of “Precipitation in Microcentrifuge Tubes” on page 3-44.
Performing DNA Sequencing Reactions 3-45
Preparing Dye Primer Reaction Products for Electrophoresis
Ethanol
Precipitation for
Fluorescein/
Rhodamine Dye
Primers
Reagents and equipment required for these methods:
♦
1.5-mL microcentrifuge tubes
♦
Benchtop microcentrifuge, capable of reaching at least 14000 ¥ g
♦
Vacuum centrifuge
♦
95% Ethanol (ACS reagent grade), non-denatured
Note
These procedures do not use salt.
IMPORTANT
Use non-denatured 95% ethanol rather than absolute (100%) ethanol.
Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration.
This can lead to inaccurate final concentrations of ethanol, which can affect some protocols.
Method 1
Step
1
Action
Add 80 µL of 95% ethanol to a clean microcentrifuge tube.
Note
2
The use of sodium acetate is not necessary for precipitation.
Pipet the extension reactions from the bottom of each of the four tubes into the
ethanol mixture. Mix thoroughly.
Note
If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling,
remove the reactions from the tubes as shown in step 1 on page 3-37.
3
Place the tube on wet ice or leave it at room temperature for 10–15 minutes to
precipitate the extension products.
4
Spin the tube in a microcentrifuge for 10–20 minutes at maximum speed. Carefully
aspirate the supernatant and discard. At this point, a pellet may or may not be
visible.
5
Optional: Rinse the pellet with 250 µL of 70% ethanol and spin for 5 minutes in a
microcentrifuge. Again, carefully aspirate the supernatant and discard. This may
remove some of the salts from the pellet, but doing so is often not necessary.
Note
If you use sodium acetate, you must rinse the pellet. This will reduce the
carryover of salt.
6
Dry the pellet in a vacuum centrifuge for 1–3 minutes, or until dry. Do not overdry.
Method 2
Step
1
Action
Add 80 µL of 95% ethanol to the “A” reaction tube.
Note
This method will not work if the TC1 or DNA Thermal Cycler 480 was used
for thermal cycling.
2
Transfer the contents of the “A” reaction tube into the “C” reaction tube.
3
Pipet that mixture into the “G” reaction tube and so on until the contents of all four
reaction tubes have been transferred into a single 1.5-mL microcentrifuge tube.
4
Spin the tube in a microcentrifuge for 10–20 minutes at maximum speed. Carefully
aspirate the supernatant and discard. At this point, a pellet may or may not be
visible.
3-46 Performing DNA Sequencing Reactions
Method 2
Step
(continued)
Action
5
Optional: Rinse the pellet with 250 µL of 70% ethanol and spin for 5 minutes in a
microcentrifuge. Again, carefully aspirate the supernatant and discard. This may
remove some of the salts from the pellet, but doing so is often not necessary.
Note
If you use sodium acetate, you must rinse the pellet. This will reduce the
carryover of salt.
6
Dry the pellet in a vacuum centrifuge for 1–3 minutes, or until dry. Do not overdry.
Ethanol Reagents and equipment required for this method:
Precipitation for ♦ 1.5-mL microcentrifuge tubes
BigDye Primers
♦
Benchtop microcentrifuge, capable of reaching at least 14000 ¥ g
♦
Vacuum centrifuge
♦
95% Ethanol (ACS reagent grade), non-denatured
Note
This procedure does not use salt.
IMPORTANT
Use non-denatured 95% ethanol rather than absolute (100%) ethanol.
Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration.
This can lead to inaccurate final concentrations of ethanol, which can affect some protocols.
Method 1
Step
1
Action
Add 53 µL of 95% ethanol (100 µL if sequencing BAC DNA or other high-sensitivity
reactions) to a clean microcentrifuge tube.
Note
2
The use of sodium acetate is not necessary for precipitation.
Pipet the extension reactions from the bottom of each of the four tubes into the
ethanol mixture. Mix thoroughly.
Note
If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling,
remove the reactions from the tubes as shown in step 1 on page 3-37.
3
Place the tube on wet ice or leave it at room temperature for 10–15 minutes to
precipitate the extension products.
4
Spin the tube in a microcentrifuge for 10–20 minutes at maximum speed. Carefully
aspirate the supernatant and discard. At this point, a pellet may or may not be
visible.
5
Optional: Rinse the pellet with 250 µL of 70% ethanol and spin for 5 minutes in a
microcentrifuge. Again, carefully aspirate the supernatant and discard. This may
remove some of the salts from the pellet, but doing so is often not necessary.
Note
If you use sodium acetate, you must rinse the pellet. This will reduce the
carryover of salt.
6
Dry the pellet in a vacuum centrifuge for 1–3 minutes, or until dry. Do not overdry.
Performing DNA Sequencing Reactions 3-47
Method 2
Step
1
Action
Add 53 µL of 95% ethanol (100 µL if sequencing BAC DNA or other high-sensitivity
reactions) to the “A” reaction tube.
Note
This method will not work if the TC1 or DNA Thermal Cycler 480 was used
for thermal cycling.
2
Transfer the contents of the “A” reaction tube into the “C” reaction tube.
3
Pipet that mixture into the “G” reaction tube and so on until the contents of all four
reaction tubes have been transferred into a single 1.5-mL microcentrifuge tube.
4
Spin the tube in a microcentrifuge for 10–20 minutes at maximum speed. Carefully
aspirate the supernatant and discard. At this point, a pellet may or may not be
visible.
5
Optional: Rinse the pellet with 250 µL of 70% ethanol and spin for 5 minutes in a
microcentrifuge. Again, carefully aspirate the supernatant and discard. This may
remove some of the salts from the pellet, but doing so is often not necessary.
Note
If you use sodium acetate, you must rinse the pellet. This will reduce the
carryover of salt.
6
Dry the pellet in a vacuum centrifuge for 1–3 minutes, or until dry. Do not overdry.
3-48 Performing DNA Sequencing Reactions
Express Load Option Note
This procedure is for use only with the BigDye primers.
for BigDye Primers
Run on 36-Lane Gels Reagents and equipment required for this method:
♦ 0.2-mL PCR tubes
♦
Benchtop microcentrifuge, capable of reaching at least 14000 ¥ g
♦
Vortexer
♦
Ethylenediaminetetraacetic acid, disodium salt (Na2EDTA), 5 mM
♦
25 mM EDTA (pH 8.0)with 50 mg/mL blue dextran (P/N 402055)
♦
Deionized formamide
To perform express load with BigDye primers:
Step
Action
1
Combine the four reactions (A, C, G, T) with 5 µL of 5 mM EDTA (25 µL total
volume).
2
Vortex briefly, then spin in a microcentrifuge.
3
Prepare a loading buffer by combining the following in a 5:1 ratio:
♦
deionized formamide
♦
25 mM EDTA (pH 8.0) with blue dextran (50 mg/mL)
! WARNING ! CHEMICAL HAZARD Formamide is a teratogen and is
harmful by inhalation, skin contact, and ingestion. Use in a well-ventilated
area. Use chemical-resistant gloves and safety glasses when handling.
4
Combine 4 µL of each 25-µL reaction/EDTA mixture with 4 µL of loading buffer in a
fresh tube.
Store the remaining reaction/EDTA mixture at –15 to –25 °C. This mixture can be
concentrated by ethanol precipitation if the Express Load procedure does not yield
enough signal.
5
Heat the samples at 98 °C for 5 minutes with the lids open to denature and
concentrate the sample. Place on ice until ready to load.
6
Load 2.5 µL of each sample onto the gel.
Performing DNA Sequencing Reactions 3-49
Preparing and Loading Samples for Gel Electrophoresis
Loading The amount of sample to load depends on many factors, including:
Recommendations ♦ Sequencing chemistry used
♦
Quality and nature of the DNA template
♦
Primer performance
♦
Instrument configuration (i.e., which instrument and the number of gel lanes)
In general, a very clean template with a perfectly matched primer of good cycle
sequencing characteristics will produce enough signal. However, GC-rich templates
tend to give weaker signals, so more of the sample should be loaded.
The optimal range of total signal strength is 400–4000. For dRhodamine-based
chemistries, the lower limit can be 200. The total signal is calculated by adding the
signals for all four bases. These numbers are located in the annotation view of the
analyzed sample file (see page 7-8) and at the top of the electropherogram printout.
Acceptable sequence can be obtained from data with total signal strength below these
numbers. However, the background will be more noticeable and can interfere with
basecalling, especially at the end of the run. If the total signal strength is below 400
(200 for dRhodamine-based chemistries) and the quality of the data is unacceptable,
more of the reaction needs to be loaded. If 100% of the reaction was already loaded,
try any of the following:
♦
Check the quantitation and purity of the template. Adjust the amount of template.
Sometimes adding 25–50% more template can raise signals.
♦
Use a high-sensitivity sequencing method (2X volume reaction).
♦
Repurify the template to ensure that salt and ethanol contaminants are removed.
♦
Ensure that the primer is suitable for the template and has good melting
characteristics (ideally having a Tm of approximately 50–60°C).
If you are running these chemistries for the first time, use the upper limit of the load
amount (Table 3-4 through Table 3-6 on page 3-51). Having more signal than you
need is better than having an insufficient amount of signal to analyze the data.
Preparing Prepare the loading buffer by combining the following in a 5:1 ratio:
Loading Buffer ♦ Deionized formamide (see page A-16 for preparation)
♦
25 mM EDTA (pH 8.0) with 50 mg/mL blue dextran (P/N 402055)
! WARNING ! CHEMICAL HAZARD. Formamide is a teratogen and is harmful by
inhalation, skin contact, and ingestion. Use in a well-ventilated area. Use
chemical-resistant gloves and safety glasses when handling.
Note
With ABI™ 373 instruments, you can use 50 mM EDTA (with or without blue dextran) in
place of 25 mM EDTA with blue dextran. Do not use 50 mM EDTA on ABI PRISM 377
instruments.
3-50 Performing DNA Sequencing Reactions
Sample Loading ABI 373 and ABI 373 with XL Upgrade
Volumes
Table 3-4 Loading Amounts for Different Comb Sizes and Chemistries
Chemistry
Volume (µL)
18-well
24-well
36-well
48-well
64-well
Rhodamine
Dye Terminator
Resuspend
4–6
4–6
3–4
2
2
Load
1–2
1–2
3–4 (24, 34 cm)
1–2
1–1.5
2–4 (48 cm)
Fluorescein/
Rhodamine
Dye Primer
Resuspend
4–6
4–6
3–4
2
2
Load
4–6
4–6
3–4 (24, 34 cm)
1–2
1–1.5
2–4 (48 cm)
ABI 373 and ABI 373XL with BigDye Filter Wheel
Table 3-5 Loading Amounts for Different Comb Sizes and Chemistries
Chemistry
Volume (µL)
dRhodamine
Terminator
Resuspend
BigDye™
Terminator
Resuspend
BigDye™
18-well
Load
Load
Primer Resuspend
Load
24-well
36-well
48-well
64-well
4
4
4
2
2
2–4
2–4
2–4
1–2
1–2
4
4
4
4
4
1–2
1–2
1–2
1–2
1–2
4
4
4
4
4
1–2
1–2
1–2
1–2
1–2
ABI PRISM 377 (All Models)
Table 3-6 Loading Amounts for Different Comb Sizes and Chemistries
Chemistry
Volume (µL)
Rhodamine
Dye Terminator
Resuspend
dRhodamine
Terminator
BigDye
Terminator
Load
18-well
36-well
48-well
64-well
96-well
6
6
4–6
4–6
4–6
1.5
1.5
1.5
1.5
1.5
Resuspend
4
4
2
2
2
Load
2
2
1
1
1
6–8
6–8
4–6
4–6
4–6
0.75–1.5
0.75–1.5
0.5–1
0.5–1
0.5–1
1X reactions
Resuspend
Load
0.5X reactions
2X reactions
Fluorescein/
Rhodamine
Dye Primer
Load
BigDye Primer
1X reactions
Resuspend
2
2
2
2
2
Load
2
2
1.5–2
1–1.5
1–1.5
Resuspend
2
2
1.5
1.5
1.5
Load
2
2
1.5
1.5
1–1.5
Resuspend
6
6
4–6
4–6
4–6
1.5
1.5
1.5
1.5
1.5
6
6
4–6
4–6
4–6
1–1.5
1–1.5
1
1
1
2–4
2–4
1–2
1–1.5
1–2
Load
2
2
1
1
1
Resuspend
2
2
1.5
1
1.5
Load
2
2
1.5
1
1.5
Resuspend
Load
0.5X reactions
2X reactions
Resuspend
Performing DNA Sequencing Reactions 3-51
Loading Samples
Step
Action
1
Resuspend each sample pellet in the appropriate volume of loading buffer
according to Table 3-4 through Table 3-6 on page 3-51.
2
Vortex and spin the samples.
3
Heat the samples at 95 °C for 2 minutes to denature, then place on ice.
4
Load the appropriate volume of each sample into a separate lane of the gel
according to Table 3-4 through Table 3-6 on page 3-51.
3-52 Performing DNA Sequencing Reactions
Preparing and Loading Samples for Capillary Electrophoresis
Minimum Sample The minimum sample volume is 10 µL.
Volume
One sample can be injected several times, because very little sample volume is used
for each injection.
Preparing the
Samples
Step
1
Action
Add 12–25 µL of TSR (see page 5-2) to each sample pellet.
Note
Because injection is electrokinetic, the resuspension volume is not as
important as for slab gel electrophoresis.
2
Mix thoroughly on a vortex mixer and heat for 2 minutes at 95 °C.
Note
tray.
If you are using a 96-well tray, samples can be denatured directly in the
3
Chill on ice, vortex thoroughly, then spin briefly in a microcentrifuge.
4
Hold on ice until ready to load on the instrument.
5
Transfer the samples to 0.5-mL or 0.2-mL sample tubes and cover with tube septa.
Note
You must use tube septa to prevent evaporation of samples, especially if
samples are put in the autosampler more than six hours before analysis.
Preparing a Portion Occasionally, you may want to prepare only a portion of a sequencing reaction mixture
of a Reaction for analysis on the ABI PRISM® 310 Genetic Analyzer and reserve the rest of the
Mixture for Analysis sample for analysis later or elsewhere.
Step
Action
1
Add 6 µL of TSR to the dried DNA sequencing reaction.
2
Vortex to dissolve the extension products.
3
Heat for 1 minute at 95 °C to ensure denaturation.
4
Add 2 µL of the sample to 10 µL of TSR in a sample tube.
5
Cover the tube with a septum and vortex well.
6
Heat the mixture for 2 minutes at 95 °C, then place it on ice until ready to use.
Although not recommended on a routine basis, you can keep samples prepared in
TSR frozen for several weeks before running on the ABI PRISM 310 Genetic Analyzer
with no detectable loss in resolution or base calling.
Performing DNA Sequencing Reactions 3-53
Loading the Samples Move the samples into the autosampler as follows:
Step
1
Action
Transfer the denatured samples to a 48- or 96-well tray.
Note
tray.
If you are using a 96-well tray, samples can be denatured directly in the
IMPORTANT
The tube arrangement and order of the samples in the tray and
on the Sample Sheet must be the same. Make note of the tube arrangement you
use, so that you can prepare the Sample Sheet correctly.
2
Seal each tube with a septum, and place the tray into the autosampler.
3
Refer to the ABI PRISM 310 Genetic Analyzer User’s Manual for electrophoresis
procedures.
3-54 Performing DNA Sequencing Reactions
Optimizing Gel
Electrophoresis
4
4
Introduction
In This Chapter This chapter describes the following:
♦
Theory of polyacrylamide gels
♦
Reagents used to make sequencing gels
♦
Avoiding problems with sequencing gels
Refer to page 7-44 for information on troubleshooting gel electrophoresis.
Theory of Many variables are involved in determining the number of bases you can expect to
Polyacrylamide Gels read when sequencing DNA. One of the most important of these is the polyacrylamide
gel.
Polyacrylamide gels are formed by co-polymerization of acrylamide and
bisacrylamide. The reaction is a vinyl addition polymerization initiated by a free radical
generating system:
♦
Polymerization is initiated by TEMED (tetramethylethylenediamine) and APS
(ammonium persulfate). The TEMED acts as an electron carrier to activate the
acrylamide monomer, providing an unpaired electron to convert the acrylamide
monomer to a free radical.
♦
The activated monomer then reacts with an unactivated monomer to begin the
polymer chain reaction.
♦
The elongating polymer chains are randomly crosslinked by bisacrylamide,
resulting in closed loops and a complex web polymer with a reproducible porosity
that depends on the polymerization conditions and monomer concentration.
Polymerization depends on factors such as concentration of initiator, temperature, pH,
additives, breakdown products and impurities in the chemicals and water used. Light
and high temperature can cause autopolymerization of linear acrylamide, which
hinders the reproducibility of gel formation.
When used in conjunction with the ABI™ 373 or ABI PRISM® 377 DNA Sequencer, a
typical gel made with good reagents and properly polymerized can separate DNA
fragments from 1–600 bases in length easily. An exceptional gel used on the ABI 373
or ABI PRISM 377 DNA Sequencer can yield basecalling beyond 900 bases.
For more information, refer to Acrylamide Polymerization—A Practical Approach on
the Bio-Rad Laboratories World Wide Web site (http://www.bio-rad.com/38973.html).
Optimizing Gel Electrophoresis 4-1
Reagents
Acrylamide ! WARNING ! CHEMICAL HAZARD. Acrylamide and bisacrylamide are poisons,
neurotoxins, irritants, carcinogens, and possible teratogens. Acrylamide and
bisacrylamide sublime (the solids release toxic vapor) and are harmful if swallowed,
inhaled, or absorbed through the skin. Effects are cumulative. When handling, always
wear protective equipment (lab coat, safety glasses, and chemical-resistant gloves) and
use in a well-ventilated area. On a routine basis, thoroughly clean surfaces subject to
contamination.
Use fresh, high-quality acrylamide. Poor quality acrylamide contains acrylic acid (a
deamidation product) and linear polyacrylamide, which will copolymerize and cause
local pH changes in the gel. This causes streaking and smearing of bands
(Figure 7-50 on page 7-44).
Store acrylamide solutions at 2–6 °C up to 1 month. During storage, especially in
water, acrylamide breaks down into acrylic acid. It decomposes more quickly at room
temperature.
Urea ! WARNING ! CHEMICAL HAZARD. Urea is a potential mutagen. Dangers cited in
toxicity studies show reproductive and tumorigenic effects. Urea can cause irritation to
the skin, eyes, and respiratory tract. Avoid inhalation and contact with skin, eyes, and
clothing. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer.
Wear appropriate protective eyewear, clothing, and gloves.
Urea, (NH2)2CO, slowly decomposes in water, forming ammonia and cyanate ions
that can interfere with electrophoresis. Avoid heating solutions containing urea as
much as possible. Cyanate formation is accelerated with increasing temperature. Urea
solutions can be pretreated with mixed-bed resins to reduce the amount of ions in
solution.
TBE Buffer Prepare Tris-borate-EDTA buffer with Tris base (see page A-15 for recipe).
If Tris-HCl is used to prepare buffers, the concentration of salt will be too high and
nucleic acids will migrate anomalously, yielding extremely diffuse bands.
APS ! WARNING ! CHEMICAL HAZARD. Ammonium persulfate (APS) is harmful if
swallowed, inhaled, or absorbed through the skin. It is extremely destructive to mucous
membranes, eyes, and skin. Inhalation can be fatal. Always work in a fume hood. Obtain
a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear,
clothing, and gloves.
APS is very hygroscopic and reactive by nature and is prone to reduction and
decomposition. It begins to break down almost immediately when dissolved in water.
Optimally, APS should be prepared fresh daily. At the very least, store 10% stocks at
–15 to –25 °C and replace every week. Listen for a crackling sound when adding
water to dry APS. Material that has broken down due to high humidity or liquid
contamination will no longer crackle when moistened.
Persulfate is consumed in the polymerization because it provides the free radicals that
drive polymerization, but excess APS can cause oxidation of proteins and nucleic
acids. Prerunning gels keeps excess APS traveling ahead of the sample.
4-2 Optimizing Gel Electrophoresis
TEMED ! WARNING ! CHEMICAL HAZARD. TEMED (Tetramethylethylenediamine) is
extremely flammable, and can be very destructive to the skin, eyes, nose, and
respiratory system. Keep TEMED in a tightly closed container. Avoid inhalation and
contact with the skin, eyes, and clothing. Always work in a fume hood. Obtain a copy of
the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and
gloves.
TEMED oxidizes readily, which causes a gradual loss in catalytic activity. It is
hygroscopic and accumulates water which accelerates oxidative decomposition.
Discard it as chemical waste when oxidation products begin to give it a yellow color.
For this reason, use colorless tips to add TEMED.
TEMED introduces fixed charges into the gel matrix that cause gradient drift towards
the anode. Unreacted TEMED will focus at the cathode and produce a more alkaline
pH gradient. Because TEMED is only active as a free base, polymerization is inhibited
at low pH. Use of more than the recommended volume of TEMED will result in brittle
gels.
TEMED and APS The properties of the gel depend on the concentrations of APS and TEMED:
Concentrations ♦ Optimal amounts of APS/TEMED result in long chain lengths, low turbidity, and
gel elasticity. These are desirable properties.
–
If concentrations of APS/TEMED are too low, polymerization will be too slow.
When polymerization is too slow, oxygen can enter the monomer solution
during the process and inhibit polymerization further. Too little polymerization
leads to extension products migrating too quickly.
–
If concentrations of APS/TEMED are too high, chain initiation events increase,
resulting in shorter chain lengths, higher gel turbidity, and decreased gel
elasticity. Low percentage acrylamide gels are most sensitive to excess
initiator concentrations.
♦
Excess TEMED increases buffer pH.
♦
Excess APS acts as a buffer between pH 8 and 9.
♦
If you use degraded TEMED or APS, the concentration of initiators will be less
than that recommended in the protocol.
Optimizing Gel Electrophoresis 4-3
Avoiding Problems with Sequencing Gels
Formulation Use the correct gel formulation for your instrument and application. See Appendix A
for gel preparation protocols.
Contaminants Contaminants cause a variety of problems that affect the quality of sequence data
adversely. Contaminants in gels can come from many sources:
♦
Glassware used to make the gel
♦
Sinks used to clean gel plates and glassware
♦
Sponges used to clean gel plates
♦
Colored paper towels
♦
Water used for cleaning or gel formulation
♦
Solvents used for cleaning glass plates
♦
Marking pens
Background Fluorescence
Background fluorescence on the gel is a problem because it masks the signals from
the dyes used to detect sequencing extension products. It can be avoided by careful
handling of glass plates, spacers, combs, and buffer chambers.
Do not use any kind of ink on plates, spacers, combs, and buffer chambers. Ink
fluoresces strongly, obscuring data (Figure 7-52 on page 7-46).
Washing plates in communal sinks where fluorescent products are disposed can also
lead to background fluorescence problems.
Polymerization The properties of the gel depend on the rate of polymerization. The rate of
polymerization is affected by temperature, initiator (APS and TEMED) concentrations,
oxygen, and contaminants in the reagents used to formulate gels.
Temperature
Controlling the temperature is crucial for achieving reproducible gels because it
directly affects the polymerization time and thus affects the gel properties. A gel
formed in a cold environment (such as a room at 2–6 °C) will be turbid, porous,
inelastic, and the run-to-run reproducibility will be greatly compromised.
Polymerization in a 20–25 °C room is optimal.
In addition to the temperature of the room, it is important that the gel solution and
glass plates also be at a temperature of 20–25 °C. Gels formed at this temperature
will be transparent, less porous, more elastic, and more reproducible. If the
temperature is too high, the polymer chains will be shorter and the resulting gel
inelastic.
4-4 Optimizing Gel Electrophoresis
TEMED and APS Concentrations
See page 4-3 for information about TEMED and APS concentrations.
Oxygen
Oxygen acts as a free radical trap, thereby inhibiting polymerization. The result is a
porous gel. To prevent the problems caused by oxygen, the following conditions
should be met:
♦
Polymerization must be fast enough to prevent too much oxygen from dissolving
into the gel solution during polymerization.
As long as you use fresh high-quality reagents and follow a standard protocol, this
should not be a problem.
♦
Minimize the amount of oxygen dissolved in the gel solution prior to casting the
gel since it can interfere with the rate of polymerization.
Partial degassing can be accomplished during the vacuum filter step of gel
preparation. It is important to keep the vacuum strength and time constant during
this step for run-to-run reproducibility. Since cold solutions have a greater capacity
for dissolved oxygen, vacuum filtering (and gel casting) should be done with the
solution at room temperature. Also, be careful not to introduce bubbles during the
stirring and pouring steps of gel casting.
♦
Air bubbles trapped while casting the gel should be eliminated as they occur.
Ideally, gels should be poured carefully and gently so that bubbles never form.
Tapping gently on the plates while pouring the gel solution will help prevent
bubbles from forming.
Using Fresh Gels For consistent results, use gels within 2–6 hours after casting. Be sure to wait at least
2 hours after casting the gel to ensure complete polymerization, but not longer than
6 hours, as resolution begins to noticeably deteriorate after this time.
Gels that stand overnight can show significantly slower DNA migration due to the slow
hydrolysis of urea to ammonium carbonate. Because the amide groups of the polymer
slowly hydrolyze into carboxylate groups, gels that stand more than 48 hours may also
show significant loss in resolution beyond 350 bases.
If read length is not important for your application, gels can be stored overnight. Wrap
the ends of the gels in plastic wrap to prevent drying.
Red Rain The ABI PRISM 377 DNA Sequencer sometimes produces a gel image having intense,
vertical red streaks. This phenomenon, called “red rain,” is usually found near the end
of a run (top of the gel image), but can begin much earlier (Figure 7-55 on page 7-49).
Red rain is caused by gel destruction in the read region of the gel. The damaged gel
matrix scatters the laser light, with the effect on red light the greatest. Gel destruction
often results from drying out of the gel, and is exacerbated by extreme run conditions,
i.e., high voltage, high power, high temperature, and long run times.
Therefore, 36-cm, 2400-scan/hr and 48-cm runs are expected to suffer from red rain
more readily than 36-cm, 1200-scan/hr runs. However, red rain has been reported to
occur in all three standard ABI PRISM 377 run conditions.
Optimizing Gel Electrophoresis 4-5
The following can be used to help prevent red rain:
♦
Wrap the gel plates to prevent the gel from drying out.
♦
Lower the run temperature from 51 °C to 48 °C.
–
A lower temperature results in a slower run speed. Less data will be collected
in the same run time.
–
A lower temperature also means less denaturing power in the gel, which can
lead to more compressions.
Gel Plate Quality Use high-quality gel plates. Plates from vendors other than Applied Biosystems may
not have the same quality control. Several problems result from poor quality plates,
including warping. Figure 7-58 on page 7-52 shows data from plates that warped after
6 months of use.
When plates become warped, the pathlength of the light changes and the laser no
longer focuses correctly on the gel. When this occurs on the ABI 373 DNA Sequencer,
laser light is scattered back to the detector, causing the gel image to appear blue and
green and obscuring data. On the ABI PRISM 377 DNA Sequencer, there is a filter to
keep out most of the scattered laser light, but data quality will still suffer.
Gel Plate Cleaning Cleaning gel plates properly is very important to prevent plate contamination and
obtain good data. Plate contamination can cause the following problems with gels:
♦
Gel extrusion (see page 4-9)
♦
Artifacts and background from fluorescent contamination (see Figure 7-52 on
page 7-46)
♦
Poor resolution
♦
Temporary loss of signal (see page 4-9)
Regular Cleaning
We recommend using a laboratory dishwasher with a hot deionized water (90 °C)
rinse for regular cleaning.
♦
The quality of the water and its temperature, pressure, and volume are critical for
effective cleaning.
♦
Rinse residual gel material before placing plates in the dishwasher.
♦
Use the longest deionized water rinse cycle initially, followed by a drying cycle.
After some experimentation, you may be able to reduce the rinse time.
♦
The use of detergents is not necessary when a dishwasher is used.
If a dishwasher is not available, then wash plates with a dilute solution of Alconox
detergent. Rinse with hot water, then rinse again with deionized water.
4-6 Optimizing Gel Electrophoresis
The following dishwashers have been found to work well:
Dishwasher
P/N
Supplier
Lancer 1600 dishwasher
with facility for drying
Lancer 1600 UP
Lancer USA Inc.
705 West Highway 434
Longwood, Florida 32750
Telephone: 407-332-1855
Lancer UK Ltd.
1 Pembroke Avenue
Waterbeach, Cambridge
CB5 9QR
Telephone: 44-01223-861665
Fax: 44-01223-861990
Sequencing plate rack (50
plate capacity) for Lancer
dishwasher
SPR 16
Lancer USA Inc. as listed
above
Labconco Undercounter
Glassware
15-352-801
Fisher Scientific
U.S. Headquarters
585 Alpha Drive
Pittsburgh, Pennsylvania 15238
Customer Service:
1-800-766-7000
Fax: 1-800-926-1166
Internet:
http://www.fishersci.com
We also recommend the following for preventing contamination of gel plates:
♦
Clean plates as soon as possible following electrophoresis.
♦
Once dry, avoid excessive handling of the plates with ungloved hands.
Removing Contaminants
The following procedures are not meant to be used for regular gel plate maintenance,
but for decontamination. For regular plate cleaning, we recommend using a
dishwasher with a hot, deionized water rinse (see page 4-6).
! WARNING ! Preparation of all solutions should be carried out in a hood using
safety glasses, gloves, and other appropriate protective clothing.
To perform an alcoholic KOH wash:
Step
1
Action
Add 30–35 g of potassium hydroxide (KOH) or sodium hydroxide (NaOH) pellets to
a plastic bottle.
! WARNING ! Potassium hydroxide is hygroscopic and caustic. It can
cause severe burns and blindness if it comes in contact with the skin or eyes.
Always work in a fume hood. Obtain a copy of the MSDS from the
manufacturer. Wear appropriate protective eyewear, clothing, and gloves.
2
Add 200 mL of absolute ethanol to the bottle.
! WARNING ! CHEMICAL HAZARD. Ethanol is a flammable chemical and
is irritating to the skin, eyes, respiratory system. It can cause nerve and liver
damage, CNS depression, nausea, vomiting, and headache. Always work in a
fume hood. Obtain a copy of the MSDS from the manufacturer. Wear
appropriate protective eyewear, clothing, and gloves.
Optimizing Gel Electrophoresis 4-7
To perform an alcoholic KOH wash:
Step
3
(continued)
Action
Mix the solution well. It will take at least 15 minutes for most of the pellets to
dissolve.
Note
This recipe is for a saturated solution, so some pellets will remain. Store
the solution with the bottle capped tightly. During storage, the color of the solution
will turn dark red-brown. The solution can still be used, and is good for 1 year.
4
Place some uncolored absorbent towels or other covering in the hood to catch
spills.
5
Place the gel plates on the towels with the inside surfaces facing up.
Note
The plates should be nearly level so that the cleaning solution does not
run off onto the bench. Only the inside (gel side) surface of the plates need be
cleaned, though the outside surfaces can be cleaned similarly.
6
Pour approximately 15 mL of the cleaning solution onto the center of each plate to
be cleaned. Spread the solution over the surface of plate.
7
Allow the solution to remain on the plates for 5 minutes.
CAUTION
8
Longer times can harm the plates.
Rinse the plates thoroughly with distilled, deionized water. Allow plates to dry.
Note
Avoid other cleaning procedures or solutions that may reintroduce
contaminants to the plates.
An alcoholic KOH wash can also be used to remove buffer chamber gasket marks
from the plates.
Step
Action
1
Perform steps 1–5 above.
2
Pour approximately 15 mL of the cleaning solution onto the area of the plate where
the gasket mark is.
3
Allow the solution to remain on the plates for 10 minutes.
CAUTION
Longer times can harm the plates.
4
Repeat steps 2 and 3.
5
Rinse thoroughly with deionized water.
6
Clean plates as usual.
To perform a 3 M HCl wash:
Step
Action
1
Place some uncolored absorbent towels or other covering in the hood to catch
spills.
2
Pour 10 mL of concentrated HCl (12 N, 37%) carefully into 30 mL of water and mix
thoroughly.
! WARNING ! Hydrochloric acid (HCl) is a very corrosive liquid. Always
work in a fume hood to avoid inhalation. Obtain a copy of the MSDS from the
manufacturer. Wear appropriate protective eyewear, clothing, and gloves.
4-8 Optimizing Gel Electrophoresis
To perform a 3 M HCl wash:
Step
3
(continued)
Action
Place the plates on the towels with the inside surfaces facing up.
Note
The plates should be nearly level so that the cleaning solution does not
run off onto the bench. Only the inside (gel side) surface of the plates need be
cleaned, though the outside surfaces can be cleaned similarly.
4
Pour approximately 15 mL of the cleaning solution in the center of each plate to be
cleaned. Spread the solution over the surface of plate.
5
Allow the solution to remain on the plates for 5 minutes.
Note
6
Longer times will not harm the plates but are unnecessary.
Rinse the plates thoroughly with distilled, deionized water. Allow plates to dry.
Note
Avoid other cleaning procedures or solutions that can reintroduce
contaminants to the plates.
Gel Extrusion When voltage is applied on the ABI PRISM 377 DNA Sequencer, the polyacrylamide
gel sometimes moves from between the glass gel plates toward the cathode (upper
electrode) and into the upper buffer chamber. Up to about five centimeters of gel in a
folded sheet can be deposited in the chamber.
This “gel extrusion” usually begins at the start of a run or even during the prerun. It is
believed to be caused by a buildup of charge on the surface of the glass plate such
that the gel is not bound to the plate after pouring. As the voltage is applied, the gel
migrates toward the upper electrode.
The gel image can show a variety of anomalous effects, including catastrophic loss of
resolution, lane splitting, extreme band tilt, and band distortion (Figure 7-56 on
page 7-50).
Almost all known cases of gel extrusion have been resolved by alcoholic KOH washing
(see page 4-7) or acid washing (see page 4-8).
Temporary Loss of This problem manifests itself as a band of little or no signal across the entire width of
Signal the gel image. It usually occurs between 150 and 250 bases (see Figure 7-57 on
page 7-51). Temporary loss of signal has been traced to contaminants on the gel
plates. These contaminants include surfactants, fatty acids, and long chain polymers.
Rinsing glass plates in a dishwasher with hot deionized water (90 °C) has been found
in most cases to remove the contaminants that cause temporary loss of signal (see
page 4-6).
In a few cases where a dishwasher did not work well, soaking the plates overnight in a
5% solution of Multiterge detergent (VWR Scientific, P/N 34171-010) eliminated the
temporary loss of signal.
Optimizing Gel Electrophoresis 4-9
Optimizing Capillary
Electrophoresis
5
5
Introduction
In This Chapter This chapter describes the following:
♦
Differences between capillary and gel electrophoresis
♦
Capillary electrophoresis consumables
♦
Optimizing capillary electrophoresis conditions
Refer to page 7-55 for information on troubleshooting capillary electrophoresis.
Capillary The large surface area of a capillary allows heat generated during electrophoresis to
Electrophoresis be dissipated efficiently, allowing high-voltage electrophoresis. The result is rapid,
high-resolution separation of DNA fragments. Applied Biosystems takes advantage of
this with the ABI PRISM® 310 Genetic Analyzer, a highly automated capillary
electrophoresis instrument.
Advantages of Capillary Electrophoresis
Capillary and slab gel electrophoresis both separate DNA fragments by size through a
sieving matrix, but there are important differences between the two techniques:
♦
There is no gel pouring. The ABI PRISM 310 Genetic Analyzer uses a liquid
polymer that is pumped automatically into the capillary.
♦
There is no manual sample loading. The instrument uses electrokinetic injection.
♦
Run times are shorter.
♦
Quantitation is more accurate.
♦
Very little sample is injected, which allows the sample to be run several times if
necessary.
Refer to the ABI PRISM 310 Genetic Analyzer User’s Manual (P/N 903565) for more
information.
Optimizing Capillary Electrophoresis 5-1
Capillary Electrophoresis Consumables
Polymer The polymer is the medium used to separate DNA fragments. There are two types of
polymer available for DNA sequencing on the ABI PRISM 310 Genetic Analyzer:
♦
DNA Sequencing Polymer (DSP)
♦
Performance Optimized Polymer 6 (POP-6™)
POP-6 provides superior performance over DSP because it can be run at a higher
temperature and yields greater read lengths.
Note
We do not recommend using the POP-6 polymer with fluorescein/rhodamine dye
primer chemistry.
IMPORTANT
Do not leave polymer on the instrument more than 5 days.
Introduction of any kind of dust into the polymer can cause spikes in the data. We
recommended that you minimize any actions that could introduce particles into the
polymer:
♦
Do not leave the POP-6 polymer exposed to the air by leaving the vial with the lid
open.
♦
Do not clean the syringe and gel block with unfiltered water.
♦
Do not install a capillary that has been sitting on a bench exposed to dust.
Genetic Analyzer Genetic Analyzer Buffer is used for electrophoresis. It is supplied in 10X concentration
Buffer and should be diluted to 1X concentration for use.
♦
Use 10X Genetic Analyzer Buffer with EDTA (P/N 402824) with uncoated
capillaries. If you do not use buffer with EDTA, capillary life will be shortened
greatly.
♦
Change the buffer every 2–3 days.
Note
Use 10X Genetic Analyzer Buffer without EDTA with coated capillaries, which are used
only with DNA Sequencing Polymer.
TSR The cycle sequencing protocols on the ABI PRISM 310 Genetic Analyzer use a sample
preparation reagent called Template Suppression Reagent (TSR). TSR is used to
prevent high molecular weight species from being injected into and clogging the
capillary.
♦
Store TSR at 2–6 °C.
♦
At room temperature, samples in TSR are stable for a maximum of 48 hours.
♦
Although not recommended on a routine basis, you can keep samples prepared in
TSR frozen for several weeks before running on the ABI PRISM 310 Genetic
Analyzer with no detectable loss in resolution or base calling.
5-2 Optimizing Capillary Electrophoresis
Capillaries The capillary has an opaque, polyimide external coating except in the window area.
The laser and detector read samples during electrophoresis through the window in the
coating. Capillaries are very fragile in the uncoated window area.
Capillaries should last at least 100 runs if treated properly. You may be able to get
more injections from a capillary, depending on your template preparation methods and
run conditions.
♦
Do not let capillaries with polymer in them dry out. Store their ends in buffer or
deionized water when not in use.
♦
Store unused capillaries in a dust-free environment.
♦
Do not touch capillary windows. If you do touch a window accidentally, clean it
with 95% ethanol.
Signs of capillary failure are the following:
♦
Decreased resolution
♦
High baseline
♦
Noisy data
♦
Trailing peaks in data (Figure 7-59 on page 7-55)
Information about the capillaries used on the ABI PRISM 310 Genetic Analyzer is given
in Table 5-1.
Table 5-1 Capillary Types
Capillary
Type
Marking
Color
Polymer Used
Coated/
Uncoated
10X Genetic
Analyzer
Buffer Type
Length
(cm)
Length to
Detector
(cm)
Internal
Diameter
(µm)
POP-6™
internally
uncoated
with EDTA
47
36
50
pink
POP-6
internally
uncoated
with EDTA
61
50
50
silver
DNA
Sequencing
Polymer (DSP)
internally
coated
without EDTA
47
36
75
Rapid
sequencing
green
Long-read
sequencing
Sequencing
Optimizing Capillary Electrophoresis 5-3
Optimizing Electrokinetic Injection
Introduction Optimizing electrokinetic injection can greatly improve data quality and run-to-run
reproducibility. The goal is to inject sufficient DNA to yield peaks of adequate height
(that is, data with a good signal-to-noise ratio) while maintaining resolution and read
length.
The ABI PRISM 310 run modules have preset values for injection times and voltages.
These values are adequate for most applications. However, you should consider
modifying the injection parameters if the signal is too strong or too weak or if the
resolution is poor.
Signal Too Strong
♦ Decrease the injection time.
♦
Decrease the injection voltage.
Signal Too Weak
♦ Increase the injection time.
The default injection time is 30 seconds. We do not recommend injection times
>120 seconds.
♦
Increase the injection voltage.
Increase the voltage in 1–2-kV increments. The maximum possible injection
voltage is 15 kV.
♦
Reduce the amount of salt in the sample.
IMPORTANT
Negative ions, e.g., EDTA and acetate, compete with DNA for injection. To
reduce the amount of salt in a sequencing reaction, use a spin column (see page 3-34).
Poor Resolution
♦ Decrease the injection time.
This will decrease the signal strength, so you may need to increase the injection
voltage.
Modifying Injection When you modify the injection time, you will encounter a tradeoff between signal
Time strength and resolution.
♦
Signal strength (as measured both by peak height and by peak area) increases
linearly with increasing injection time for most applications (Figure 5-1 and
Figure 5-2 on page 5-5).
♦
However, an n-fold increase in injection time does not result in an n-fold increase
in peak height. In Figure 5-1, no improvement is seen after 10 seconds for the
larger fragment. The signal decreases dramatically after 40 seconds for the
smaller fragment.
♦
As the injection time increases, the resolution decreases because of increasing
peak widths (Figure 5-3 on page 5-5). There is too much sample to move as a
discrete, well-resolved band.
5-4 Optimizing Capillary Electrophoresis
Figure 5-1 Peak height vs. injection time for two different-sized fragments (90 bp and 300 bp)
Figure 5-2 Peak area vs. injection time for two different-sized fragments (150 bp and 340 bp)
Figure 5-3 Resolution vs. injection time for different-sized fragments
Optimizing Capillary Electrophoresis 5-5
Modifying Injection Injection voltage has little effect on peak resolution. Resolution with injection voltages
Voltage of 319 V/cm (the highest possible setting) is often indistinguishable from resolution
with injection voltages of 53 V/cm (a typical value for a 47-cm capillary).
Peak height and peak area increase linearly with increasing injection voltage.
Setting For information on setting electrokinetic injection values, refer to the ABI PRISM 310
Electrokinetic Genetic Analyzer User’s Manual.
Injection Values
5-6 Optimizing Capillary Electrophoresis
Optimizing Electrophoresis Conditions
Introduction Optimizing electrophoresis conditions (run time, run voltage, and run temperature)
can greatly improve data quality, run-to-run reproducibility, and/or throughput. When
selecting values for these parameters, consider the following factors:
♦
Read length desired
♦
Required degree of resolution
Run Time Determining Required Run Time
To ensure that you collect sufficient data to perform analysis, set the electrophoresis
run time approximately 10% higher than the migration time of the longest fragment
you want to detect.
♦
For sequencing samples using DSP, the standard run voltage is 160 volts/cm. For
a 47-cm capillary, this translates to 7.5 kV. The current at this voltage is 7–10 µA.
The time required for a 450-base fragment to reach the detector window is about
135 minutes with these run conditions.
♦
For sequencing samples using POP-6 and long-read sequencing, the standard
run voltage is 200 volts/cm. For a 61-cm capillary, this translates to 12.2 kV. The
current at this voltage is 4–6 µA. The time required for a 600-base fragment to
reach the detector window is about 120 minutes with these run conditions.
♦
For sequencing samples using POP-6 and rapid sequencing, the standard run
voltage is 320 volts/cm. For a 47-cm capillary, this translates to 15 kV. The current
at this voltage is 5–8 µA. The time required for a 400-base fragment to reach the
detector window is about 35 minutes with these run conditions.
Changing Run Time
You can change the data collection time for special requirements. For example, you
can shorten the data collection time if you only need information about short extension
products, e.g., in PCR sequencing.
Run Temperature Protocols for sequencing applications with POP-6 specify a 50 ∞C electrophoresis
temperature.
For templates that do not denature readily, the run temperature can be increased by
1–2 °C, however, there is a tradeoff between run temperature and resolution.
Laboratory The laboratory temperature should be maintained between 15 and 30 °C. It should not
Temperature and fluctuate more than ±6 °C during a run for optimal results.
Humidity
The ABI PRISM 310 Genetic Analyzer can tolerate up to 80% non-condensing relative
humidity. Avoid placing the instrument near heaters, cooling ducts, or windows.
For More For information on setting electrophoresis parameters, refer to the ABI PRISM 310
Information Genetic Analyzer User’s Manual.
Optimizing Capillary Electrophoresis 5-7
Run Parameters for Specific Sequencing Chemistries
Table 5-2 Reference Table for Specific Sequencing Chemistries
Run
Module
Dye Set/Primer File
Base
Caller
Capillary
Size
Capillary
Mark
DSP
Seq Run
(250 uL) A
DT5%CEHV{A Set–
any primer}
CE-1
47 cm x
75 µm
silver
1.0 mL
POP-6™
Seq POP6
(1.0 mL) A
DT POP 6
CE-2
61 cm x
50 µm
pink
Rhodamine
Dye Terminator,
rapid sequencing
1.0 mL
POP-6
Seq POP6
(1.0 mL)
Rapid A
DT POP 6
CE-2
47 cm x
50 µm
green
dRhodamine
Terminator
250 µL
DSP
Seq Run
(250 uL) E
DT5%CEHV{dR Set–
any primer}
CE-1
47 cm x
75 µm
silver
dRhodamine
Terminator,
long-read
sequencing
1.0 mL
POP-6
Seq POP6
(1.0 mL) E
DT POP6 {dR Set–
any primer}
CE-1
61 cm x
50 µm
pink
dRhodamine
Terminator,
rapid sequencing
1.0 mL
POP-6
Seq POP6
(1.0 mL)
Rapid E
DT POP6 {dR Set–
any primer}
CE-1
47 cm x
50 µm
green
BigDye™
Terminator,
long-read
sequencing
1.0 mL
POP-6a
Seq POP6
(1.0 mL) E
DT POP6 {BD Set–
any primer}
CE-1
61 cm x
50 µm
pink
BigDye Terminator,
rapid sequencing
1.0 mL
POP-6
Seq POP6
(1.0 mL)
Rapid E
DT POP6 {BD Set–
any primer}
CE-1
47 cm x
50 µm
green
Fluorescein/
Rhodamine
Dye Primer
250 µL
DSP
Seq Run
(250 uL) A
DP5%CEHV{-21M13}
or
DP5%CEHV{M13rev}
CE-1
47 cm x
75 µm
silver
BigDye™ Primer,
long-read
sequencing
1.0 mL
POP-6a
Seq POP6
(1.0 mL) E
DP POP6 {BD
Set-21M13} or
DP POP6 {BD
Set-M13rev}
CE-1
61 cm x
50 µm
pink
BigDye Primer,
rapid sequencing
1.0 mL
POP-6
Seq POP6
(1.0 mL)
Rapid E
DP POP6 {BD
Set-21M13} or
DP POP6 {BD
Set-M13rev}
CE-1
47 cm x
50 µm
green
Chemistry
Syringe
Polymer
Rhodamine
Dye Terminator
250 µL
Rhodamine
Dye Terminator,
long-read
sequencing
a. The BigDye terminator and BigDye primer chemistries are not used with DNA Sequencing Polymer (DSP) on the ABI PRISM 310
instrument.
5-8 Optimizing Capillary Electrophoresis
Optimizing Software
Settings
6
6
Introduction
In This Chapter This chapter describes the following:
Topic
See page
Choosing a Run Module
6-2
Choosing a Dye Set/Primer (Mobility) File
6-3
Choosing the Correct Basecaller
6-6
Creating an Instrument (Matrix) File
6-7
Setting the Data Analysis Range
6-15
Optimizing Software Settings 6-1
Choosing a Run Module
Overview A run module file contains all the parameters required for a particular function or
application (see page 1-12). On the ABI PRISM® 310, ABI™ 373 with XL Upgrade, and
ABI PRISM® 377 instruments, choosing a run module automatically chooses the filter
set used to collect the data. If an incorrect run module is chosen for a particular
chemistry, the data will be poor, with low resolution and miscalled bases (Figure 7-62
on page 7-62). If this happens, rerun the samples using the correct run module.
On ABI 373 instruments there are no run modules, so the filter set and electrophoresis
parameters must be chosen manually. For fluorescein/rhodamine dye primer and
rhodamine dye terminator chemistries, Filter Set A is used. For dRhodamine-based
chemistries on instruments with the BigDye™ Filter Wheel, Filter Set A is also used,
but the filters have different wavelengths (see page 1-8).
List of Run Modules
Table 6-1 Run Modules
Instrument
Filter Set A
Filter Set E
ABI PRISM 310
Seq Fill Capillary
Seq Fill Capillary
Seq POP6 (1 mL) A
Seq POP6 (1 mL) E
Seq POP6 Rapid (1 mL) A
Seq POP6 (250 uL) E
Seq Run (250 uL) A
Seq POP6 Rapid (1 mL) E
Test CCD 4-Color
Seq Run (250 uL) E
Test CCD 4-Color
ABI 373 with
XL Upgrade
Plate Check
Pre Run
Seq Run
ABI PRISM 377 (All
Models)a
Plate Check
Plate Check
Plate Check A
Plate Check Ab
Seq PR 36A-1200
Plate Check Ec
Seq PR 36A-2400
Seq PR 36A-1200b
Seq Run 36A-1200
Seq PR 36A-2400b
Seq Run 36A-2400
Seq PR 36E-1200c
Seq Run 48A-1200
Seq PR 36E-2400c
Seq Run 36E-1200
Seq Run 36E-2400
Seq Run 48E-1200
a. Older versions of the ABI PRISM 377 Collection Software may use different nomenclature for run modules,
e.g., PR 2X A = Seq PR 36A-1200, Run 4X A = Seq Run 36A-2400.
b. Any plate check and prerun module can be used on the ABI PRISM 377 DNA Sequencer.
c. For the ABI PRISM 377 DNA Sequencer with 96-Lane Upgrade only.
6-2 Optimizing Software Settings
Choosing a Dye Set/Primer (Mobility) File
Overview The different dyes affect the electrophoretic mobility of cycle sequencing extension
products to varying degrees, especially on shorter fragments. However, these mobility
shifts are consistent for each dye set and can be corrected readily during analysis.
The analysis software is able to compensate for these mobility differences by applying
mobility shifts to the data so that evenly spaced peaks are presented in the analyzed
data. The files that contain the mobility shift information are called dye set/primer
(mobility) files.
The dye set/primer files available are listed on page 6-5. The files are located in the
ABI folder within the System folder on the Macintosh® or Power Macintosh® computer.
They are selected in the sample sheet during instrument setup. They can also be
selected in the Sequencing Analysis software if samples need to be reanalyzed.
Using the Wrong The seriousness of choosing the wrong dye set/primer file (mobility) depends on
Dye Set/Primer File which mobility file you choose to analyze the data:
♦
Analyzing fluorescein/rhodamine dye primer data with the wrong fluorescein/
rhodamine dye primer mobility file causes shifted peaks.
♦
Analyzing fluorescein/rhodamine dye primer data with rhodamine dye terminator
mobility files or vice versa causes both shifted peaks and miscalled bases,
because the dyes used for the fluorescein/rhodamine dye primer and rhodamine
dye terminator chemistries are different.
♦
Analyzing fluorescein/rhodamine dye primer data with BigDye primer mobility files
causes shifted peaks.
♦
Analyzing BigDye™ primer data with a mobility file for dRhodamine terminator or
BigDye™ terminator chemistry (or vice versa) causes both shifted peaks and
miscalled bases (Figure 7-63 on page 7-62). These three chemistries use the
same dyes for fluorescence emission, but on different bases from each other. See
Chapter 2, especially page 2-14, for more information.
♦
Analyzing dye terminator chemistry data with the wrong type of dye terminator
mobility file can cause both shifted peaks and miscalled bases. The dRhodamine
terminators and BigDye terminators use different dyes for different bases, i.e., the
dyes for the C and T bases are switched (see page 2-14).
The dye set/primer file used for data analysis is shown in the annotation view of the
sample file (see page 7-8) and is also part of the header information on the sequence
electropherogram printout. If you do analyze with incorrect information, the data can
be reanalyzed with the correct dye set/primer file as described in your user’s manual.
Optimizing Software Settings 6-3
Chemistry-Specific Fluorescein/Rhodamine Dye Primers
Mobility The four fluorescent dyes used for fluorescein/rhodamine dye primer sequencing are
Information of two structural types: 5-FAM and JOE molecules are fluorescein dyes, and TAMRA
and ROX are rhodamine dyes (see Figure 2-5 on page 2-8). Rhodamine dyes migrate
more slowly during electrophoresis than fluorescein dyes.
The dyes affect the mobilities of different primers in ways specific to each primer. The
analysis software contains mobility files for the following sequencing primers:
♦
–21 M13 Forward
♦
T7
♦
M13 Reverse
♦
PI(+)a
♦
SP6
♦
PI(–)a
♦
T3
a. Used with the Primer Island Transposition Kit.
IMPORTANT
For correct data analysis, the dye set/primer file must be specified in the
sample sheet before data collection and automatic analysis. If you choose the wrong dye set/
primer file, the data can be reanalyzed with a different file after automatic analysis.
The fluorescent dyes appear to interact with the first five bases on the 5´ end of the
oligonucleotide to which they are attached. This interaction is responsible for their
effect on primer mobility.
If you use custom dye-labeled primers, synthesize them so that their 5´ ends contain
the same five bases as the 5´ end of the M13 Reverse primer, CAGGA. Analyze the
sequence data with M13 Reverse dye set/primer files. This works well in most cases,
as long as the primers are made with the 5-FAM, JOE, TAMRA, and ROX dyes.
Contact the Applied Biosystems Custom Oligonucleotide Synthesis Service (see
page 3-19 for contact information).
BigDye Primers
–21 M13 Forward and M13 Reverse primers use the same dye set/primer file on the
ABI PRISM 377 DNA Sequencer. This is possible because the mobility shifts are very
similar for each primer.
Rhodamine Dye Terminators
Rhodamine dye terminator chemistry uses a set of four rhodamine dyes (see
Figure 2-1 on page 2-2). Rhodamine dye-labeled DNA fragments migrate true to
molecular weight and no mobility correction is needed. However, the dye set/primer
file is needed to tell the software which matrix file is used for analysis and which color
is associated with each of the four bases.
dRhodamine Terminators and BigDye Terminators
dRhodamine- and BigDye-labeled DNA fragments do not necessarily migrate true to
molecular weight. Some mobility correction is required.
Mobility shifts and dye set/primer file names for the dRhodamine Terminators are
similar to those for the BigDye Terminators. If a mobility file for the wrong sequencing
chemistry is used, C and T bases will be miscalled because of differences in which
terminators are labeled with which dyes (see page 2-14).
6-4 Optimizing Software Settings
List of Dye Set/Primer Files
Table 6-2 Dye Set/Primer (Mobility) Files
Sequencing Chemistry
Instrument
Rhodamine
Dye Terminator
ABI PRISM 310
DT POP6
DT5%CEHV{A
Set-AnyPrimer}
dRhodamine
Terminator
BigDye
Terminator
DT POP6{dR
Set-Any Primer}
DT POP6{BD
Set-Any Primer}
DT DSP{dR
Set-AnyPrimer}
Fluorescein/
Rhodamine
Dye Primer
DP5%CEHV{-21M13}
DP5%CEHV{M13Rev}
DP5%CEHV{SP6}
DP5%CEHV{T3}
BigDye Primer
DP POP6{BD
Set-21M13}
DP POP6{BD
Set-M13
Reverse}
DP5%CEHV{T7}
ABI 373 and
ABI 373 with
XL Upgrade
DT4%Ac{A
Set-AnyPrimer}
DP4%Acv2{M13Rev}
DT6%Ac{A
Set-AnyPrimer}
DP4%Ac{SP6}
DP4%Ac{-21M13}
DP4%Ac{T3}
DP4%Ac{T7}
DP6%Ac{-21M13}
DP6%Ac{M13Rev}
DP6%Ac{SP6}
DP6%Ac{T3}
DP6%Ac{T7}
PI+.mob
PI-.mob
373 dRDT
ABI 373 and
ABI 373XL
with BigDye
Filter Wheel
ABI PRISM 377
(All Models)
373 BDT
373 BDP(-21)
373 BDP rev
DT4%Ac{A
Set-AnyPrimer}
DT {dR Set
Any-Primer}
DT {BD Set
Any-Primer}
DP4%Acv2{M13Rev}
DP4%Ac{-21M13}
DP4%Ac{SP6}
DP5%LR{BD
M13 FWD &
REV}
DP4%Ac{T3}
DP4%Ac{T7}
PI+.mob
PI-.mob
Refer to your instrument user’s manual and the ABI PRISM DNA Sequencing Analysis
Software User’s Manual for more specific information on choosing a dye set/primer
file.
l
Optimizing Software Settings 6-5
Choosing the Correct Basecaller
Choosing a The basecaller is the program that determines the individual base identities in a
Basecaller sequence. The Applied Biosystems basecallers differ from each other primarily in the
shape of the internal spacing curves.Choosing the most effective basecaller for any
given sample file depends on the quality of the data, the type of run, and the run and
gel conditions.
Table 6-3 shows basecaller for each run type. You can try other basecallers with your
data to see which works best.
Table 6-3 Choosing the Correct Basecaller
If the samples are from a...
Then use...
ABI PRISM 310, rhodamine dye terminator chemistry with POP-6
CE-2
ABI PRISM 310, all other applications
CE-1
24- or 34-cm well-to-read gel on the ABI 373
ABI50
BaseSprinter or 373-18 run on the ABI 373
ABI100
Typical 1200-scan/hr, 36-cm well-to-read gel on the ABI PRISM 377
ABI100
Typical 2400-scan/hr, 36-cm well-to-read gel on the ABI PRISM 377
ABI200
48-cm well-to-read gel on the ABI 373
ABI50
48-cm well-to-read, 5.25% PAGE-PLUS gel on the ABI PRISM 377
ABI50
48-cm well-to-read, Long Ranger or 19:1 or 29:1 polyacrylamide gel
on the ABI PRISM 377
ABI100
Run with many insertions or deletions near the end of the run
SemiAdaptive
If the spacing is a negative number
SemiAdaptive
If the spacing is still a negative number with SemiAdaptive
Adaptive
If you noticed problems with run conditions
Adaptive
IMPORTANT
Although each basecaller is optimized for a specific type of run, depending on
your run conditions you might get better data using a different basecaller. Analyze your data
with different basecallers to determine which one works best for your run conditions.
IMPORTANT
If you reanalyze a sample file, the previous analysis results are overwritten by
the new results. To avoid erasing the previous analysis results, save a copy of the sample file
under a different name before you reanalyze.
For more information, refer to the ABI PRISM DNA Sequencing Analysis Software
User’s Manual.
6-6 Optimizing Software Settings
Creating an Instrument (Matrix) File
Overview You must use an appropriate instrument (matrix) file to get good data. The instrument
file contains the information that allows the Sequencing Analysis software to
compensate for the spectral overlap between the dyes when data is analyzed. In
some cases, using an incorrect instrument file causes analysis to fail. Instrument files
vary between different instruments, and between filter sets on a single instrument. An
instrument file must be made for each filter set on each instrument.
The appropriate matrix file can be applied to data on subsequent capillary runs or gels
on the same instrument, as long as the same filter set is used. This is because the
spectral overlap between the four dyes is very reproducible.
When an You will need to make a new instrument file if:
Instrument File ♦ Different dye set used
Needs to be Remade
♦
Aging filter wheel on an ABI 373 DNA Sequencer
♦
Changes to any optics occur, e.g., new filter wheel (on an ABI 373 DNA
Sequencer) or CCD camera (on an ABI PRISM 310 or ABI PRISM 377 instrument)
If you use the wrong instrument file (Figure 7-65 on page 7-63), you will need to
reanalyze the data with the correct instrument file. However, if you collect data using
the wrong filter set, you should rerun the samples using the correct filter set.
Refer to the ABI PRISM DNA Sequencing Analysis Software User’s Manual for
instructions on creating a new instrument file or follow the instructions on page 6-8 for
creating an instrument file for dRhodamine-based chemistries.
Data Utility The Data Utility software is used to make instrument files. You must put the correct
Software data file for each matrix standard into the correct “box” in the Data Utility application.
Table 6-4 shows the correct placement for making a Filter Set E instrument file (Filter
Set A on the ABI 373 DNA Sequencer with BigDye Filter Wheel).
Table 6-4 Placement of Standards for dRhodamine-Based Chemistries
Dye Primer
Matrix
Taq Terminator
Matrix
T7 Terminator
Matrix
C…
dR110
dROX
dR6G
A…
dR6G
dR6G
dTAMRA
G…
dTAMRA
dR110
dROX
T…
dROX
dTAMRA
dR110
Box
When creating a Filter Set E instrument file, you need to make all three matrix files,
even if you are only using one dRhodamine-based chemistry. The data collection
software will not run with only a Taq or T7 terminator matrix in the file.
The T7 Terminator Matrix file is needed to analyze dRhodamine terminator and
BigDye terminator sequencing data. It has a baselining algorithm associated with it
that works well with these chemistries. The dRhodamine terminator and BigDye
terminator dye set/primer files have tags in them that tell the Sequencing Analysis
software to select this matrix file.
Optimizing Software Settings 6-7
Making a
Filter Set E
Instrument File
from Matrix
Standards
This procedure is for making instrument files for dRhodamine-based chemistries
(Filter Set E on the ABI PRISM 310 and ABI PRISM 377 instruments, Filter Set A on the
ABI 373 DNA Sequencer with BigDye Filter Wheel). Refer to your instrument user’s
manual for general instructions on creating instrument files.
To make the Dye Primer Matrix:
Step
Action
1
Launch the Data Utility software (located in the Utilities folder within the Sequencing
Analysis folder).
2
From the Utilities menu, choose Make Matrix…
The Make Matrix dialog box appears as shown below. Verify that the Dye Primer
Matrix button at the lower left is selected.
3
4
Click on the box for each nucleotide base and select the sample file that
corresponds to the correct matrix standard as shown in the table below.
Box
Dye Primer
Matrix
C…
dR110
A…
dR6G
G…
dTAMRA
T…
dROX
For each matrix standard sample, start with the default value of 2000 for the start
point. Start with the default value of 1500 for the number of data points to analyze.
Note
If the default values do not work, follow the instructions for using other
values in steps 8 and 9 below.
6-8 Optimizing Software Settings
To make the Dye Primer Matrix:
Step
5
(continued)
Action
Click New File…
A dialog window appears as shown below. Name the file dRhod_BigDye (or another
appropriate name) and save it in the ABI folder within the System folder.
6
The Make Matrix dialog box should look like that shown below.
a.
Click OK.
The computer makes the matrix. When finished, a dialog window appears with the
message “Make matrix successfully completed.”
b.
7
Click OK.
If the computer is unable to make a matrix, examine the raw data again in the
Sequencing Analysis software. If you used the default values, then select new start
points as directed in steps 8 and 9 below. If many peaks are off-scale, dilute the
matrix standards and rerun them.
Optimizing Software Settings 6-9
To make the Dye Primer Matrix:
Step
8
9
(continued)
Action
If the matrix cannot be made with the default values, proceed with steps a, b, and c
below.
a.
In the Sequencing Analysis software, open a matrix standard sample and
examine the raw data. An example is shown below.
b.
Select a starting point where there are no peaks and the baseline is flat.
c.
Select a number of data points to analyze such that no peaks in the range are
off-scale, i.e., above 4000 relative fluorescence units (RFU), and that the
baseline at the end of the range is flat. A typical number of data points is 1500.
Repeat step 8 for each matrix standard sample. Record the results for later use.
IMPORTANT
The number of data points analyzed is the same for each matrix
standard. Choose starting points for each sample such that all peaks are less than
4000 RFU and that both the starting and ending points have flat baselines and no
peaks.
6-10 Optimizing Software Settings
To make the Taq Terminator Matrix:
Step
Action
1
In the Data Utility application, choose Make Matrix… from the Utilities menu. The
Make Matrix dialog box appears.
2
In the Make Matrix dialog box, click the Taq Terminator Matrix button at the lower
left.
3
Click on the box for each nucleotide base and enter the data file that corresponds to
the correct matrix standard as shown in the table below.
Box
Taq Terminator
Matrix
C…
dROX
A…
dR6G
G…
dR110
T…
dTAMRA
IMPORTANT
The order of matrix standard data files is different from that in the
Dye Primer Matrix (see Table 6-4 on page 6-7).
4
Enter the same numbers for each matrix standard sample in the Start at and Points
boxes as were used for the Dye Primer Matrix.
5
Click Update File… A dialog window appears.
6
Choose dRhod_BigDye from the ABI folder within the System folder and click
Open.
The Make Matrix dialog box should look like that shown below.
Note
The numbers in the Start at and Points boxes are default values. Your
numbers may vary.
7
a.
Click OK.
The computer makes the matrix. When finished, a dialog window appears with the
message “Make matrix successfully completed.”
b.
Click OK.
Optimizing Software Settings 6-11
To make the T7 Terminator Matrix:
Step
Action
1
In the Data Utility application, choose Make Matrix… from the Utilities menu. The
Make Matrix dialog box appears.
2
In the Make Matrix dialog box, click the T7 Terminator Matrix button at the lower left.
3
Click on the box for each nucleotide base and enter the data file that corresponds to
the correct matrix standard as shown in the table below (note the order of the matrix
standard files).
Box
T7 Terminator
Matrix
C…
dR6G
A…
dTAMRA
G…
dROX
T…
dR110
4
Enter the same numbers for each matrix standard sample in the Start at and Points
boxes as were used in the Dye Primer Matrix and Taq Terminator Matrix.
5
Click Update File… A dialog window appears.
6
Choose dRhod_BigDye from the ABI folder within the System folder and click
Open.
The Make Matrix dialog box should look like that shown below.
Note
The numbers in the Start at and Points boxes are default values. Your
numbers may vary.
7
a.
Click OK.
The computer makes the matrix. When finished, a dialog window appears with the
message “Make matrix successfully completed.”
b.
6-12 Optimizing Software Settings
Click OK.
To check the instrument file:
Step
Action
1
From the Utilities menu, choose Copy Matrix…
2
Under Source, select Instrument file and choose dRhod_BigDye from the ABI folder
within the System folder.
The three matrix files within the dRhod_BigDye instrument file appear as shown
below.
3
Make sure that all three matrix files have numbers that range from 0–1. The
numbers on the diagonals from top left to bottom right should be 1, and the
off-diagonal numbers should decrease moving away from the diagonal in any
direction.
If not, then repeat the matrix-making procedure starting with “To make the Dye
Primer Matrix:” on page 6-8.
Note
The corresponding numbers for all three matrix files will be the same (or
within 0.001 because of rounding).
4
Click Cancel.
5
Open or restart the Sequencing Analysis software and use dRhod_BigDye as the
instrument file to analyze your sequencing data.
Optimizing Software Settings 6-13
Making an An instrument file can be made from matrix standards as explained above, or it can be
Instrument File from made from a sample file. This procedure requires fewer steps than running matrix
a Sample File standards, however, the matrix made from a sample file may not be as good as one
made from matrix standards. The quality of a matrix file made from a sample file
depends on the quality of the sample file used.
The best samples to choose for making a matrix have approximately 25% each of A,
C, G, and T. A good example of this is the pGEM® control DNA that is included in
every Applied Biosystems cycle sequencing kit. To create a matrix from a sample file,
follow the steps below.
Step
Action
1
Before making the matrix, verify that lane tracking is accurate. Adjust if necessary.
2
Duplicate the unanalyzed sample file three times. Use the Duplicate command from
the File menu in the Finder. You will have a total of four copies of the same sample
file with the following names:
♦
Sample name
♦
Sample name Copy 1
♦
Sample name Copy 2
♦
Sample name Copy 3
3
These four sample files can now be used in the same way as the four matrix
standard samples. The same instructions can be used with these four samples as
with the four matrix standard samples.
4
Follow the directions in your instrument user’s manual for creating an instrument
file.
Wherever the protocol indicates a specific matrix standard to be used, follow the
table below:
Matrix Standard
6-14 Optimizing Software Settings
Standard File
C…
Sample name
A…
Sample name Copy 1
G…
Sample name Copy 2
T…
Sample name Copy 3
Setting the Data Analysis Range
Overview Three values are important in setting the data analysis range:
♦
Peak 1 Location
♦
Start Point
♦
Stop Point
Peak 1 Location
The Peak 1 Location is the data point that marks the beginning of the first base peak
in the data. This data point is the reference point for the spacing and mobility
corrections performed by the basecalling software. Correct identification of this
position is very important. If the Peak 1 Location value is wrong due to low signal,
excess dye peaks, or other pathology, your data can show bad spacing or strange
mobility shifts.
The Peak 1 Location value is calculated by the Sequencing Analysis software. In the
2.1 version of the software the term “Primer Peak Location” is used to designate this
value.
Occasionally the basecalling software assigns the Peak 1 Location value either too
early or too late. In either case, the correct Peak 1 Location should be identified and
changed for optimal basecalling. The procedure used to change the Peak 1 Location
(Primer Peak Location) will depend on which version of Sequencing Analysis software
and the type of chemistry, i.e., dye primer or dye terminator, you are using.
Table 6-5 below and Table 6-6 on page 6-16 show typical Peak 1 Location values for
various instrument configurations and run conditions.
Table 6-5 Approximate Peak 1 Locations for the ABI 373 and ABI PRISM 377 DNA Sequencers
Instrument
ABI 373b
ABI PRISM 377c
Run Type
Gel Typea
Approximate
Peak 1 Location
(scan number)
24
Full or XL scan
6% Ac19
700
70
24
BaseSprinter
4.75% Ac19
1000
50
34
Full or XL scan
4.75% Ac19
800
80
34
BaseSprinter
4.25% Ac19
1200
60
48
Full or XL scan
4% Ac19
1000
100
36
1200 scans/hr
4% Ac19, 4.5% Ac29,
5% LR, 4.8% PP
800
40
36
2400 scans/hr
4% Ac19, 4.5% Ac29,
5% LR
800
20
48
1200 scans/hr
4% Ac19, 4.25% Ac29,
4.75% LR, 5.25% PP
1200
60
Well-to-Read
Length (cm)
Approximate
Time to
Peak 1 (min)
a. Ac19 = 19:1 polyacrylamide, Ac29 = 29:1 polyacrylamide, LR = Long Ranger, PP = PAGE-PLUS.
b. ABI 373 and ABI 373 with XL Upgrade.
c. All models.
Note
For the ABI 373 and ABI PRISM 377 instruments, scan numbers correspond to data
points. These terms are sometimes used interchangeably.
Optimizing Software Settings 6-15
Table 6-6 Approximate Peak 1 Locations for the ABI PRISM 310 Genetic Analyzer
Capillary
Length (cm)
Approximate
Peak 1 Location
(data points)
Approximate
Time to Peak
1 (min)
Run Type
Polymer Type
47
rapid sequencing
POP-6
1050–1150
25
61
long-read
sequencing
POP-6
1700–2000
40
Note
Peak 1 Locations and run times on the ABI PRISM 310 instrument can be affected by
the laboratory temperature.
Start Point
The Start Point is the starting point for data analysis. The Start Point is normally the
same as the Peak 1 Location value. However, the Start Point can be set later than the
Peak 1 Location if desired (see page 7-64).
Note
The Start Point can never be less than the Peak 1 Location value.
Stop Point
The Stop Point specifies the last raw data point to be included in the basecalling. If the
default Stop Point is used, this endpoint is the last data point in the file. However, the
basecalling can be stopped earlier if there is unusable raw data at the end of the file or
if you want to analyze only a portion of the raw data in the file.
Peak 1 Location for The Peak 1 Location (Primer Peak Location) is the position of the first base peak. In
Dye Primer dye primer chemistries this peak is found on the downward slope of the primer peak
Chemistries (Figure 6-1).
If you need to change the Peak 1 Location, use one of the following procedures
depending on which version of the Sequencing Analysis software you are using (refer
to Figure 6-1 during the procedure).
Figure 6-1 Electropherogram of raw data with the dashed vertical line showing the right edge
of the primer peak, which is the recommended position of the Peak 1 Location for this sample
file, i.e., 1109 scans.
6-16 Optimizing Software Settings
Using Sequencing Analysis Version 2.1
Step
Action
1
Launch the Sequencing Analysis software if it is not already open.
2
In the Sample File Queue display, double-click the first file to be analyzed to view
the raw data.
Zoom in completely. From the Window menu, choose Actual Size (or use the c ]
keys on the keyboard).
3
Starting at the beginning of the raw data file, scroll along the data by clicking and
holding the right direction arrow at the bottom of the window.
Continue scrolling until the primer peak is approximately in the center of the
window.
4
5
a.
Click and drag in the window to change the cursor to a cross-hair.
b.
Move the cursor along the data until the vertical dotted line is aligned at the
right edge of the primer peak (Figure 6-1 on page 6-16).
c.
Read the scan number (data point) that is reported at the top of the dialog box
(1109 in the example shown in Figure 6-1). Use this number as the Primer
Peak Location for the file.
Return to the Sample File Queue display.
a.
Highlight the name of the file just inspected and click the Custom Settings
window.
b.
Select the Change Primer Peak check box. The Use Start Point and Change
Primer Peak radio buttons become active. The Use Start Point radio button is
selected by default, since the Primer Peak Location and the Start Point are the
same in most cases.
c.
Click the Change Primer Peak radio button to display the entry field. The
number in the data field is the number used for the Primer Peak Location
during the last analysis.
d.
Enter the new number.
Note
If the value assigned for the Primer Peak location is greater than that
assigned for the Start Point, the Start Point value needs to be changed to that of the
Primer Peak Location. If you want the Primer Peak location to be used as the Start
Point, changing the Start Point value and leaving the Change Primer Peak radio
button as the default (Use Start Point) should also work. In Sequencing Analysis
version 2.1, there is a link between the Start Point and the Primer Peak Location.
Using Sequencing Analysis Version 3.0 or 3.2
Step
Action
1
Launch the Sequencing Analysis software if it is not already open.
2
In the Sample Manager window, click the Add Files button and choose the sample
to be analyzed. Click Done.
3
Highlight the sample name and click the Open Files button to display the raw data,
or double-click the sample file name.
Zoom in completely. From the Window menu, choose Actual Size (or use the c ]
keys on the keyboard).
Optimizing Software Settings 6-17
Using Sequencing Analysis Version 3.0 or 3.2
Step
4
(continued)
Action
Starting at the beginning of the raw data file, scroll along the data by clicking and
holding the right direction arrow at the bottom of the window.
Continue scrolling until the primer peak is approximately in the center of the dialog
window.
5
6
a.
Click and drag in the window to change the cursor to a cross-hair.
b.
Move the cursor along the data until the vertical dotted line is aligned at the
right edge of the primer peak (Figure 6-1 on page 6-16).
c.
Read the scan number (data point) that is reported at the top of the dialog box
(1109 in the example shown in Figure 6-1). Use this number as the Peak 1
Location of the file.
Return to the Sample Manager window.
Highlight the Peak 1 Location box and enter the information. If you want to use the
Peak 1 location value for the Start Point, enter the new Start Point as well.
Note
The Start Point value must be equal to or greater than the Peak 1 Location
value or an error will occur. If the Start Point is not greater than the Peak 1 Location,
highlight the Start Point box and enter a number greater than or equal to the
number used for the Peak 1 Location.
Peak 1 Location for As with dye primer chemistries, the Peak 1 Location value (Primer Peak Location) is
Dye Terminator the position that marks the beginning of the first base in the file. To determine the Peak
Chemistries 1 Location in terminator chemistry the software looks for an increase in signal followed
by several peaks.
With dye terminator chemistries the raw data can show peaks between scan points.
These peaks can be designated erroneously as the Peak 1 Location value by the
software. These peaks are due to unincorporated dye terminators that are not
removed during the purification of the dye terminator reactions.
If your data has excess dye peaks or if the software has not chosen the Peak 1
Location correctly, you will need to set this manually. To change the Peak 1 Location,
use one of the following procedures depending on which version of the Sequencing
Analysis software you are using (refer to Figure 6-2 on page 6-19 during the
procedure).
6-18 Optimizing Software Settings
Figure 6-2 Electropherogram of raw data with the dashed vertical line showing the left edge of
the first data peak, which is the recommended position of the Peak 1 Location for this sample
file, i.e., 960 scans.
Using Sequencing Analysis Version 2.1
Step
Action
1
Launch the Sequencing Analysis software if it is not already open.
2
In the Sample File Queue display, double-click the first file to be analyzed to view
the raw data.
Zoom in completely. From the Window menu, choose Actual Size (or use the c ]
keys on the keyboard).
3
Starting at the beginning of the raw data file, scroll along the data by clicking and
holding the right direction arrow at the bottom of the window.
Continue scrolling until the fist data peak is approximately in the center of the
window.
4
a.
Click and drag in the window to change the cursor to a cross-hair.
b.
Move the cursor along the data until the vertical dotted line is aligned at the left
edge of the first data peak (Figure 6-2).
c.
Read the scan number (data point) that is reported at the top of the dialog box
(960 in the example shown in Figure 6-2). Use this number as the Primer Peak
Location of the file.
Optimizing Software Settings 6-19
Using Sequencing Analysis Version 2.1
Step
5
(continued)
Action
Return to the Sample File Queue display.
a.
Highlight the name of the file just inspected and click the Custom Settings
window.
b.
Select the Change Primer Peak check box. The Use Start Point and Change
Primer Peak radio buttons become active. The Use Start Point radio button is
selected by default, since the Primer Peak Location and the Start Point are the
same in most cases.
c.
Click the Change Primer Peak radio button to display the entry field. The
number in the data field is the number used for the Primer Peak Location
during the last analysis.
d.
Enter the new number.
Note
If the value assigned for the Primer Peak location is greater than that
assigned for the Start Point, the Start Point value needs to be changed to that of the
Primer Peak Location. If you want the Primer Peak location to be used as the Start
Point, changing the Start Point value and leaving the Change Primer Peak radio
button as the default (Use Start Point) should also work. In Sequencing Analysis
version 2.1, there is a link between the Start Point and the Primer Peak Location.
Using Sequencing Analysis Version 3.0 or 3.2
Step
Action
1
Launch the Sequencing Analysis software if it is not already open.
2
In the Sample Manager window, click the Add Files button and choose the sample
to be analyzed. Click Done.
3
Highlight the sample name and click the Open Files button to display the raw data,
or double-click the sample file name.
Zoom in completely. From the Window menu, choose Actual Size (or use the c ]
keys on the keyboard).
4
Starting at the beginning of the raw data file, scroll along the data by clicking and
holding the right direction arrow at the bottom of the window.
5
a.
Click and drag in the window to change the cursor to a cross-hair.
b.
Move the cursor along the data until the vertical dotted line is aligned at the left
edge of the first data peak (Figure 6-2 on page 6-19).
c.
Read the scan number (data point) that is reported at the top of the dialog box
(960 in the example shown in Figure 6-2). Use this number as the Peak 1
Location of the file.
6
Return to the Sample Manager window.
Highlight the Peak 1 Location box and enter the information. If you want to use the
Peak 1 Location value for the Start Point, enter the new Start Point as well.
Note
The Start Point value must be equal to or greater than the Peak 1 Location
value or an error will occur. If the Start Point is not greater than the Peak 1 Location,
highlight the Start Point box and enter a number greater than or equal to the
number used for the Peak 1 Location.
6-20 Optimizing Software Settings
Stop Point The Stop Point specifies the last raw data point to be including in the base calling. If
the default Stop Point is used, this endpoint is the last data point in the file.
Basecalling can be stopped earlier if there is clearly unusable raw data at the end of
the file, or if you want to analyze only a portion of the raw data in the file.
Most often, this is done for short PCR products to eliminate the unusable data at the
end of the run. With dye primer chemistries there will be a large peak at the end of the
PCR product. Set the Stop Point just in front of this full-length PCR peak (Figure 6-3).
With dye terminator chemistries there is no full-length PCR peak, but there will be an
abrupt end to sequence peaks in the data file. Set the Stop Point there.
For other samples, the Stop Point can be set where the data becomes too weak to
give useful information or after a position where the sequence data stopped abruptly
due to secondary structure in the template or for some other reason.
The inclusion of large amounts of data points with little or no signal at the end of the
sequence will affect the scaling of the analyzed data. Their removal may be necessary
to get better data in the region where there is good signal.
If you need to change the Stop Point, use the following procedure (refer to Figure 6-3
during the procedure).
Stop Point
Figure 6-3 Electropherogram of raw data from a fluorescein/rhodamine dye primer
sequencing reaction run on a short PCR product. The dashed vertical line shows the
recommended position of the Stop Point.
Optimizing Software Settings 6-21
Using Sequencing Analysis Version 2.1
Step
Action
1
Launch the Sequencing Analysis software if it is not already open.
2
In the Sample File Queue display, double-click the first file to be analyzed to view
the raw data.
Zoom in completely. From the Window menu, choose Actual Size (or use the c ]
keys on the keyboard).
3
Starting at the beginning of the raw data file, scroll along the data by clicking and
holding the right direction arrow at the bottom of the window.
4
a.
Click and drag in the window to change the cursor to a cross-hair.
b.
Move the cursor along the raw data until the region of the desired Stop Point is
in view.
For a PCR product this would be in front of the full-length PCR peak
(Figure 6-3 on page 6-21) or at the end of the sequence peaks.
5
c.
Align the cross-hair just before the peak.
d.
Read the scan number (data point) that is reported on the top of the window.
Use this number as the Stop Point.
Return to the Sample File Queue display.
a.
Highlight the name of the file just inspected and click the Custom Settings
window.
b.
Select the Sample file or files you wish to change.
c.
Click Custom Settings to display the Analysis Settings dialog box.
d.
Select the Change Stop Point check box.
e.
Enter a new number in the entry field.
Using Sequencing Analysis Version 3.0 or 3.2
Step
Action
1
Launch the Sequencing Analysis software if it is not already open.
2
In the Sample Manager window, click the Add Files button and choose the sample
to be analyzed. Click Done.
3
Highlight the sample name and click the Open Files button to display the raw data,
or double-click the sample file name.
Zoom in completely. From the Window menu, choose Actual Size (or use the c ]
keys on the keyboard).
4
Starting at the beginning of the raw data file, scroll along the data by clicking and
holding the right direction arrow at the bottom of the window.
5
a.
Click and drag in the window to change the cursor to a cross-hair.
b.
Move the cursor along the raw data until the region of the desired Stop Point is
in view.
For a PCR product this would be in front of the full-length PCR peak
(Figure 6-3 on page 6-21) or at the end of the sequence peaks.
6-22 Optimizing Software Settings
c.
Align the cross-hair just before the peak.
d.
Read the scan number (data point) that is reported on the top of the window.
Use this number as the Stop Point.
Using Sequencing Analysis Version 3.0 or 3.2
Step
6
(continued)
Action
Return to the Sample Manager window.
Highlight the Stop Point box and enter the information.
With the Sequencing Analysis software versions 3.0 and 3.2, you can set an earlier
Stop Point either manually in the Sample Manager window, or automatically by
changing the values on the Basecaller Settings page of the Preferences dialog box.
During basecalling, the basecaller considers both the Basecaller Settings in the
Preferences and the Stop Point value in the Sample Manager window, and stops at
the earliest designated endpoint.
Optimizing Software Settings 6-23
Data Evaluation and
Troubleshooting
7
7
Overview
In This Chapter This chapter describes how to use the tools in the Sequencing Analysis software to
evaluate and troubleshoot sequencing data. The chapter has three parts:
♦
Evaluating data using the information in the gel file and sample files (page 7-2)
♦
Practical examples of evaluating data (page 7-10)
♦
Troubleshooting sequencing data
–
DNA sequencing reactions (page 7-16)
–
DNA sequence composition (page 7-30)
–
Gel electrophoresis (page 7-44)
–
Capillary electrophoresis (page 7-55)
–
Software settings (page 7-62)
Data Evaluation and Troubleshooting 7-1
Data Evaluation
Introduction There are many variables associated with DNA sequencing that can affect data
quality. Understanding the data evaluation tools that are available in the Sequencing
Analysis software can help in determining where problems may have occurred in the
sequencing process. This section provides an introduction to these tools.
Gel Files The gel file stores the raw data collected during the entire run of an ABI™ 373 or
ABI PRISM® 377 instrument. Initially, the file contains the raw data collected during the
run, a gel image, a copy of the data collection sample sheet, and a copy of the
instrument file. After lane tracking and editing, the file also contains the lane tracking
information and any changes made to the original information in the file.
Note
The ABI PRISM® 310 Genetic Analyzer does not use gel files because samples are run
consecutively in a capillary, not on a gel.
After the gel image has been generated and the lanes tracked, you should perform the
following steps:
♦
Check the gel image for accurate lane tracking and assignment.
♦
If necessary, adjust the lane markers to correct lane numbering errors.
♦
If necessary, adjust the placement of the tracker lines.
Note
The gel image can be magnified to aid in lane tracking. The gel contrast also can be
adjusted to make the lanes easier to see if necessary.
♦
If you change any of the gel file or sample sheet information after extracting the
sample file data, re-extract the data from the edited lanes to regenerate the
information in the sample files.
It is helpful to look at the gel image, not only for correct lane assignment and tracking,
but also for the following information:
♦
An overall impression of the run
–
Background noise in the gel
–
Buffer leaks
–
Instrument problems, such as a bad laser
–
Smeared or streaked lanes
–
Wavy lanes
–
Pinched lanes near beginning of run (bottom of gel image)
–
Band tilt (when the gel image is magnified)
–
Gel resolution (when the gel image is magnified)
♦
Excess dye peaks from dye terminator reactions
♦
Signal strength of individual samples
–
Failed reactions
–
Weak reactions
7-2 Data Evaluation and Troubleshooting
Figure 7-1 on page 7-4 shows a portion of a 96-lane gel with 52 lanes loaded. The
samples include the following:
♦
Full-length plasmids sequenced with BigDye™ terminator chemistry (lanes 1–4)
♦
Short PCR products sequenced with BigDye terminator chemistry (lanes 5–26)
♦
Long and short PCR products sequenced with dRhodamine terminator chemistry
(lanes 27–52, except 42 and 43)
Note
Lanes 42 and 43 were not loaded because the wells were damaged.
This gel file has several problems, including the following:
♦
Background noise
A horizontal yellow band runs across the loaded region of the gel. It is clearly
visible in lanes 27–52. This band might show up as noise in the analyzed data:
blue in dRhodamine chemistry and red in BigDye terminator chemistry (see
“Dye/Base Relationships for Sequencing Chemistries” on page 2-14 and
Figure 7-36 on page 7-29).
♦
Excess dye peaks
Lanes 5–52 show excess dye peaks, which are more pronounced in lanes 5–26.
The dye peaks result from poor ethanol precipitation. Figure 7-2 on page 7-4
shows a closeup view of the excess dye peaks at the bottom of the gel image.
Using the closeup view can give information about peak shape as well as excess
dye peaks. Refer to the ABI PRISM DNA Sequencing Analysis Software User’s
Manual for information on magnifying the gel image.
Excess dye peaks obscure data at the beginning of the sequence.
♦
Weak and failed reactions
Lanes 8–11, 18–22, 27, 29, 31, 33, and 48 have weak signal strengths that can
cause the analyzed data to be noisy. Lanes 28, 30, 32, 34–47, and 49–52 show
failed reactions, i.e., they have signal too low to be analyzed or no usable data.
Observing the overall gel image or magnified areas of the gel image can help you
identify features that can cause problems in data analysis.
Data Evaluation and Troubleshooting 7-3
Figure 7-1 96-lane gel run on an ABI PRISM 377 DNA Sequencer at 1200 scans/hr
Figure 7-2 Zoomed-in view of the excess dye peaks in lanes 4–51 of Figure 7-1
7-4 Data Evaluation and Troubleshooting
Sample Files There are six different fields within the sample file that can be used to display
information about the sample. Three of these fields are useful for data evaluation:
♦
Electropherogram view
♦
Raw data view
♦
Annotation view
Refer to the ABI PRISM DNA Sequencing Analysis Software User’s Manual for more
information about the other three views and their uses.
For ABI PRISM 310 users, the sample file contains all of the necessary information for
data evaluation.
Using the Electropherogram View
If your data was analyzed successfully, this is the default window that appears when
opening the sample file. Scrolling through the data provides the following information
for evaluating performance:
♦
Whether the Peak 1 Location and Start Point for data analysis are set correctly
♦
Presence of any extraneous dye peaks from unincorporated terminators or other
fluorescent contaminants
♦
Peak shape and resolution
♦
Quality of mobility correction
♦
Match of basecalling with electropherogram peaks
♦
Point at which basecalling accuracy declines (number of Ns increases
significantly)
♦
Signal-to-noise ratio
♦
Anomalies in the data that require further review
Figure 7-3 on page 7-6 shows an example of dRhodamine terminator data run on an
ABI PRISM 377 DNA Sequencer. This sample was run in lane 29 of the gel shown in
Figure 7-1 on page 7-4, so we already know that the sample has weak signal and
excess dye peaks. The electropherogram view provides the following additional
information:
♦
Excess dye peaks
The dye peaks at the beginning of the sequence cause the Peak 1 Location and
Start Point to be chosen incorrectly by the software (see page 7-64). The dye
peaks also obscure data up to base 40.
♦
Poor peak shape and resolution
The peaks in the data are broad and asymmetric. They are not well resolved from
each other, which leads to miscalled bases. For example, the C peak at base 243
obscures the T peak at base 244, giving an ambiguous basecall (N).
♦
Poor signal-to-noise
The data in the fourth panel is weak and noisy, causing many Ns.
Data Evaluation and Troubleshooting 7-5
Figure 7-3 Electropherogram from the sample run in lane 29 of the gel in Figure 7-1 on
page 7-4. The arrow points to the miscalled base at position 244 caused by poor resolution.
Using the Raw Data View
If the electropherogram is acceptable, it is not necessary to look at the raw data.
However, looking at the raw data can be helpful in the following cases:
♦
The data has a poor signal-to-noise ratio
♦
The data has anomalies
♦
The data was not analyzed
♦
The Peak 1 Location and Start Point for data analysis were chosen incorrectly or a
different Start Point is desired
The raw data view shows the following:
♦
Signal balance during the course of the run
♦
Abrupt signal changes during the run
♦
Elevated baselines
♦
Spikes (on the ABI PRISM 310 Genetic Analyzer)
Figure 7-4 on page 7-7 shows the raw data from lane 29 of the gel in Figure 7-1 on
page 7-4. The raw data is weak and has excess dye peaks, which we already know
from looking at the gel image and electropherogram.
7-6 Data Evaluation and Troubleshooting
Figure 7-4 Raw data from the sample run in lane 29 of the gel in Figure 7-1 on page 7-4
If analysis fails, the raw data is the default window that appears when opening the
sample file. Figure 7-5 shows a failed BigDye terminator reaction on the
ABI PRISM 310 Genetic Analyzer. Only unincorporated dye terminator peaks are seen.
Figure 7-5 Raw data from a failed reaction
Figure 7-6 shows raw data from a successful BigDye terminator reaction on the
ABI PRISM 310 Genetic Analyzer.
Figure 7-6 Raw data from a successful reaction
Data Evaluation and Troubleshooting 7-7
Figure 7-7 shows an example of BigDye terminator raw data from an ABI PRISM 377
DNA Sequencer run. The data shows signal imbalance. The signal is top heavy, i.e.,
stronger at the beginning of the sequence, then tapering off.
Figure 7-7 Top heavy data
Using the Annotation View
The annotation view shows data collection and analysis information associated with a
sample file. The most useful information for evaluating data is the following:
♦
Dye set/primer (mobility) file
♦
Instrument (matrix) file
♦
Signal strength
Signal strength indicators are a useful guide for determining whether signal
strength is sufficient to obtain good data. Signal strengths for each nucleotide
should usually be from 100–1000.
–
For fluorescein/rhodamine dye primers and rhodamine dye terminators, the
signal strength for each base should be >100.
–
For BigDye™ primers, dRhodamine terminators, and BigDye terminators, the
signal strength for each base should be >50. Signal limits are lower for these
chemistries because of their better signal-to-noise ratios.
♦
Basecaller setting
♦
Base spacing
The base spacing value indicates the speed of electrophoresis. The higher the
base spacing, the slower the run, as there are more data points detected per
peak. For basecalling, the software requires values between 8–16. If the base
spacing falls outside this range, a default value is assigned (a red 9.0 in the
Sample Manager in Sequencing Analysis version 3.0 and higher, and a -12 in
Sequencing Analysis 2.1 and lower).
Refer to the ABI PRISM DNA Sequencing Analysis Software User’s Manual for a
more detailed discussion of base spacing.
♦
Analysis Start Point and Peak 1 Location
These should be checked against those of other samples to determine whether
data analysis started too early or too late.
7-8 Data Evaluation and Troubleshooting
♦
Run module (on the ABI PRISM 310 Genetic Analyzer)
This is useful for determining whether the correct filter set was used to collect
data.
Note
All of the information described above can also be obtained from the top of the
electropherogram printout.
Figure 7-8 shows the annotation view for lane 29 of the gel in Figure 7-1 on page 7-4.
Useful items are highlighted.
Figure 7-8 Annotation view
In the annotation view for lane 29, the signal strength values are G: 104, A: 94, C: 136,
and T: 103. These values are twice the minimum usually required for successful data
analysis of dRhodamine terminator reactions, but are probably artificially raised by the
excess dye peaks. The annotation view also shows that the correct dye set/primer file,
instrument file, and basecaller were used.
Data Evaluation and Troubleshooting 7-9
Practical Examples of Data Evaluation
Overview The following are common features of poor sequencing data:
♦
No recognizable sequence
♦
Noise
♦
Poor mobility correction
♦
Early signal loss
Examples of these problems are given below. Possible causes for them are listed.
Some of the causes are described in more detail in the troubleshooting section of this
chapter along with potential solutions.
No Usable Sequence Figure 7-9 shows analyzed data with a high level of noise and lack of well-defined
peaks. In this reaction, pGEM® control DNA was sequenced using the T3 primer. The
pGEM control has no T3 annealing site.
For many failed reactions, analyzed data is not present because the signal strength is
below the threshold for analysis. If excess dye peaks are present, they can raise
signal levels artificially above this threshold. The data can be analyzed, but will not
have usable sequence.
The raw data for this sample shows flat lines (Figure 7-5 on page 7-7). In the
annotation view (not shown), signal strengths are very low (G:20, A:21, T:20, C:23).
Base spacing for this sample is 8.93, but in failed reactions is often set to the default
value.
Figure 7-9 Analyzed data from a reaction that had very low signal due to the absence of a
priming site in the template for the primer that was used
Possible causes of failed reactions:
♦
Primer has no annealing site, as in the example above
♦
Insufficient template
♦
Contaminated template
♦
Insufficient primer
♦
Poor primer design, e.g., low melting temperature
♦
Old, mishandled, or missing reagents
♦
Thermal cycler failure
♦
Extension products lost during reaction cleanup
7-10 Data Evaluation and Troubleshooting
♦
Extension products not resuspended
♦
Lane tracking failure (ABI 373 and ABI PRISM 377 DNA Sequencers)
♦
Electrokinetic injection failure (ABI PRISM 310 Genetic Analyzer)
Noise Some background noise is always present in sequencing data. Noisy data is
characterized by a high background and peaks under peaks. Noise can be grouped
into several categories, including the following:
♦
Noise throughout the sequence
♦
Noise up to or after a specific point in the sequence
♦
Noise caused by incorrect or poor quality instrument (matrix) file
Noise Throughout the Sequence
Figure 7-10 shows an example of noisy data. The background is high enough to cause
ambiguities in basecalling, e.g., the N at base 377. For this sample, the annotation
view (not shown) indicates the cause to be low signal strengths (C: 47, A: 35, G: 30, T:
38). The raw data for this sample, shown in Figure 7-11, confirms the low signal
strength. Other than the primer peak, the data is very weak.
Figure 7-10 Analyzed data from a BigDye primer reaction run on an ABI PRISM 310 Genetic
Analyzer
Figure 7-11 Raw data view for the sample file shown in Figure 7-10
Data Evaluation and Troubleshooting 7-11
Not all noisy data is caused by low signal. In other cases, the signal strength and the
raw data can appear normal, so other possibilities should be considered.
Potential causes for noise throughout the sequence include the following:
♦
Low signal strength, as in the example above
♦
High signal strength, saturating the detector
♦
Contaminated template
♦
Expired or mishandled reagents
♦
Multiple templates in sequencing reaction
♦
Multiple priming sites
♦
Multiple primers
♦
N–1 primer
♦
Thermal cycler failure
♦
Lane tracking failure (ABI 373 and ABI PRISM 377 DNA Sequencers)
♦
Electrokinetic injection problem (ABI PRISM 310 Genetic Analyzer)
♦
Incorrect run module used to collect the data
♦
Incorrect instrument (matrix) file used to analyze the data
Noise Up To or After a Specific Point in the Sequence
Figure 7-12 shows data from a plasmid clone sequenced with the BigDye terminators
on an ABI PRISM 377 DNA Sequencer. Because the noise starts after the multiple
cloning region of the vector (base 62, see arrow below), the probable cause was
picking a colony that was not well isolated and also had bacteria with no insert or a
different insert in the plasmid.
The raw data (not shown) appears normal. For this sample, the annotation view shows
normal base spacing. In other cases, it could show default base spacing if the noise
occurred at the beginning of the sequence, because base spacing is calculated from
the early data.
Figure 7-12 Noisy data after a specific point
This kind of noise can have the following causes:
♦
Mixed plasmid or PCR preparation, as in the example above
♦
Frame shift mutation
♦
Primer-dimer contamination in PCR sequencing
♦
Slippage after homopolymer region in template
7-12 Data Evaluation and Troubleshooting
Noise Caused by an Incorrect or Poor Quality Instrument (Matrix) File
Figure 7-13 on page 7-13 shows BigDye terminator data, collected on an
ABI PRISM 377 DNA Sequencer, with specific peaks under peaks throughout the run.
For example, every black peak has a smaller red peak underneath it.
The annotation view (not shown) indicates that an incorrect instrument file, one for
Filter Set A instead of Filter Set E, was used to analyze the data. If your data looks like
this, you should check that the correct run module was used to collect the data. If the
correct run module and instrument file were used, you may have a poor quality matrix.
In this case, the instrument file should be remade (see page 6-7).
For this type of noise, the raw data appears normal because a matrix is not applied to
raw data.
Figure 7-13 BigDye terminator data analyzed with an incorrect (Filter Set A) instrument file
Potential causes of this type of noise are the following:
♦
Incorrect instrument file used, as in the example above
♦
Poor quality instrument file
♦
Choosing the wrong run module, which causes the wrong filter set to be used to
collect the data
\
Poor Mobility Figure 7-14 shows BigDye terminator sequencing data with poor mobility correction.
Correction Some peaks are very close together, while others have large gaps between them. The
annotation view (not shown) reveals that the BigDye primer mobility file was used to
analyze the data.
Looking at the raw data is not helpful because mobility corrections are not applied to
raw data.
Figure 7-14 BigDye terminator data analyzed with a BigDye primer dye set/primer (mobility)
file, which results in poor mobility correction
Data Evaluation and Troubleshooting 7-13
There are three potential causes of poor mobility correction:
♦
Choosing the incorrect dye set/primer (mobility) file, as in the example above
♦
Incorrect Peak 1 Location for data analysis
If the Peak 1 Location is not set correctly, the mobility algorithm is not applied
correctly (see “Setting the Data Analysis Range” on page 6-15).
♦
Using a gel matrix on the ABI 373 or ABI PRISM 377 DNA Sequencer with very
different separation properties from the gel matrices that were used to construct
the dye set/primer (mobility) files
For information on selecting the correct dye set/primer file for your gel type, refer
to the ABI PRISM DNA Sequencing Analysis Software User’s Manual.
Early Signal Loss There are two kinds of early signal loss:
♦
Gradual
♦
Abrupt
Gradual Signal Loss
Figure 7-15 shows BigDye terminator sequencing data where the signal gradually
dies after a region of CTT trinucleotide repeats starting at base 280. In this example,
the loss of signal is enough to cause some miscalls, but there is still usable data.
Bases were called accurately to 650. Figure 7-16 on page 7-15 shows raw data from
the same sample. As in the analyzed data, the signal in the raw data dies out
gradually in the repeat region.
In this example, the annotation view does not provide useful information. If the signal
loss had occurred early in the sequence, signal strengths would be low and default
base spacings might be assigned.
Figure 7-15 Gradual signal loss
7-14 Data Evaluation and Troubleshooting
Figure 7-16 Raw data view from the sample shown in Figure 7-15 on page 7-14
Abrupt Signal Loss
Figure 7-17 shows BigDye terminator sequencing data that has an abrupt loss of
signal. The signal dies out so completely that the basecaller fails after base 640.
The raw data view in this example looks like that in Figure 7-16, but with flatter lines
after the signal loss. The annotation view would be uninformative, as it was in the case
of gradual signal loss.
Figure 7-17 Abrupt signal loss
Gradual and abrupt signal losses have many causes, including the following:
♦
Region of secondary structure in the template
♦
Template sequence idiosyncrasies
♦
Poor lane tracking, such that the tracker line diverges from the data
♦
Poor quantitation of primer and/or template, leading to top heavy data
♦
Buffer leak on the ABI 373 or ABI PRISM 377 DNA Sequencer
Data Evaluation and Troubleshooting 7-15
Troubleshooting Sequencing Reactions
Overview This section shows common examples of unsatisfactory sequencing data caused by
poor template preparation, sequencing reaction setup, or reaction cleanup
procedures. Refer to the table on page 7-39 for a more complete guide to
troubleshooting sequencing data.
Poor Template Poor template quality is one of the most common causes of sequencing problems (see
Quality “DNA Template Quality” on page 3-15). Template quality can be affected by:
♦
Residual salts or organic chemicals carried over from the template preparation
♦
Incomplete removal of cellular components such as RNA, proteins,
polysaccharides, and contaminating chromosomal DNA
The presence of residual RNA or chromosomal DNA in the template preparation
will affect the quantitation of the DNA if this is done spectrophotometrically. The
presence of such contaminants can be determined by analysis of the template
preparations on agarose gels (see “Determining DNA Quality” on page 3-16).
♦
Degradation of DNA in storage
♦
More than one template DNA in the sequencing reaction
The appearance of the sequence data will vary depending on the source of the
problem.
Contaminants
The presence of various types of contaminants in the template preparation can result
in inhibition of the sequencing reaction, giving weak signal. This may or may not be
accompanied by significant noise.
Figure 7-18 on page 7-17 shows data from a template preparation that gave fairly
clean data with BigDye terminator chemistry, but with weak signal. Although the
sequence data is fairly good, the background is apparent because of the low signal
and the increased scaling of the noise by the software. Increasing the amount of
template from 0.25 µg to 1.0 µg resulted in only a slight increase in total signal
strength, from 144 to 189.
The template was precipitated with ethanol and resequenced (Figure 7-19 on
page 7-17). After ethanol precipitation the signal was much stronger, with 0.25 µg
having signal strength of 558 and 1.0 µg having signal strength of 1383. The ethanol
precipitation probably removed a contaminant that was present in the original
template preparation and was inhibiting the sequencing reaction (see “Salt
Contamination” on page 7-25).
7-16 Data Evaluation and Troubleshooting
Figure 7-18 Sequence data obtained from a contaminated template preparation. 0.25 µg of
template was sequenced. The total signal strength was 144.
Figure 7-19 Same template after ethanol precipitation. 0.25 µg of template was sequenced.
The total signal strength was 558.
Degraded DNA
Nuclease contamination in a template preparation and repeat freeze-thaw cycles can
result in degradation of DNA over time. Figure 7-20 shows sequence data from a
BigDye primer reaction done with an old template preparation. There is a large stop
peak present in the sequence data after base 320.
Note
Stop peaks are common to all chemistries, but are detected only in dye primer
chemistries (see page 7-30).
When the DNA was reisolated and the new template DNA sequenced, the data was
clean in this region (Figure 7-21 on page 7-18).
Figure 7-20 Sequence data obtained using BigDye primers with an old template preparation
Data Evaluation and Troubleshooting 7-17
Figure 7-21 Sequence data obtained using BigDye primers with a newer template preparation
Multiple Templates
The presence of more than one template in a reaction will result in multiple,
overlapping sequences in the data. This can happen with both PCR templates and
cloned DNA templates. For PCR reactions, lack of complete specificity can result in
more than one product being produced (Figure 7-22).
The majority of cleanup procedures for PCR products are designed to remove
unincorporated nucleotides and residual PCR primers, not secondary PCR products.
The presence of secondary PCR products can be detected by agarose gel
electrophoresis (see page 3-16).
Optimization of PCR conditions and/or use of a Hot Start method usually result in
generation of a clean PCR product. If necessary, the PCR product can be gel purified
before sequencing.
The sequence data shown in Figure 7-22 is from a PCR product that showed three
bands on an agarose gel, even though the PCR product was cleaned up by
ultrafiltration. After gel purification, good sequence data was obtained for the product
of interest (Figure 7-23).
Figure 7-22 Rhodamine dye terminator data from a contaminated PCR product
Figure 7-23 Rhodamine dye terminator data from the same PCR product after gel purification
7-18 Data Evaluation and Troubleshooting
For cloned DNA the presence of two or more sequences generally results when mixed
plaques or colonies are picked. In Figure 7-24 the plasmid DNA used in the
sequencing reaction was isolated from a mixed culture that contained bacteria with
only vector DNA and bacteria with vector containing the insert of interest. With a
mixed clone such as this, the early sequence data is clean because this is the
sequence of the multiple cloning site. After the cloning site the data is noisy, with
peaks under peaks.
Figure 7-24 Sequence data obtained with BigDye Terminators and a template preparation
that contained two different plasmid templates
When the DNA was reisolated from a pure colony, clean sequence data was obtained
(Figure 7-25). When picking bacterial colonies for growth and DNA isolation select a
colony that is well isolated. With M13 plaques, fresh plates should be used for plaque
picking. Check the DNA template purity on an agarose gel (see page 3-16).
Figure 7-25 Sequence data obtained with BigDye Terminators and a template isolated from a
pure colony
Primer-Related There are several primer-related problems that can affect the data obtained in
Problems sequencing reactions. These can be divided into three categories based on the type of
problem seen in the data:
♦
No recognizable sequence
♦
Very weak signal
♦
Two or more sequences present in the electropherogram
No Recognizable Sequence
If there is no priming site for the primer in the template, no sequence data will be
obtained. The raw data will show only a flat line except for the primer peak in primer
reactions or sometimes excess terminator peaks in the case of terminator reactions
(Figure 7-9 on page 7-10).
Data Evaluation and Troubleshooting 7-19
This would also happen if the wrong primer is used for a particular vector, or if a
mutation is present in the primer binding site in the vector that results in the primer not
working effectively.
Such a mutation is present in some samples of pUC18 and vectors such as pUC118
that were derived from it (Lobet et al., 1989). These vectors are missing a C in the lacZ
region that is complementary to the 3´ base for some reverse sequencing primers.
Very Weak Signal
The signal in sequencing reactions can be very weak if the primer anneals poorly
because of a low melting temperature (Tm). Generally primers should have a Tm
above 45 °C (see“Primer Design” on page 3-18). In some cases, however, lowering
the annealing temperature in the reaction can help in obtaining good signal.
The primer used to generate the sequence data in Figure 7-26 was 15 bases long and
had a Tm of 41 °C. The same template was resequenced with a 30-mer primer that
had a melting temperature of 58 °C (Figure 7-27).
Figure 7-26 Rhodamine dye terminator data using a 15-mer primer and AmpliTaq® DNA
Polymerase
Figure 7-27 Rhodamine dye terminator data from the same template using a 30-mer primer
and AmpliTaq DNA Polymerase
Weak signal can also result if the primer anneals poorly due to secondary structure,
particularly at the 3´ end. Whether or not a primer is likely to have significant
secondary structure can be determined by analyzing its sequence with one of several
primer design programs that are available, such as Primer Express™ software
(P/N 402089).
7-20 Data Evaluation and Troubleshooting
Two or More Sequences Present
More than one sequence can be present in an electropherogram if:
♦
The primer used in the reaction is contaminated with N–1 primer (primer that is
one base shorter than the desired primer).
♦
More than one primer is present in the sequencing reaction.
♦
There is a secondary hybridization site for the primer in the template.
In each case, there will be two or more sequences in the electropherogram. The data
will look similar to that shown in Figure 7-28, with peaks under peaks except where
the bases in both sequences are the same.
Figure 7-28 shows data from a sequencing reaction where the primer was
contaminated with N–1 primer. Careful examination of the data from such a reaction
shows that the two sequences are identical, except that the sequence from the N–1
primer is one base shorter than the sequence from the full length primer. For example,
the G at base 63 is shadowed by a smaller G underneath the A at base 62 (see
arrow). No additional peak is seen at position 61 because the extension products from
both the full length primer and the N–1 primer have an A at this position.
N–1 sequence can be detected in a sequencing reaction if the N–1 primer is present
at the level of 5–10% of the correct primer concentration. However, somewhat higher
levels can be tolerated, depending on the particular chemistry used.
In the sequence data shown in Figure 7-28, the contamination by N–1 primer is 40%.
This results in ambiguities (Ns) in the basecalling. In a high quality synthesis, the N–1
contamination should be slight. The concentration of N–1 sequence in the primer can
be further reduced by HPLC purification.
Figure 7-28 pGEM control DNA sequenced with BigDye terminator chemistry and –21 M13
primer contaminated by 40% N–1 primer
The presence of more than one primer in a sequencing reaction can be a problem
when sequencing PCR products. Since two primers are present in the PCR reaction,
failure to completely remove the unincorporated primers from the PCR product will
result in the carryover of some of these primers into the sequencing reaction.
When using dye primer chemistries, fragments that extend from these residual PCR
primers will be unlabeled. If the concentration of these fragments is not too high, there
should not be a significant impact on the reaction. With dye terminator chemistries,
however, the extension products from the residual primers will also be labeled and will
result in a second sequence being present in the data.
Data Evaluation and Troubleshooting 7-21
Complete removal of the unincorporated primers from the PCR amplification before
sequencing will prevent this. Ultrafiltration in a Centricon®-100 column is an effective
way to remove the unincorporated primers as well as the unused dNTPs (see
page 3-12).
If a secondary hybridization site for the primer is present in the template, two
sequences will be detected, resulting in noisy data.
Hybridization at a secondary site that is not a perfect match for the primer will happen
more readily at lower annealing temperatures and at higher concentrations of primer.
To minimize this, keep the annealing temperature as high as possible and do not use
excessively high concentrations of primer.
If the secondary hybridization site is a perfect match for the primer due to the
presence of homologous sequence, you will have to redesign the primer or use a
different sequencing strategy.
Pull-up Peaks Figure 7-29 shows rhodamine dye terminator data collected on an ABI PRISM 377
DNA Sequencer. The data has pull-up peaks (also known as bleedthrough), multiple
peaks in the same position at some points.
Pull-up peaks are caused by very strong signals that saturate the detector. These
signals are therefore clipped digitally (truncated). The software underestimates the
amount of signal at these positions, so it also underestimates the amount of spectral
overlap to correct. Hence pull-up peaks are seen.
These extra peaks are consistently observed at places in the electropherogram where
there is a tall peak. Usually, they are of only one color. Occasionally, when the signals
are very strong, more than one pull-up peak color is observed, as in Figure 7-29.
Figure 7-29 Pull-up peaks with rhodamine dye terminator chemistry
In the sample file from Figure 7-29, the total signal strength shown in the annotation
view is 6077, which is much higher than the recommended maximum of 4000 (1000
for each base).
You can also look in the raw data to determine if signals are too strong. After zooming
in, if any peaks are truncated at the top (i.e., off-scale) then pull-up peaks may be
observed in the analyzed data.
7-22 Data Evaluation and Troubleshooting
Very strong signals are common when sequencing short PCR fragments, as the
sequencing reaction is often very efficient. You may need to load less of this type of
sample to compensate for the increased signal.
Another influence on pull-up peaks is the sequence-specific peak patterns associated
with each chemistry. The rhodamine dye terminators have several peak patterns in the
electropherogram data where very strong signals occur. For example, an A after G, a
T after G, and a C after one or more Ts all display an enhanced signal. These base
composition effects increase the chance of a pull-up peak being observed at these
positions (Figure 7-29 on page 7-22).
The dRhodamine terminators and BigDye terminators have more uniform peak
heights that lessen the potential for individual signals to go off-scale (Figure 7-30).
However, if combined signals are above 4000, then pull-up peaks are likely to be
observed.
Figure 7-30 Same template as in Figure 7-29, but sequenced with dRhodamine terminators.
The arrows point to bases where pull-up peaks were seen with the rhodamine dye terminators
Data Evaluation and Troubleshooting 7-23
Stop Peaks in PCR Stop Peaks in Dye Primer Chemistry Caused by Primer-Dimer Formation
Sequencing Sometimes during PCR amplification, the forward and reverse primers form a
primer-dimer. If one of the PCR primers is used for sequencing, that primer can
anneal to and extend both the PCR fragment and the primer-dimer. In dye primer
sequencing, the multiple annealing can create noise in bases up to the end of the
primer-dimer and a large stop peak at the end of the primer-dimer (Figure 7-31). This
kind of artifact is typically seen in the first 25–60 bases of the sequence, but can
extend as far as 100 bases.
The sequence data after the primer-dimer stop peak is often unaffected unless very
significant amounts of primer-dimer are present in the reaction. In this case, much of
the sequencing primer is used in priming primer-dimer products rather than template
molecules and short reads are observed.
Figure 7-31 Stop peak in dye primer chemistry caused by primer-dimer formation
Stop Peaks in Dye Primer Chemistry Caused by Default Fragments
Sometimes a default fragment is generated from free vector included in the PCR along
with the cloned target (vector with insert). During PCR, the vector’s multiple cloning
region is amplified as well as the intended insert. The resulting default fragment is
extended by the dye primers during sequencing, creating a large stop peak the size of
the multiple cloning region.
Eliminating Stop Peaks
♦ Use careful design for your PCR primers to avoid stop peaks caused by
primer-dimer formation:
♦
–
Make sure there is no sequence complementarity between the two PCR
primers, especially at the 3´ ends.
–
Use a sequencing primer that is different from either of the PCR primers.
–
Ensure that your sequencing primer does not overlap the sequence of the
PCR primers.
–
Use a Hot Start technique for the PCR amplification used to generate the
sequencing template, e.g., AmpliTaq Gold® DNA Polymerase.
You can also use dye terminator chemistries to eliminate stop peaks caused by
primer-dimers or default fragments.
With dye terminators, extension products only appear in the sequencing data
when a dye-labeled ddNTP is incorporated. Stops are invisible because they are
not labeled.
7-24 Data Evaluation and Troubleshooting
Salt Contamination Salts used in template preparation can decrease signal strength and read length if not
completely removed before sequencing.
Figure 7-32 shows the effect of adding increasing concentrations of sodium chloride
(NaCl) to BigDye terminator sequencing reactions before cycle sequencing. Lane 1
has no added salt. Lanes 2–11 have added salt in 10-mM increments from
10–100 mM. At 40 mM NaCl (lane 5), the reduction in read length is apparent.
Figure 7-32 Effect of contaminating NaCl on sequencing data
Figure 7-33 on page 7-26 shows the effect of EDTA on BigDye terminator cycle
sequencing reactions. The impact on read length is not as great as that of NaCl
(Figure 7-32), but there is a steady decrease in signal as EDTA concentration
increases (right to left on gel image).
Data Evaluation and Troubleshooting 7-25
Lane
[EDTA] (mM)
14
1
15
0.8
16
0.6
17
0.4
18
0.3
19
0.2
20
0.1
At EDTA concentrations of 1 mM or higher (up to 6 mM was examined—data not
shown), no signal is obtained.
Figure 7-33 Effect of contaminating EDTA on BigDye terminator sequencing data
Salt is not seen in the usual template quality determination methods, such as agarose
gel electrophoresis and spectrophotometry. If your data has a short read length, odd
peak shape, and low signal strength, the template may be contaminated by salt. See
“Cleaning Up Dirty Templates” on page 3-16 for information about removing salt from
templates.
7-26 Data Evaluation and Troubleshooting
Excess Dye Peaks A common issue occurring with dye terminator cycle sequencing chemistries stems
from incomplete removal of unincorporated, fluorescently labeled ddNTPs during
alcohol precipitation. In the worst case, residual unincorporated dye can obscure the
entire sequence in the first 40 bases (Figure 7-34) and cause scaling problems
throughout the sequence (Figure 7-68 on page 7-65).
Figure 7-34 Unincorporated dye terminator peaks from a BigDye terminator sequencing
reaction
There are several alcohol precipitation methods available for each chemistry (see
page 3-33). This is to provide as much flexibility as possible. Use the method that
gives the best results in your laboratory.
To avoid excess dye peaks:
♦
Use only room-temperature alcohol.
Cold alcohol will also precipitate unincorporated dye terminators.
♦
Do not use denatured alcohol.
Denatured alcohol has inconsistent quality. The concentration of the alcohol and
purity of the additives can vary.
♦
Use the correct concentration of alcohol recommended for the method you have
chosen as described in “Removing Unincorporated Dye Terminators” on
page 3-34.
Figure 7-35 on page 7-28 shows the effect of ethanol concentration on the
precipitation of BigDye terminator sequencing reactions. A 60% concentration
removes most of the unincorporated dye terminators without decreasing signal
strength. Similar results are obtained with isopropanol.
♦
Use an appropriate precipitation method for your sequencing chemistry.
Data Evaluation and Troubleshooting 7-27
70%
65%
60%
55%
Figure 7-35 Effect of ethanol concentration on BigDye terminator sequencing reaction
precipitation
Second- or Third-Panel T Terminator Peak
When removing excess dye terminators from rhodamine dye terminator or BigDye
terminator reactions, a broad red peak sometimes appears in the second or third
panel of analyzed data (Figure 7-36 on page 7-29).
Often this peak is the result of poor spin column purification procedure. To avoid this
problem, load the sample in the center of the column bed. Make sure that the sample
does not touch the sides of the column. See page 3-34 for more information on spin
column purification.
This peak can also result from poor ethanol precipitation procedures if all of the
supernatant is not aspirated after the first centrifugation.
7-28 Data Evaluation and Troubleshooting
Figure 7-36 The second- or third-panel T peak in rhodamine dye terminator chemistry with
AmpliTaq® DNA Polymerase. Two examples are shown.
Data Evaluation and Troubleshooting 7-29
Troubleshooting DNA Sequence Composition Problems
Overview DNA sequence compositions cause different problems depending on the sequencing
chemistry used. No single chemistry works with all sequences. Modification of a
particular method or use of an alternative chemistry may be necessary. This section
describes some sequence contexts that cause problems and some of the common
sequence-related problems:
♦
False stops in dye primer chemistry
♦
Compressions
♦
GC-rich (>70%) regions of sequence
♦
Overall GC-rich sequences
♦
Regions of pronounced secondary structure
♦
GT-rich regions in BigDye terminator chemistry
♦
Homopolymer regions
♦
Repetitive DNA
False Stops in Dye One of the advantages of cycle sequencing is that the high extension temperature
Primer Chemistry discourages the formation of template secondary structures. Certain templates,
particularly GC-rich sequences, can still form intrastrand complexes through which
AmpliTaq® DNA Polymerase, FS has difficulty extending.
In dye primer sequencing, when the DNA polymerase dissociates from a partially
extended fragment without incorporating a dideoxynucleotide terminator, a false stop
is seen. If this occurs in all four dye primer reactions, a peak appears in the
electropherogram at that position in all four colors (Figure 7-37). In the most severe
cases, sequence data stops abruptly.
In dye terminator sequencing, extension products are labeled only if a dye-labeled
dideoxynucleotide is incorporated. If the enzyme falls off the template at a region of
secondary structure and no dye-labeled dideoxynucleotide is incorporated, the
fragment is not detected (Figure 7-38 on page 7-31).
Figure 7-37 A false stop in pGEM control DNA sequenced using fluorescein/rhodamine dye
primer chemistry. A peak is seen in all four colors at the position of the N on the
electropherogram where secondary structure creates a false stop for the polymerase.
7-30 Data Evaluation and Troubleshooting
Figure 7-38 The same region of pGEM control DNA sequenced with dye terminators. The site
of the false stop evident in Figure 7-37 on page 7-30 is not seen when using dye terminators.
An arrow marks the base that was called as an N in Figure 7-37.
Ways to Obtain the Sequence
There are two ways to obtain the sequence at false stops when Ns are called:
♦
Sequence the region using a dye terminator chemistry (Figure 7-38).
♦
Sequence the opposite strand. False stops rarely occur at exactly the same base
position on both strands.
Compressions Band compressions in DNA sequencing result from the formation of secondary
structures in the DNA fragments that are not eliminated by the denaturing conditions
of the gel (Mills and Kramer, 1979). The fragments do not migrate according to their
size, and more than one fragment can migrate at the same position.
In dye primer chemistries, 7-deaza-dGTP is used to minimize problems with
compressions (Barr et al., 1986; Mizusawa et al., 1986), but it is not effective at
eliminating all of them (Figure 7-39).
In the fluorescein/rhodamine dye primer sequencing data shown in Figure 7-39,
compressions are present at bases 257 and 323 (see arrows).
Note
Stop peaks are present at bases 304 and 308.
Figure 7-39 Compressions in pGEM control DNA sequenced with the M13 Reverse primer
using fluorescein/rhodamine dye primer chemistry on an ABI PRISM 377 DNA Sequencer
In dye terminator reactions, dITP is used in place of dGTP. This eliminates most
compressions (Figure 7-40 on page 7-32).
Data Evaluation and Troubleshooting 7-31
Figure 7-40 Same template as in Figure 7-39 on page 7-31, but sequenced using BigDye
terminator chemistry
When problems are encountered with compressions in dye primer data, there are
three ways to resolve them:
♦
Sequence the DNA template using a dye terminator chemistry.
♦
Sequence the complementary strand, if possible.
Compressions rarely occur at the same position in both strands of a template.
♦
Increase the denaturing power of the gel or polymer by using a higher run
temperature and/or by increasing the concentration of denaturant. An additional
denaturant such as formamide can be used in slab gels.
These changes can affect the resolution of the gel or polymer and tend to
decrease the read length, which makes them less than ideal.
GC-Rich Templates Templates with a GC content greater than 70% can be difficult to sequence when
or GC-Rich Regions using the standard reaction conditions. This is probably related to the higher melting
temperature of the DNA caused by the higher proportion of GC base pairs. Even a
template that has a fairly average base composition overall can have a very GC-rich
region that affects its ability to be sequenced.
The most common problem seen with GC-rich templates is weak signal. Figure 7-41
shows data that was obtained with GC-rich DNA using BigDye terminators and the
standard cycling conditions. The signal strengths of the four bases are G: 63, A: 34,
T: 26, and C: 64. The increased T noise (red) is due to the software scaling up the low
T signal.
Figure 7-41 GC-rich template sequenced using BigDye terminators under standard
conditions
When the denaturation temperature was raised from the standard 96 °C to 98°C, the
signals obtained were increased to G: 481, A: 241, T: 181, and C: 498 (Figure 7-42 on
page 7-33).
7-32 Data Evaluation and Troubleshooting
Figure 7-42 GC-rich template sequenced using BigDye terminators with a 98 °C denaturation
temperature
Suggested Approaches for GC-Rich Templates
♦ Increase the denaturation temperature to 98 °C.
♦
Add DMSO to a final concentration (v/v) of 5% (Burgett et al., 1994; Landre et al.,
1995).
Addition of a mixture of 5% DMSO and 5% glycerol has also been used
successfully for some templates.
♦
Incubate the reaction at 96 °C for 10 minutes before cycling.
♦
Add betaine to a final concentration of 1M (Henke et al., 1997; Baskaran et al.,
1996).
♦
Double all reaction components and incubate at 98 °C for 10 minutes before
cycling.
♦
Add 5–10% formamide or 5–10% glycerol to the reactions.
♦
Linearize plasmids with a restriction enzyme.
♦
Shear the insert into smaller fragments (<200 bp) and subclone.
♦
Amplify the DNA with substitution of 7-deaza-dGTP for 75% of the dGTP in the
PCR, then sequence the PCR product (Innis, 1990; Fernandez-Rachubinski et al.,
1990).
Secondary Structure The presence of secondary structure in the template strand often results in difficulty
in the Template obtaining good sequence data beyond the region of secondary structure. Many of the
same approaches that are useful for sequencing GC-rich templates are also useful for
templates that have regions of strong secondary structure. An approach that
sometimes works is to use a primer that anneals close to the region of signal loss. The
use of short-insert libraries has also been used to solve problems of secondary
structure in a genome sequencing project (McMurray et al., 1998).
Data Evaluation and Troubleshooting 7-33
Sequence of a
GT-Rich Template
Obtained with
BigDye Terminators
Figure 7-43 shows a portion of sequence data obtained with BigDye terminators. The
insert in this clone is from Dityostelium japonicum. The base composition is
approximately 30% GC. However, the distribution of bases is significantly different in
the two strands.
In the strand from which the data shown here was obtained, there is a higher
proportion of Cs and As, which results in a high proportion of Gs and Ts in the
synthesized strand. In particular in the region of bases 192–250, 52 of 58 bases are
either G or T, i.e., 90%. After this region the sequence data obtained with BigDye
terminator chemistry dies (Figure 7-43).
Figure 7-43 A template with a very GT-rich region sequenced using BigDye terminator
chemistry. Note the lack of signal after the GT-rich region.
When the same template was sequenced with dRhodamine terminators, good data
was obtained beyond the GT-rich region (Figure 7-44).
Figure 7-44 Same template as in Figure 7-43, but sequenced with dRhodamine terminators
In both chemistries dITP is used in place of dGTP, but in the BigDye terminator
chemistry the dTTP has been replaced with dUTP as well. Both dITP and dUTP will
lower the melting temperature of DNA.
Presumably with such a high concentration of Gs and Ts in this region, the duplex
formed by the extended primer and the template is less stable. The polymerase has
difficulty extending through the region.
7-34 Data Evaluation and Troubleshooting
Further studies with this template showed that replacement of either the dITP with
dGTP or the dUTP with dTTP allowed good extension through this region (data not
shown).
The following methods were found useful at Applied Biosystems to improve the
sequencing results for this template:
♦
Lowering the extension temperature from 60°C to 55°C or 50°C
Note
Lowering the extension temperature will result in some loss of signal strength.
♦
Addition of 1 mM magnesium chloride to the reaction, which increases the final
concentration in the BigDye terminator reaction from 2 mM to 3 mM magnesium
chloride.
♦
Using dRhodamine terminator chemistry (Figure 7-44 on page 7-34)
At the present time the exact composition of a GT-rich region that is sufficient to cause
this problem with BigDye Terminators is unknown.
Homopolymer With Cloned DNA
Regions Long homopolymer T regions (or A regions) can cause problems in DNA sequencing
reactions due to “slippage” in the region of the homopolymer. Although the sequence
data can be clean through the homopolymer region, the data after this region is noisy
due to the presence of multiple sequences (Figure 7-45).
The exact mechanism of slippage is not known. Presumably, the two strands do not
stay paired correctly during polymerization through the homopolymer region. This
generates fragments with homopolymer regions of different length that have the same
sequence after that region.
Figure 7-45 Homopolymer region sequenced using BigDye terminator chemistry
The occurrence of slippage is length dependent and short homopolymer regions are
rarely problematic in sequencing reactions. Slippage is more of a problem with T
regions (A in the template strand) in BigDye terminator reactions due to the use of
dUTP in the deoxynucleotide mixture.
Data Evaluation and Troubleshooting 7-35
When the same template shown in Figure 7-45 is sequenced with dRhodamine
terminators, the sequence data is much cleaner (Figure 7-46).
Figure 7-46 Homopolymer A region sequenced using dRhodamine terminator chemistry
Good sequence data immediately past a polyA region can be obtained by using an
anchored primer, a sequencing primer that is polyT containing an A, C, or G base at
its 3´ end (Figure 7-47). The 3´ base anchors the primer into place at the end of the
homopolymer region (Khan et al., 1991; Thweatt et al., 1990; Thomas et al., 1993).
Note
When sequencing with a polyT primer that has a “wobble” (A, C, or G at the 3´ end),
use 3.2 pmol of each primer, i.e., 9.6 pmol total of primer.
Figure 7-47 Sequencing past a polyA region in the template using a primer with the sequence
T25G. The sequence past the homopolymer region is improved markedly over that shown in
Figure 7-45 on page 7-35).
With PCR Templates
Slippage also occurs in PCR amplification and is a common problem when
sequencing PCR products. Slippage can occur at regions that normally are not
problematic to sequence with cloned DNA.
Sequence data is shown below from a plasmid clone in which the sequencing
template was prepared by amplification of the insert (Figure 7-48 on page 7-37) or by
isolation of the plasmid DNA (Figure 7-49 on page 7-37). After the homopolymer G
region, the sequence data is unusable for the amplified template. When the plasmid
7-36 Data Evaluation and Troubleshooting
DNA was sequenced directly, the sequence data was weak but unambiguous after the
homopolymer G stretch.
Figure 7-48 Fluorescein/rhodamine dye primers with AmpliTaq® DNA Polymerase, CS+ were
used to sequence a template obtained from a plasmid clone by PCR amplification of the insert
Figure 7-49 BigDye terminator data from the same plasmid clone as in Figure 7-48, but the
template was obtained by isolation of the plasmid DNA
At the present time there is no easy solution for the problem of slippage in PCR
amplifications. There are three approaches that can be used to obtain the sequence
data after such a region in PCR-generated templates:
♦
Anchored primers can sometimes be used to obtain sequence data after
homopolymer T regions (see page 7-36).
♦
Sequence the complementary strand.
This will give good sequence for the ambiguous region up to the homopolymer
region, but the same problem will occur afterwards. It can be difficult to determine
the exact number of bases present in the homopolymer region.
♦
Clone the PCR product.
In many cases the cloned product will not show any evidence of slippage when
sequenced. Multiple clones need to be sequenced to be certain that the correct
species has been identified. The main disadvantage of this is the work required to
isolate and sequence a reasonable number of clones.
Data Evaluation and Troubleshooting 7-37
Repetitive DNA DNA containing short repetitive regions (up to 200-300 bases) is generally not difficult
to sequence unless the base composition of the repeat is problematic. For example,
with a relatively short CGG repeat consisting of 38 repeat units, we have had difficulty
getting good sequence data beyond the repeat. In this case, the DNA was handled as
for GC rich templates.
When the length of the repeat is more than 500 bases it can be difficult to get good
sequence data from both strands. Since there is generally no unique sequence in
these repeats, synthesis of walking primers is not an option. Two approaches have
been used successfully with such clones:
♦
Use of directed deletions
♦
Use of an in vitro transposon system, such as the Primer Island Transposition Kit
(Devine and Boeke, 1994; Devine et al., 1997)
Refer to the Primer Island Transposition Kit Protocol (P/N 402920) for more
information about this kit.
7-38 Data Evaluation and Troubleshooting
Troubleshooting Sequencing Data
Troubleshooting Sequencing Data
Observation
Possible Causes
Recommended Actions
No recognizable
sequence (see
page 7-10)
Insufficient template
Quantitate the DNA template. Increase the amount
of DNA in the sequencing reactions. See
page 3-17.
Inhibitory contaminant in template
Clean up the template. See page 3-16.
Insufficient primer
Quantitate the primer. Increase the amount of
primer in the sequencing reactions. See page 3-19.
Primer has no annealing site
Use a primer that is complementary to the
template.
Poor primer design or incorrect
primer sequence
Redesign the primer. See page 3-18.
Missing reagent
Repeat reactions following the protocol carefully.
See page 3-21.
Old or mishandled reagents
Use fresh reagents. See page 3-20.
Thermal cycler power failure
Repeat reactions.
Thermal cycling conditions
Calibrate the thermal cycler regularly.
Use the correct thermal cycling parameters.
Use the correct tube for your thermal cycler.
Set ramp rates to 1 °C/second.
Extension products lost during
reaction cleanup
Ensure that correct centrifugation speeds and
times are used for precipitation and spin column
procedures. See page 3-33
Extension products not resuspended
Resuspend sample pellet in loading buffer or TSR
carefully.
Lane tracking failure (ABI 373 or
ABI PRISM 377 DNA Sequencer)
Check lane tracking. Retrack and reextract lanes if
necessary.
Electrokinetic injection failure
(ABI PRISM 310 Genetic Analyzer)
Repeat injections.
Data Evaluation and Troubleshooting 7-39
Troubleshooting Sequencing Data
(continued)
Observation
Possible Causes
Recommended Actions
Noisy data throughout
sequence, with low
signal strength (see
page 7-11)
Not enough DNA in the sequencing
reactions
Use more DNA in the sequencing reactions.
GC-rich template or GC-rich region in
template
Increase the denaturation temperature to 98 °C.
Load or inject more of the resuspended sequencing
reactions. See “Preparing and Loading Samples for
Gel Electrophoresis” on page 3-50 or “Preparing
and Loading Samples for Capillary Electrophoresis”
on page 3-53.
Add DMSO to a final concentration (v/v) of 5%.
Incubate the reaction at 96 °C for 10 minutes
before cycling.
Double all reaction components and incubate at
98 °C for 10 minutes before cycling.
Add 5–10% glycerol or 5–10% formamide to the
reactions.
Linearize the DNA with a restriction enzyme.
Shear the insert into smaller fragments (<200 bp)
and subclone.
Amplify the DNA using 7-deaza-dGTP in the PCR,
then sequence the PCR product.
Expired or mishandled reagents
Use fresh reagents. See page 3-20.
Thermal cycling conditions
Calibrate the thermal cycler regularly.
Use the correct thermal cycling parameters.
Use the correct tube for your thermal cycler.
Set ramp rates to 1 °C/second.
Noisy data throughout
sequence, with good
signal strength (see
page 7-11
Lane tracking failure
Check lane tracking. Retrack and reextract lanes if
necessary.
Contaminated template
Clean up the template. See page 3-16.
Multiple templates in sequencing
reaction
Examine your template on an agarose gel to see
that only one template is present. See page 3-16.
Multiple priming sites
Ensure that your primer has only one priming site.
Redesign the primer if necessary. See page 3-18.
Multiple primers when sequencing
PCR products
Purify your PCR template to remove excess
primers. See page 3-12.
Primer with N–1 contamination
Use HPLC-purified primers.
High signal saturating detector
Use less DNA in the sequencing reactions or load
less on the gel or into the capillary.
Incorrect run module
Use the correct run module. See page 6-2.
Incorrect instrument (matrix) file
Use the correct instrument file for your sequencing
chemistry. See page 6-7 for information on creating
instrument files.
7-40 Data Evaluation and Troubleshooting
Troubleshooting Sequencing Data
(continued)
Observation
Possible Causes
Recommended Actions
Noise up to or after a
specific point in the
sequence (see
page 7-12)
Mixed plasmid preparation
Ensure that you have only one template. See
“Plasmid DNA Templates” on page 3-6 and
“Determining DNA Quality” on page 3-16.
Multiple PCR products
Ensure that you have only one template. See
“Preparing PCR Products for Sequencing” on
page 3-12 and “Determining DNA Quality” on
page 3-16.
Primer-dimer contamination in PCR
sequencing
Optimize your PCR amplification. See page 3-10.
Make sure there is no sequence complementarity
between the two PCR primers.
Use a sequencing primer that is different from
either of the PCR primers.
Ensure that your sequencing primer does not
overlap the sequence of the PCR primers.
Use a Hot Start technique, e.g., AmpliTaq Gold
DNA Polymerase.
Slippage after repeat region in
template
Try an alternate sequencing chemistry. See
page 2-15.
Use an anchored primer. See page 7-36.
Poor mobility correction
(see page 7-13)
Early signal loss (see
page 7-14)
Incorrect dye set/primer (mobility) file
Use the correct mobility file. See page 6-5.
Incorrect Peak 1 Location for data
analysis
Choose a new Peak 1 Location. See page 6-15.
Gel with very different separation
properties from the gel matrices that
were used to construct the dye
set/primer (mobility) files
Use the correct dye set/primer file for your gel type.
Refer to the ABI PRISM DNA Sequencing Analysis
Software User’s Manual.
Region of secondary structure in the
template
Sequence the opposite strand.
Use a sequencing primer that anneals at a different
position.
Try an alternate sequencing chemistry. See
page 2-15.
Incubate the reaction at 96 °C for 10 minutes
before cycling.
Increase the denaturation temperature to 98 °C.
Increase the extension temperature by 2–3 °C.
GT-rich regions with BigDye
terminators (see page 7-34)
Decrease the extension temperature in cycle
sequencing to 55 °C or 50 °C.
Increase the magnesium ion concentration by
1 mM.
Sequence the opposite strand.
Try an alternate sequencing chemistry. See
page 2-15.
Data Evaluation and Troubleshooting 7-41
Troubleshooting Sequencing Data
(continued)
Observation
Possible Causes
Recommended Actions
Early signal loss (see
page 7-14)
GC-rich region in template
Increase the denaturation temperature to 98 °C.
Add DMSO to a final concentration (v/v) of 5%.
Incubate the reaction at 96 °C for 10 minutes
before cycling.
Double all reaction components and incubate at
98 °C for 10 minutes before cycling.
Add 5–10% glycerol or 5–10% formamide to the
reactions.
Linearize the DNA with a restriction enzyme.
Shear the insert into smaller fragments (<200 bp)
and subclone.
Amplify the DNA using 7-deaza-dGTP in the PCR,
then sequence the PCR product.
Excess dye peaks at
the beginning of the
sequence in dye
terminator chemistries
(see page 7-27)
Poor lane tracking, such that tracker
line diverges from the data
Check lane tracking. Retrack and reextract lanes if
necessary.
Poor quantitation of primer
Quantitate the primer. See page 3-19.
Poor quantitation of template
Quantitate the DNA template, especially with PCR
products. See page 3-17.
Poor removal of unincorporated dye
terminators
Choose the Start Point for data analysis to exclude
the excess dye peaks. See page 6-18.
Follow the protocols for excess dye terminator
removal carefully. See page 3-33.
Refer also to the Precipitation Methods to Remove
Residual Dye Terminators from Sequencing
Reactions User Bulletin (P/N 4304655). This
document can be obtained from the Applied
Biosystems WWW site
(www.appliedbiosystems.com/techsupport).
When using Centri-Sep spin columns, be careful to
load the sample onto the center of the gel surface.
Do not touch the gel surface with the pipet tip. See
page 3-34.
IMPORTANT
When using BigDye terminators,
be sure to hydrate the column for at least 2 hours.
Spin samples in the centrifuge for the
recommended times. Spinning too long precipitates
more dyes with the sample.
When working with microcentrifuge tubes, aspirate
the supernatant rather than decant it. Decanting
leaves excess ethanol on the sides of the tube.
7-42 Data Evaluation and Troubleshooting
Troubleshooting Sequencing Data
(continued)
Observation
Possible Causes
Recommended Actions
Broad, red peak
between base 200 and
350 (see page 7-28)
Poor removal of unincorporated dye
terminators
Follow the protocols for excess dye terminator
removal carefully. See page 3-33.
Refer also to the Precipitation Methods to Remove
Residual Dye Terminators from Sequencing
Reactions User Bulletin (P/N 4304655). It can be
obtained from the Applied Biosystems WWW site
(www.appliedbiosystems.com/techsupport).
When using Centri-Sep spin columns, be careful to
load the sample onto the center of the gel surface.
Do not touch the gel surface with the pipet tip. See
page 3-34.
IMPORTANT
When using BigDye terminators,
be sure to hydrate the column for at least 2 hours.
Pull-up peaks/
bleedthrough (see
page 7-22)
Total signal strength above 4000
Stop peaks in dye
primer chemistry
Primer-dimer contamination in PCR
sequencing (see page 7-24)
Quantitate the DNA template (see page 3-17). Use
less template.
Load or inject less of the resuspended sequencing
reactions. See “Preparing and Loading Samples for
Gel Electrophoresis” on page 3-50 or “Preparing
and Loading Samples for Capillary Electrophoresis”
on page 3-53.
Optimize your PCR amplification. See page 3-10.
Make sure there is no sequence complementarity
between the two PCR primers, especially at the 3´
end.
Use a Hot Start technique for the PCR amplification
used to generate the sequencing template, e.g.,
AmpliTaq Gold DNA Polymerase.
Use a dye terminator sequencing chemistry.
Default fragments in PCR sequencing
of plasmid inserts (see page 7-24)
Ensure that you have only one template. See
“Plasmid DNA Templates” on page 3-6 and
“Determining DNA Quality” on page 3-16.
DNA sequence composition (see
page 7-30)
Use a dye terminator sequencing chemistry. See
page 2-2.
Sequence the opposite strand.
Compressions (see
page 7-31)
Sequence-dependent region of
anomalous mobility, particularly with
dye primer chemistries
If using dye primer chemistry, try a dye terminator
sequencing chemistry. See page 2-2.
Sequence the opposite strand.
Increase the denaturing ability of the gel or polymer
by using higher run temperatures or denaturing
agents such as formamide.
Note
This can decrease the resolution of the gel
or polymer and give shorter read lengths.
Poor data following a
long homopolymer
region (see page 7-35)
Slippage
Try an alternate sequencing chemistry.
Use an anchored primer to determine sequence
after a homopolymer T region in the sequence
(A region in the template strand). See page 7-36.
Data Evaluation and Troubleshooting 7-43
Troubleshooting Gel Electrophoresis on the ABI 373 and ABI PRISM 377
Overview This section shows examples of common problems that can occur with gel
electrophoresis. Refer to the table on page 7-53 for a more complete guide to
troubleshooting gel electrophoresis.
Poor-Quality Use fresh, high-quality acrylamide. Poor quality acrylamide contains acrylic acid (a
Acrylamide deamidation product) and linear polyacrylamide, which will copolymerize and cause
local pH changes in the gel. This causes streaking and smearing of bands
(Figure 7-50).
During storage at room temperature, especially in water, acrylamide breaks down into
acrylic acid. Prepare only as much acrylamide-bisacrylamide solution as you will need
in a month.
Figure 7-50 Effect of poor quality acrylamide on sequencing data
7-44 Data Evaluation and Troubleshooting
Salt Excess salt in the wells can cause pinching of lanes toward the center of the gel
(Figure 7-51).
Note
Lanes 3–8 are short PCR products. Lane 11 was not loaded.
When performing ethanol precipitation, remove all of the ethanol by aspiration after
the first spin. If residual ethanol is dried down with the sample, the pinching and
bending of lanes is worsened. Performing a 70% ethanol wash after ethanol
precipitation of dye terminator reactions also helps to alleviate this problem.
Figure 7-51 Effect of excess salt on an ABI PRISM 377 gel. The overall signal strength is also
lowered.
Data Evaluation and Troubleshooting 7-45
Fluorescent Contaminating fluorescent species can obscure sequencing data completely. A
Contaminants common cause of fluorescent contamination is ink from marker pens. Do not write on
the gel plates, spacers, combs, or buffer chambers.
In Figure 7-52, a large green band is seen shortly after the first fragments are
detected. The band is from a marking pen that was used to label a spacer. Even
though the spacer was cleaned before use, enough ink remained to ruin the
sequencing data.
Figure 7-52 Fluorescent contamination from a marking pen
7-46 Data Evaluation and Troubleshooting
Buffer Leaks If buffer spills or leaks onto the read region of the gel plates, it can cause blue or green
artifacts on the gel image (Figure 7-53).
To avoid buffer leaks, make sure that the plates are clamped correctly and that the
upper buffer chamber gasket makes a proper seal. Do not spill buffer behind the upper
buffer chamber, as wicking can occur.
Figure 7-53 Buffer leak in the read region of the plates
Buffer leaks or evaporation also can cause electrophoresis failure if there is not
enough buffer for electrophoresis. Note that electrophoresis fails at the same point in
each sample (Figure 7-54 on page 7-48), causing diffuse bands to appear throughout
the rest of the run.
Data Evaluation and Troubleshooting 7-47
Figure 7-54 Electrophoresis failure caused by a buffer leak
To prevent this from happening:
♦
Clean the front plate well, so the gasket will make a good seal.
♦
Use the lid on the upper buffer chamber.
♦
Take care when filling the upper buffer chamber not to spill buffer behind it.
♦
Do not fill the upper buffer chamber to the top because buffer will wick over the
ears of the notched plate and run down the sides or back of the gel plates.
♦
Check gasket for leaks before starting the run.
7-48 Data Evaluation and Troubleshooting
Red Rain Gel destruction in the read region of the gel can cause red streaks in the data, often
near the end of the run (and therefore near the top of the gel image). This effect,
shown in Figure 7-55, is known as “red rain.”
Gel destruction often results from drying out of the gel, and is exacerbated by extreme
run conditions, e.g., high voltage, high power, high temperature, and long run times.
The following can be used to help prevent red rain:
♦
Wrap the gel plates to prevent the gel from drying out.
♦
Lower the run temperature from 51 °C to 48 °C.
–
A lower temperature results in a slower run speed. Less data is collected in
the same run time.
–
A lower temperature also means less denaturing power in the gel, which can
lead to more extension product secondary structure in the gel. This can result
in more compressions, particularly with dye primer chemistries (see
page 7-31).
Figure 7-55 “Red rain” on a 48-cm, 36-lane ABI PRISM 377 gel
Data Evaluation and Troubleshooting 7-49
Gel Extrusion When voltage is applied on the ABI PRISM 377 DNA Sequencer, the polyacrylamide
gel sometimes moves from between the glass gel plates toward the cathode (upper
electrode) and into the upper buffer chamber. In extreme cases, up to about five
centimeters of gel in a folded sheet can be deposited in the chamber.
This “gel extrusion” usually begins at the start of a run or even during the prerun. It is
believed to be caused by a buildup of charge on the surface of the glass plate such
that the gel is not bound to the plate after pouring. As the voltage is applied, the gel
migrates toward the upper electrode.
The gel image can show a variety of anomalous effects, including catastrophic loss of
resolution, lane splitting, extreme band tilt, and band distortion (Figure 7-56).
Almost all known cases of gel extrusion have been resolved by either acid washing or
alcoholic KOH washing. Refer to the cleaning procedures on page 4-9.
Figure 7-56 Effect of gel extrusion on sample migration
7-50 Data Evaluation and Troubleshooting
Temporary Loss of This problem usually occurs at the beginning of the gel (between 150 and 250 base
Signal pairs in the analyzed data). It manifests itself as a band of little or no signal across the
entire width of the gel image (Figure 7-57). Temporary loss of signal has been traced
to contaminants on the gel plates. These contaminants include surfactants, fatty acids,
and long chain polymers that are not removed when the plates are washed.
Rinsing glass plates with hot deionized water (90 °C) has been found to remove the
contaminants that cause temporary loss of signal. Refer to page 4-9 for more
information.
Figure 7-57 Portion of an ABI PRISM 377 gel image showing temporary loss of signal. The
vertical blue line is from dust in the read region of the gel.
Data Evaluation and Troubleshooting 7-51
Poor-Quality Plates from vendors other than Applied Biosystems may not have adequate quality
Gel Plates control for use on our instruments. Several problems result from poor quality plates,
including warping. Figure 7-58 shows data from plates that warped after 6 months of
use.
When plates become warped, the laser is no longer focused correctly on the gel.
When this occurs on the ABI 373 DNA Sequencer, laser light is scattered back to the
detector, causing the gel image to appear blue and green and obscuring data. On the
ABI PRISM 377 DNA Sequencer, there is a filter to keep out most of the scattered laser
light. Data quality still suffers because the scattering results in less excitation of the
dyes by the laser.
Always use high-quality gel plates.
Figure 7-58 Effect of warped gel plates on ABI 373 sequencing data
7-52 Data Evaluation and Troubleshooting
Troubleshooting Gel Electrophoresis
Observation
Gel runs too quickly
Possible Causes
Recommended Actions
Total polymer concentration too low
Check reagents. Prepare new solutions
using fresh reagents.
Bisacrylamide concentration too low
Gel runs too slowly
Buffer concentration too high
Note
Do not use TBE buffer if it has
precipitate in it.
Total polymer concentration too high
Check reagents. Prepare new solutions
using fresh reagents.
Bisacrylamide concentration too high
Buffer concentration too low
Old gel
Use gels within 2–6 hours of casting for
the ABI PRISM 377 DNA Sequencer.
Use gels within 18–24 hours of casting for
the ABI 373 DNA Sequencer.
IMPORTANT
Poor resolution caused by gel
Do not refrigerate.
Poor quality reagents, especially
acrylamide (see Figure 7-50 on
page 7-44), APS, and TEMED
Use fresh reagents from a reliable source.
Small bubble between load and read
region
Clean plates thoroughly.
Well shape not flat
Assure that no air bubbles are trapped by
casting comb at gel surface.
Cast gel carefully. Remove bubble by
tapping plates while pouring.
Do not push the sharktooth comb too far
into the gel.
Old gel
Use gels within 2–6 hours of casting for
the ABI PRISM 377 DNA Sequencer.
Use gels within 18–24 hours of casting for
the ABI 373 DNA Sequencer.
IMPORTANT
Visible non-homogeneity
(Schlieren pattern or “swirl” in
gel)
Do not refrigerate.
Variation in spacers
Use spacers and comb sets that are equal
thickness.
Temperature of room, gel solution, or
glass too warm or cool during
polymerization
20–23 °C is optimal.
Excessive TEMED or APS
Check reagents. Prepare new solutions
using fresh reagents.
Temperature too high
Polymerize at 20–23 °C.
Insufficient reagent mixing
Mix reagents gently, but thoroughly.
Data Evaluation and Troubleshooting 7-53
Troubleshooting Gel Electrophoresis
(continued)
Observation
Possible Causes
Recommended Actions
Polymerization too slow (gels
should polymerize within
15–20 minutes)
Excessive dissolved oxygen
Keep vacuum filter strength/time constant.
Stir and pour gel gently.
Filter and pour gel at 20–23 °C.
Not enough TEMED or APS (or
degraded)
Check reagents. Prepare new solutions
using fresh reagents.
Temperature too low during casting
Polymerize at 20–23 °C.
Did not use deionized water
Use only deionized or distilled water for
making all solutions.
Gel loses signal around 200 bp
(see page 7-51)
Contaminant polymers on plate
surface
Wash plates with mild detergent and hot
deionized water rinses.
Lanes appear as smears
Impure or degraded TEMED or APS
Use fresh reagents.
Samples are overloaded
Follow loading procedure.
Electrophoresis failure due to buffer
leak (see Figure 7-54 on page 7-48)
Make sure that the plates are clamped
correctly, and that the upper buffer
chamber gasket makes a proper seal.
Do not spill buffer behind the upper buffer
chamber, as wicking can occur.
Gel image contains vertical red
streaks near end of run (top of
gel image)
(“red rain,” see Figure 7-55 on
page 7-49
Gel destruction in read region
Wrap the gel to prevent drying.
Gel image contains green/blue
streaks throughout run
(see Figure 7-52 on page 7-46)
Fluorescent contaminant in gel
Vacuum filter solution. Cast gel in
dust-free environment.
Urea crystals present in gel
Use room temperature reagents. Pour at
20–23 °C.
Run at a lower temperature or voltage.
IMPORTANT
Blue or green streaks (“curtain”)
at top of gel image
(see Figure 7-53 on page 7-47)
Do not refrigerate.
Particles on outer surface of plates in
read region
Wipe read region with damp lint-free
KimWipe
Buffer leak
Make sure that the plates are clamped
correctly, and that the upper buffer
chamber gasket makes a proper seal.
Do not spill buffer behind the upper buffer
chamber, as wicking can occur.
Blue or green curtain obscuring
entire gel image
(see Figure 7-58 on page 7-52)
Warped gel plate
Use gel plates from Applied Biosystems.
Green streak through entire gel
lane
Protein in template
Clean up the template before performing
sequencing reactions.
Greenish-yellow haze
Poor gel plate alignment
Remove the gel plates and realign them
correctly.
Fluorescent contaminant in gel
Use fresh reagents.
Do not write on the gel plates with marking
pens.
Residual detergent on plates
7-54 Data Evaluation and Troubleshooting
Rinse plates thoroughly with hot deionized
water.
Troubleshooting Capillary Electrophoresis on the ABI PRISM 310
Overview This section shows examples of problems that are specific to capillary
electrophoresis. Refer to the table on page 7-57 for a more complete guide to
troubleshooting capillary electrophoresis.
Capillary Failure Figure 7-59 shows data from a capillary that had been used for more than 100
injections. Data is noisy and has trailing peaks.
We recommend that capillaries be replaced after 100 injections or when you start to
see signs of capillary failure.
Figure 7-59 Trailing peaks caused by capillary failure
Spikes Spikes are caused by particulate matter in the polymer passing through the detection
window and scattering laser light.
Figure 7-60 on page 7-56 shows the effect of a spike on analyzed sequencing data
from a BigDye primer reaction. The spike observed in the center of the panel is almost
twofold higher than the sequencing peaks. The mobility shift algorithm makes this
spike look as if the red portion precedes the blue portion, but in the raw data the spike
is a single, four-color peak (Figure 7-61 on page 7-56).
If your data shows spikes:
♦
Clean the syringe and pump block with filtered, deionized water.
♦
Use a fresh capillary that has not been exposed to dust, i.e., left on the benchtop.
♦
Filter the POP-6™ polymer with a 0.2-µm or 0.45-µm disk filter attached to a
plastic syringe.
Data Evaluation and Troubleshooting 7-55
Figure 7-60 A spike in analyzed sequencing data
Figure 7-61 A spike in raw data
7-56 Data Evaluation and Troubleshooting
Troubleshooting Capillary Electrophoresis
Observation
Possible Causes
Recommended Actions
Data was not
automatically analyzed
Sample Sheet not completed or
completed incorrectly
Complete the Sample Sheet as described in your
user’s manual.
Injection List not completed or
completed incorrectly
Complete the Injection List as described in your
user’s manual.
Analysis preferences set incorrectly in
data collection program
Check the collection software preferences to make
sure that Autoanalyze with Sequencing Analysis
Software is selected under the Sequence Injection
List Defaults.
Insufficient free RAM
Restart the computer before collecting data.
Note
You should always restart the computer
before collecting data.
No current
Conflicting extensions
Choose Extensions Manager from the Control
Panels. Turn off any extensions that were not part
of the original installation and restart computer.
Too little or no buffer in anode buffer
reservoir
Replenish buffer reservoir.
Too little or no buffer in position 1 of
autosampler
Replenish buffer in position 1 of autosampler.
Electrode bent
Replace or straighten electrode and recalibrate
autosampler.
Capillary bent away from electrode
Tape capillary securely to heat plate to keep
capillary from shifting position. Place the tape on
the heat plate just above the electrode holder.
Refer to the ABI PRISM 310 Genetic Analyzer
User’s Manual.
Unfilled capillary or bubbles in
capillary
Check system for leaks. Replace capillary if
necessary and rerun module.
Major leaks in system. Polymer does
not enter capillary
Check system for leaks.
Pump blockage (pump is plugged
with urea or crystallized buffer)
Remove and clean pump block. Refer to the
ABI PRISM 310 Genetic Analyzer User’s Manual.
Loose valve fittings or syringe
Tighten valve fittings and syringe.
Anode buffer valve does not open
Open buffer valve.
Note
Filling the capillary should cause the Gel
Pump value in the Status Window to increase by
only 1–2 steps. If the instrument detects a syringe
leak, a warning message appears on the screen.
Note
The valve should depress easily when you
push the top with your finger tip. After you release
the pressure the valve should spring to the “open”
position. If the valve is stuck, it should be cleaned.
Plugged, broken, or nonconducting
capillary
Replace the capillary.
Poor quality water in buffer solutions
Remake buffer with freshly autoclaved, distilled,
deionized water
Incorrect polymer solution formulation
Make or install new polymer solution
Data Evaluation and Troubleshooting 7-57
Troubleshooting Capillary Electrophoresis
(continued)
Observation
Possible Causes
Recommended Actions
No current
Corrupted firmware
Resend firmware by performing a cold boot reset.
Syringe Pump Force too low.
Capillary is not being filled completely
Call DNA Technical Support.
Small bubble in capillary blocking
current flow
Replenish gel in capillary.
Small bubble in pump block
Remove bubble by repriming the pump block with
polymer.
Plugged, broken, or nonconducting
capillary
Replace the capillary.
Poor quality water in buffer solutions
Remake buffer with freshly autoclaved, distilled,
deionized water.
Old, defective, or incorrectly made
buffer or polymer solution
Replace buffer or polymer solution.
Low current
Fluctuating current
Too little buffer in anodic jar
Replenish buffer jar.
Small bubble in capillary blocking
current flow
Replenish gel in capillary.
Small bubble in pump block
Remove bubble by repriming the pump block with
polymer.
Broken or cracked capillary
Replace the capillary.
Arcing to conductive surface on the
instrument
Clean the hotplate and autosampler.
Position of electrode is not sufficiently
below the buffer surface
Replenish buffer.
Current is normal at
beginning of run and
then decreases rapidly
over the next several
minutes
Loss of anodic buffer capacity
Replace the buffer.
Current too high
Decomposition of urea in polymer
solution
Add fresh polymer solution to the syringe.
Incorrect buffer formulation (most
likely too concentrated)
Replace buffer with appropriate 1X running buffer.
Arcing to conductive surface on the
instrument
Clean the hotplate and autosampler.
No sample added
Add sample.
Sample not at bottom of tube
Spin sample tube in microcentrifuge.
Air bubble at bottom of sample tube
Spin sample tube in microcentrifuge to remove air
bubbles.
Capillary misaligned with electrode
Align capillary and electrode.
No signal
Ensure that the ambient temperature is between 15
and 30 °C and the humidity is below 80%. Check
for excessive condensation on the instrument.
Reposition electrode and recalibrate autosampler.
Ensure that the ambient temperature is between 15
and 30 °C and the humidity is below 80%. Check
for excessive condensation on the instrument.
Note
The capillary should be adjacent to, but
not touching, the electrode. The capillary should
protrude 0.5 mm past the electrode.
7-58 Data Evaluation and Troubleshooting
Troubleshooting Capillary Electrophoresis
(continued)
Observation
Possible Causes
Recommended Actions
No signal
Capillary bent out of sample tube
Align capillary and electrode. Recalibrate
autosampler.
Note
To verify whether a bent capillary is the
problem, watch the movement of the autosampler
tray during run operation.
Autosampler not calibrated correctly
Calibrate autosampler in X, Y, and Z directions.
IMPORTANT
The capillary should almost
touch the Z calibration point.
Sealed sample tube septum (i.e.,
septum will not open to allow
electrode into sample tube)
Replace septum.
Septum not placed in the sample tube
properly
Signal too low
Insufficient sample injected
Increase injection time.
Old Template Suppression Reagent
(TSR)
Use fresh TSR.
Ions in sample (leading to insufficient
sample injected)
Dialyze sample to remove ions.
Sample not thoroughly mixed with
TSR
Mix sample into TSR by pipetting up and down
several times.
Signal too high
Too much sample injected into
capillary.
Decrease injection time or injection voltage.
High baseline
Dirty capillary window
Clean capillary window with 95% ethanol.
Capillary moved out of position in
front of laser window
Position capillary in front of laser window.
Precipitate in polymer
Allow polymer to equilibrate to room temperature
before using.
Use fresh polymer.
Contaminant in polymer
Filter the polymer with a 0.2-µm or 0.45-µm disk
filter attached to a plastic syringe.
Incorrectly prepared and/or old buffer
or polymer solutions
Replace buffer and polymer with fresh solutions.
Improper filling of capillary
Run the Seq Fill Capillary run module to fill the
capillary.
Fluorescent contaminant in the
capillary holder
Clean the capillary holder.
Fluorescent contaminant in the
sample
Purify the sample.
Defective or old capillary
Replace the capillary.
Matrix made incorrectly resulting in
too much correction (also indicated
by troughs under peaks)
Remake matrix. Be sure to:
♦
Remove the primer peak (or aberrant off-scale
peaks) from the scan range.
♦
Pick the start and stop points on flat parts of
the baseline when viewing raw data.
♦
Make the matrix using same polymer, buffer,
and run conditions as sample injections.
Data Evaluation and Troubleshooting 7-59
Troubleshooting Capillary Electrophoresis
(continued)
Observation
Possible Causes
Recommended Actions
Noisy baseline
Incorrectly prepared and/or old buffer
or polymer solutions
Replace buffer and polymer with fresh solutions.
Dirty capillary holder aperture
Clean the capillary holder.
Defective capillary
Replace the capillary.
Precipitate in polymer
Allow polymer to equilibrate to room temperature
before adding to capillary.
Spikes in baseline (see
Figure 7-60 on
page 7-56)
Filter the polymer with a 0.2-µm or 0.45-µm disk
filter attached to a plastic syringe.
Use fresh polymer.
Extra peaks in
additional colors
displayed underneath
the position of one
strong peak
Extraneous peaks
Old polymer
Use fresh polymer.
Too much sample injected into
capillary (indicated if any peak is
greater than 4000 RFU)
Decrease injection time or injection voltage.
Incorrect matrix chosen or poor
matrix
Check matrix selection on Injection List.
Unincorporated dye primers or dye
terminators (“dye blobs”)
Purify the extension products thoroughly before
sequencing.
Fluorescent contaminant in sample
(often from marking pen ink)
Prepare new samples. Do not write on sample
tubes or septa with marking pens.
Renaturation of denatured samples
Load samples immediately following denaturation,
or store on ice until you are ready to load.
Repeat using less DNA.
If correct, create a new matrix.
IMPORTANT
Do not store samples on ice for
more than 2 hours before loading.
Stop peak (strong peak
in all four colors)
Dust or dirt in polymer
(see Figure 7-60 on page 7-56)
Filter the polymer with a 0.2-µm or 0.45-µm disk
filter attached to a plastic syringe.
Secondary structure in sequence
Use dye terminator chemistries instead of dye
primer chemistries.
Primer-dimer (primer oligomerization)
in PCR sequencing
Make sure that there is no sequence
complementarity between the two PCR primers.
Use a sequencing primer that is different from the
two PCR primers.
Ensure that the sequencing primer does not
overlap the sequence of either PCR primer.
Poor base spacing
Incorrect dye set/primer file
Check dye set/primer file used.
Incorrect polymer composition
Check urea concentration and polymer composition
against protocol.
Incorrect electrophoresis temperature
Check the Injection List for temperature setting.
If correct on Injection List, check the Log for a
recording of the actual electrophoresis
temperature.
Inconsistent peak
mobilities at beginning
of run (i.e., peaks come
off at higher scan
numbers in the first
injection)
Capillary temperature not at
equilibrium
7-60 Data Evaluation and Troubleshooting
Repeat the injection of the first sample.
Note
The run temperature can be set in the
Manual Control window while the samples are
being prepared, but we still recommend repeating
the first sample.
Troubleshooting Capillary Electrophoresis
(continued)
Observation
Possible Causes
Recommended Actions
Fragments migrate
slower than normally
Capillary allowed to dry out
Leave capillary in buffer or water when not in use.
Dirty sample
Purify extension reactions before sequencing.
Air bubbles in pump block or capillary
Check for leaks and remove air bubbles.
Incorrect buffer concentration
Remake running buffer.
Clogged pump block
Remove pump block and clean it.
Syringe pump failure
Call DNA Technical Support.
Runs get progressively
slower (i.e., primer
peaks come off at
higher and higher scan
numbers)
Leaking syringe: polymer is not filling
capillary before every injection
Clean syringe thoroughly.
Syringe out of polymer
Fill syringe with fresh polymer.
Runs get progressively
faster (i.e., primer peaks
come off at lower and
lower scan numbers)
Water in syringe
Prime syringe with small volume of polymer, invert
syringe to coat capillary walls, and discard polymer.
Poor resolution (see
Figure 7-59 on
page 7-55)
Poor capillary performance
Replace capillary.
Incorrectly prepared and/or old buffer
or polymer solutions
Replace buffer and polymer with fresh solutions.
Injection time too long (broad peaks)
Decrease injection time.
Incorrectly prepared and/or degraded
sample
Prepare new sample.
Incorrect buffer formulation
Check if buffer formulation matches protocol
requirements.
Incorrect polymer composition
Check if polymer composition matches protocol
requirements.
Electrophoresis voltage too high
Decrease electrophoresis voltage by as much as 4
kV.
Replace syringe.
Then fill syringe with fresh running polymer.
Note
Increase electrophoresis time accordingly.
Sample concentrated by evaporation
leaving excess salt behind
Do not concentrate sample by evaporation. Use an
Amicon Centricon-100 column if necessary.
Incomplete strand separation due to
insufficient heat denaturation
Make sure the samples are heated at 95 °C for 2
minutes prior to loading onto autosampler.
Wrong capillary
Verify that you are using the correct capillary (see
Table 5-2 on page 5-8).
Oil in sample (from DNA Thermal
Cycler 480)
Carefully pipette sequencing reactions without oil
carryover.
Remove oil by organic extraction.
Poor quality water
Use freshly autoclaved, distilled, deionized water.
Syringe empty or incorrect Syringe
Max Travel value
Fill syringe if necessary and recalibrate Syringe
Max Travel value.
Data Evaluation and Troubleshooting 7-61
Troubleshooting Software Settings
Overview This section shows examples of problems that can occur in sequencing data when
software settings are incorrect.
Incorrect Run Figure 7-62 shows data from a dRhodamine terminator sample, which should have
Module been collected with a Filter Set E run module, but was collected using a Filter Set A
run module.
Note
One of the most common mistakes made with the new dRhodamine-based
chemistries is to collect data using Filter Set A.
If you collect data using the wrong filter set, you should rerun the samples. If this is
impossible, a matrix can be made from the data on the ABI PRISM 310 and
ABI PRISM 377 instruments (see page 6-14). Data analyzed this way will not be free of
multicomponenting noise.
Figure 7-62 dRhodamine terminator sample run with a Filter Set A run module
Incorrect Dye If you analyze data with the wrong mobility file, the data can be reanalyzed with the
Set/Primer correct mobility file as described in your user’s manual.
(Mobility) File
Analyzing BigDye primer data with a mobility file for dRhodamine terminator or BigDye
terminator chemistry (or vice versa) causes both shifted peaks and miscalled bases
(Figure 7-63). These three chemistries use the same dyes for fluorescence emission,
but on different bases. See Chapter 2, especially page 2-14, for more information.
Figure 7-63 shows BigDye terminator data analyzed with a BigDye primer mobility file.
The C, G, and T bases are called incorrectly, and the As overlap with the following
peaks. Figure 7-64 on page 7-63 shows the same sample file reanalyzed with the
correct mobility file.
Figure 7-63 BigDye terminator data analyzed with a BigDye primer dye set/primer (mobility)
file
7-62 Data Evaluation and Troubleshooting
Note
Mobility shifts and dye set/primer file names for the dRhodamine Terminators are
similar to those for the BigDye Terminators. If a mobility file for the wrong sequencing chemistry
is used, C and T bases will be miscalled because of differences in which terminators are labeled
with which dyes (see page 2-14).
Figure 7-64 BigDye terminator data analyzed with the correct dye set/primer file
Incorrect or
Poor-Quality
Instrument
(Matrix) File
If you use the wrong instrument file (but the correct run module), the data can be
reanalyzed with the correct instrument file. Figure 7-65 shows BigDye terminator data,
collected on an ABI PRISM 377 DNA Sequencer, with specific peaks under peaks
throughout the run. For example, every black peak has a smaller red peak underneath
it.
An incorrect instrument file, one for Filter Set A instead of Filter Set E, was used to
analyze the data. If your data looks like this, you should check that the correct run
module was used to collect the data. If the correct run module and instrument file were
used, you may have a poor quality matrix. In this case, the instrument file should be
remade (see page 6-7). Figure 7-66 on page 7-64 shows the same data analyzed with
the correct instrument file.
Figure 7-65 BigDye terminator data collected with a Filter Set E run module and analyzed
with an incorrect (Filter Set A) instrument file
Data Evaluation and Troubleshooting 7-63
Figure 7-66 BigDye terminator data collected with a Filter Set E run module and analyzed
with the correct (Filter Set E) instrument file
Instrument files do not change over time, but instruments do change. You may need to
remake the instrument file as the filter wheel (on an ABI 373 DNA Sequencer) ages or
if any changes to the optics occur, e.g., the CCD camera (on an ABI PRISM 310 or
ABI PRISM 377 instrument) is replaced.
Figure 7-67 shows BigDye terminator data analyzed with a poor matrix file. Bad
multicomponenting is characterized by specific peaks under peaks throughout the run,
in this case, smaller red peaks under blue peaks.
Figure 7-67 BigDye terminator data analyzed with a poor Filter Set E matrix file
See your instrument user’s manual for instructions on creating a new instrument file or
follow the instructions on page 6-8 for creating an instrument file for Filter Set E
(dRhodamine-based chemistries).
Incorrect Peak 1 During analysis, the software assigns the Peak 1 Location and the Start Point to the
Location same scan number. Occasionally the value assigned is incorrect. This happens more
commonly with dye terminator chemistries than with dye primer chemistries. Correct
assignment of the Peak 1 Location value is important for mobility corrections to be
applied properly.
With dye primer chemistries the large primer peak helps the software to assign the
Peak 1 Location and Start Point values. With dye terminator chemistries, there is no
primer peak, so the software must detect the small sequence data peaks.
If these sequence peaks are obscured by peaks from unincorporated dye terminators
and/or other fluorescent peaks are present before the start of the real sequence, the
software can assign the Peak 1 location and Start Point too early (Figure 7-68 on
7-64 Data Evaluation and Troubleshooting
page 7-65). If the sequence data is weak, the Peak 1 location and Start Point can be
assigned too late.
See “Setting the Data Analysis Range” on page 6-15 for the procedures to change the
Peak 1 Location and Start Point.
Large peaks can also cause problems in scaling the analyzed data. For example, in
Figure 7-68 a very large red (T) peak at base 130 resulted in all other red peaks
appearing very small.
Figure 7-68 Incorrect Peak 1 Location and Start Point (both set to 963 scans)
In this case the Start Point can be set after the terminator peaks and the data
reanalyzed. When this was done, the T peaks were scaled normally (Figure 7-69).
Figure 7-69 Better Peak 1 Location (1260 scans) and Start Point (2020 scans)
In this example, the Start Point value is greater than the Peak 1 Location value. The
Peak 1 Location value should still be set to the beginning of the sequence. This
ensures that the mobility corrections and spacing are applied properly.
Data Evaluation and Troubleshooting 7-65
Gel Preparation
A
A
Introduction
Gel Formulations There are several choices of sequencing gel formulations for 34- and 36-cm
well-to-read (wtr) lengths. The 29:1 polyacrylamide, Long Ranger™, and PAGE-PLUS
gels perform similarly on both the ABI™ 373 and ABI PRISM® 377 instruments. These
gels generally perform better than 19:1 polyacrylamide gels.
For 48-cm gels, the 5.25% PAGE-PLUS gels generally provide the longest read
lengths.
For 2400-scan/hr runs on the ABI PRISM 377 DNA Sequencer, the best gel to use is
the 4.5% 29:1 polyacrylamide. This type of gel performs the best under these extreme
run conditions.
Different laboratories may obtain different results. We recommend testing several gel
formulations to see which works best under your particular run conditions.
Table A-1 lists recommended gel formulations for the ABI 373 and ABI PRISM 377
DNA Sequencers.
Table A-1 Gel Formulations
34-cm or 36-cm well-to-read (wtr)
48-cm well-to-read (wtr)
Volume
(mL)
Instrument
Gel Type
ABI 373
6% 19:1 polyacrylamidea
80
4.75% 19:1 polyacrylamide
80
ABI PRISM 377
Volume
(mL)
Gel Type
4% 19:1 polyacrylamide
100
5% 29:1 polyacrylamide
80
4.25% 29:1 polyacrylamide
100
5.75% Long Ranger
80
5% Long Ranger
100
5.75% PAGE-PLUS
80
5.25% PAGE-PLUS
100
4% 19:1 polyacrylamide
50
4% 19:1 polyacrylamide
50
4.5% 29:1 polyacrylamide
50
4.25% 29:1 polyacrylamide
50
5% Long Ranger
50
4.75% Long Ranger
50
4.8% PAGE-PLUS
50
5.25% PAGE-PLUS
50
a. Used for 24-cm well-to-read gels.
Gel Preparation A-1
Protocol and Run Conditions for 19:1 Polyacrylamide Gels
Preparing 40%
Acrylamide Stock
(19:1)
Step
Action
1
Working in a fume hood, combine the following acrylamide and bisacrylamide in a
glass beaker:
♦
Acrylamide, 57 g
♦
Bisacrylamide, 3 g
! WARNING ! CHEMICAL HAZARD. Acrylamide and bisacrylamide are
poisons, neurotoxins, irritants, carcinogens, and possible teratogens.
Acrylamide and bisacrylamide sublime (the solids release toxic vapor) and
are harmful if swallowed, inhaled, or absorbed through the skin. Effects are
cumulative. When handling, always wear protective equipment (lab coat,
safety glasses, and chemical-resistant gloves) and use in a well-ventilated
area. On a routine basis, thoroughly clean surfaces subject to contamination.
2
Dissolve the crystalline acrylamide and bisacrylamide in sufficient distilled,
deionized water to bring the total volume to 135 mL.
3
Add 15 g of mixed-bed, ion-exchange resin.
4
Stir at room temperature until all crystals dissolve. Continue stirring for
5–10 minutes.
5
Filter the mixture through a 0.2-µm cellulose nitrate filter.
6
Transfer the filtrate to a graduated cylinder and bring the total volume to 150 mL
with distilled, deionized water.
7
Store at 2–6 ∞C.
Note
40% acrylamide stock lasts for 1 month at 2–6 ∞C.
Ingredients and Run For 36-cm and 48-cm WTR Runs—4% 19:1 Polyacrylamide Gel, 6 M Urea
Conditions for the
For 50 mL
Run Conditions
ABI PRISM 377 Ingredient
urea
18.0 g
40% acrylamide stock
5.0 mL
deionized water
25 mL
Mixed-bed ion
exchange resin
0.5 g
♦
For 1200 scans/hr runs,
use standard 36-cm
1200 scans/hr run
modules.
♦
For 2400 scans/hr runs,
use standard 36-cm
2400 scans/hr run
modules.
♦
For 48-cm gels, use
standard 48-cm,
1200 scans/hr run
modules.
Filter and degas the above ingredients
before adding TBE.
10X TBE
5.0 mL
10% APS
250 µL
TEMED
35 µL
Bring to final volume (50 mL) with
deionized water.
A-2 Gel Preparation
Ingredients and Run For 24-cm WTR Runs—6% 19:1 Polyacrylamide Gel, 8.3 M Urea
Conditions for the
For 80 mL
Run Conditions
ABI 373 Ingredient
urea
40.0 g
40% acrylamide stock
12 mL
deionized water
27 mL
Mixed-bed ion
exchange resin
0.5 g
Use the standard run time of
14 hours.
Filter and degas the above ingredients
before adding TBE.
10X TBE
8.0 mL
10% APS
400 µL
TEMED
45 µL
Bring to final volume (80 mL) with
deionized water.
For 34-cm WTR Runs—4.75% 19:1 Polyacrylamide Gel, 8.3 M Urea
Ingredient
For 80 mL
urea
40.0 g
40% acrylamide stock
9.5 mL
deionized water
27 mL
Mixed-bed ion
exchange resin
0.5 g
Run Conditions
Use the standard run time of
14 hours.
Filter and degas the above ingredients
before adding TBE.
10X TBE
8.0 mL
10% APS
400 µL
TEMED
45 µL
Bring to final volume (80 mL) with
deionized water.
For 48-cm WTR Runs—4% 19:1 Polyacrylamide Gel, 8.3 M Urea
Ingredient
For 100 mL
urea
50.0 g
40% acrylamide stock
10 mL
deionized water
37 mL
Mixed-bed ion
exchange resin
0.5 g
Run Conditions
Use the standard run time of
18 hours.
Filter and degas the above ingredients
before adding TBE.
10X TBE
10 mL
10% APS
500 µL
TEMED
50 µL
Bring to final volume (100 mL) with
deionized water.
Gel Preparation A-3
Preparing 19:1 Preliminary gel preparation steps:
Polyacrylamide Gels
Step
Action
1
Referring to the appropriate list of ingredients above and the 373 DNA Sequencing
System User’s Manual or ABI PRISM 377 DNA Sequencer User’s Manual, gather all
the necessary lab equipment and ingredients.
2
Prepare all stock solutions per the list of ingredients.
3
For the ABI PRISM 377 DNA Sequencer: clean the gel plates thoroughly and mount
them in the gel pouring cassette (or alternative device).
For the ABI 373 DNA Sequencer: clean the gel plates thoroughly and prepare them
for gel pouring.
Preparing the acrylamide–urea solution:
Step
1
Action
Combine urea, 40% acrylamide stock, deionized water, and mixed-bed ion
exchange resin in a 150-mL beaker.
! WARNING ! CHEMICAL HAZARD. Urea causes eye, skin, and
respiratory irritation. Lab experiments have shown mutagenic effects. Avoid
contact. Wear chemical resistant gloves, safety goggles, and other protective
clothing.
! WARNING ! CHEMICAL HAZARD. Acrylamide and bisacrylamide are
neurotoxins. Avoid inhalation and skin contact. Wear gloves at all times, and
work in a fume hood when handling acrylamide solutions. Use appropriate
precautions to avoid inhalation of crystalline acrylamide. Read the
manufacturer’s MSDS before handling.
2
Stir the solution until all the urea crystals have dissolved.
3
Filter the solution through a 0.2-µm cellulose nitrate filter.
4
Degas for 2–5 minutes.
Note
Degas time for all gels should be constant to ensure a reproducible
polymerization rate for all gels.
5
Transfer the solution to a 100-mL graduated cylinder.
6
Add filtered 10X TBE buffer.
IMPORTANT
Always remove the mixed-bed ion exchange resin by filtration
(step 3 above) before adding the TBE buffer. Resin will destroy the effectiveness of
the buffer.
7
Add deionized water to make the final volume:
♦
50 mL for a 36-cm or 48-cm gel for the ABI PRISM 377 DNA Sequencer
♦
80 mL for a 24-cm or 34-cm gel for the ABI 373 DNA Sequencer
♦
100 mL for a 48-cm gel for the ABI 373 DNA Sequencer
IMPORTANT
If the plates are not clean and ready for gel pouring, prepare
them before adding the polymerizing agents to your solution.
A-4 Gel Preparation
Adding the polymerizing reagents:
Step
1
Action
Add freshly made 10% APS, and swirl carefully to mix without introducing air
bubbles.
Note
Be as accurate and reproducible as possible when making the 10% APS
solution. Significant variation in this reagent can produce changes in data quality.
2
Add TEMED, and swirl carefully to mix without introducing air bubbles.
! WARNING ! CHEMICAL AND FIRE HAZARD. TEMED is extremely
flammable and can be very destructive to the skin, eyes, nose, and
respiratory system. Keep it in a tightly closed container. Avoid inhalation and
contact with skin, eyes and clothing. Always work under a hood and wear
chemical resistant gloves when handling TEMED solutions. Read the MSDS in
the Safety Summary included with your instrument user’s manual.
3
Cast the gel using one of the methods described in your instrument user’s manual.
Note
Allow the gel to polymerize for a minimum of 2 hours.
Gel Preparation A-5
Protocol and Run Conditions for 29:1 Polyacrylamide Gels
Preparing 40%
Acrylamide Stock
(29:1)
Step
Action
1
Working in a fume hood, combine the following amounts of acrylamide and
bisacrylamide in a glass beaker:
♦
Acrylamide, 58 g
♦
Bisacrylamide, 2 g
! WARNING ! CHEMICAL HAZARD. Acrylamide and bisacrylamide are
poisons, neurotoxins, irritants, carcinogens, and possible teratogens.
Acrylamide and bisacrylamide sublime (the solids release toxic vapor) and
are harmful if swallowed, inhaled, or absorbed through the skin. Effects are
cumulative. When handling, always wear protective equipment (lab coat,
safety glasses, and chemical-resistant gloves) and use in a well-ventilated
area. On a routine basis, thoroughly clean surfaces subject to contamination.
2
Dissolve the crystalline acrylamide and bisacrylamide in sufficient distilled,
deionized water to bring the total volume to 135 mL.
3
Add 15 g of mixed-bed, ion-exchange resin.
4
Stir at room temperature until all crystals dissolve. Continue stirring for
5–10 minutes.
5
Filter the mixture through a 0.2-µm cellulose nitrate filter.
6
Transfer the filtrate to a graduated cylinder and bring the total volume to 150 mL
with distilled, deionized water.
7
Store at 2–6 ∞C.
Note
40% acrylamide stock lasts for 1 month at 2–6 ∞C.
Ingredients and Run For 36-cm WTR Runs—4.5% 29:1 Polyacrylamide Gel, 6 M Urea
Conditions for the This is the best formulation to use for 2400 scans/hr runs.
ABI PRISM 377
Ingredient
urea
40% acrylamide stock
For 50 mL
18.0 g
5.63 mL
deionized water
25 mL
Mixed-bed ion
exchange resin
0.5 g
For 1200 scans/hr runs:
♦
Use standard 36-cm
1200 scans/hr run
modules.
♦
Increase run time to
9 hours.
Filter and degas the above ingredients
before adding TBE.
For 2400 scans/hr runs:
10X TBE
5.0 mL
♦
10% APS
250 µL
TEMED
30 µL
Use standard 36-cm
2400 scans/hr run
modules.
♦
Increase run time to
4 hours.
Bring to final volume (50 mL) with
deionized water.
A-6 Gel Preparation
Run Conditions
For 48-cm WTR Runs—4.25% 29:1 Polyacrylamide Gel, 6 M Urea
Ingredient
urea
40% acrylamide stock
For 50 mL
18.0 g
Run Conditions
♦
Use standard 48-cm run
modules.
♦
Increase run time to
11 hours.
5.31 mL
deionized water
25 mL
Mixed-bed ion
exchange resin
0.5 g
Filter and degas the above ingredients
before adding TBE.
10X TBE
5.0 mL
10% APS
250 µL
TEMED
30 µL
Bring to final volume (50 mL) with
deionized water.
Ingredients and Run For 34-cm WTR Runs—5% 29:1 Polyacrylamide Gel, 8.3 M Urea
Conditions for the
For 80 mL
Run Conditions
ABI 373 Ingredient
urea
40.0 g
40% acrylamide stock
10 mL
deionized water
37 mL
Mixed-bed ion
exchange resin
0.5 g
Increase run time to 18 hours.
Filter and degas the above ingredients
before adding TBE.
10X TBE
8.0 mL
10% APS
400 µL
TEMED
48 µL
Bring to final volume (80 mL) with
deionized water.
Gel Preparation A-7
For 48-cm WTR Runs—4.25% 29:1 Polyacrylamide Gel, 7 M Urea
This formulation has not been tested in Applied Biosystems laboratories, but has been
used successfully in several customer laboratories.
Ingredient
urea
For 100 mL
42 g
40% acrylamide stock
Run Conditions
Run at 40 W for 17 hours.
10.65 mL
deionized water
44 mL
Mixed-bed ion
exchange resin
0.5 g
Filter and degas the above ingredients
before adding TBE.
10X TBE
10 mL
10% APS
500 µL
TEMED
70 µL
Bring to final volume (100 mL) with
deionized water.
Preparing 29:1 Preliminary gel preparation steps:
Polyacrylamide Gels
Step
Action
1
Referring to the appropriate list of ingredients above and the 373 DNA Sequencing
System User’s Manual or ABI PRISM 377 DNA Sequencer User’s Manual, gather all
the necessary lab equipment and ingredients.
2
Prepare all stock solutions per the list of ingredients.
3
For the ABI PRISM 377 DNA Sequencer: clean the gel plates thoroughly and mount
them in the gel pouring cassette (or alternative device).
For the ABI 373 DNA Sequencer: clean the gel plates thoroughly and prepare them
for gel pouring.
Preparing the acrylamide–urea solution:
Step
1
Action
Combine urea, 40% acrylamide stock, deionized water, and mixed-bed ion
exchange resin in a 150-mL beaker.
! WARNING ! CHEMICAL HAZARD. Urea causes eye, skin, and
respiratory irritation. Lab experiments have shown mutagenic effects. Avoid
contact. Wear chemical resistant gloves, safety goggles, and other protective
clothing.
! WARNING ! CHEMICAL HAZARD. Acrylamide and bisacrylamide are
neurotoxins. Avoid inhalation and skin contact. Wear gloves at all times, and
work in a fume hood when handling acrylamide solutions. Use appropriate
precautions to avoid inhalation of crystalline acrylamide. Read the
manufacturer’s MSDS before handling.
A-8 Gel Preparation
2
Stir the solution until all the urea crystals have dissolved.
3
Filter the solution through a 0.2-µm cellulose nitrate filter.
Preparing the acrylamide–urea solution:
Step
4
(continued)
Action
Degas for 2–5 minutes.
Note
Degas time for all gels should be constant to ensure a reproducible
polymerization rate for all gels.
5
Transfer the solution to a 100-mL graduated cylinder.
6
Add filtered 10X TBE buffer.
IMPORTANT
Always remove the mixed-bed ion exchange resin by filtration
(step 3 above) before adding the TBE buffer. Resin will destroy the effectiveness of
the buffer.
7
Add deionized water to make the final volume:
♦
50 mL for a 36-cm or 48-cm gel for the ABI PRISM 377 DNA Sequencer
♦
80 mL for a 34-cm gel for the ABI 373 DNA Sequencer
♦
100 mL for a 48-cm gel for the ABI 373 DNA Sequencer
IMPORTANT
If the plates are not clean and ready for gel pouring, prepare
them before adding the polymerizing agents to your solution.
Adding the polymerizing reagents:
Step
1
Action
Add freshly made 10% APS, and swirl carefully to mix without introducing air
bubbles.
Note
Be as accurate and reproducible as possible when making the 10% APS
solution. Significant variation in this reagent can produce changes in data quality.
2
Add TEMED, and swirl carefully to mix without introducing air bubbles.
! WARNING ! CHEMICAL AND FIRE HAZARD. TEMED is extremely
flammable and can be very destructive to the skin, eyes, nose, and
respiratory system. Keep it in a tightly closed container. Avoid inhalation and
contact with skin, eyes and clothing. Always work under a hood and wear
chemical resistant gloves when handling TEMED solutions. Read the MSDS in
the Safety Summary included with your instrument user’s manual.
3
Cast the gel using one of the methods described in your instrument user’s manual.
Note
29:1 polyacrylamide gels take a minimum of 2 hours to polymerize,
sometimes longer.
Gel Preparation A-9
Protocol and Run Conditions for Long Ranger and PAGE-PLUS Gels
Ingredients and Run For 36-cm WTR Runs
Conditions for the
5.0% Long Ranger Gel, 6 M Urea
ABI PRISM 377
Ingredient
For 50 mL
Run Conditions
urea
18.0 g
For 1200 scans/hr runs:
50% gel stock solution
5.0 mL
♦
10X TBE
5.0 mL
Use standard 36-cm
1200 scans/hr run
modules.
♦
Increase run time to
9 hours.
deionized water
to 50 mL
10% APS
250 µL
TEMED
25 µL
For 2400 scans/hr runs, use
standard 36-cm 2400 scans/hr
run modules.
4.8% PAGE-PLUS Gel, 6 M Urea
Ingredient
For 50 mL
Run Conditions
urea
18.0 g
For 1200 scans/hr runs:
40% gel stock solution
6.0 mL
♦
10X TBE
5.0 mL
Use standard 36-cm
1200 scans/hr run
modules.
♦
Increase run time to
9 hours.
deionized water
to 50 mL
10% APS
300 µL
TEMED
30 µL
For 2400 scans/hr runs, use
standard 36-cm 2400 scans/hr
run modules.
For 48-cm WTR Runs
This is the best formulation to use for 48-cm gels.
5.25% PAGE-PLUS Gel, 6 M Urea
Ingredient
urea
18.0 g
40% gel stock solution
6.6 mL
10X TBE
5.0 mL
deionized water
A-10 Gel Preparation
For 50 mL
to 50 mL
10% APS
250 µL
TEMED
25 µL
Run Conditions
♦
Use standard 48-cm run
modules.
♦
Increase run time to
12 hours.
4.75% Long Ranger Gel, 6 M Urea
Ingredient
urea
For 50 mL
18.0 g
50% gel stock solution
4.75 mL
10X TBE
5.0 mL
deionized water
to 50 mL
10% APS
250 µL
TEMED
25 µL
Run Conditions
♦
Use standard 48-cm run
modules.
♦
Increase run time to
11 hours.
Preparing Preliminary gel preparation steps:
PAGE-PLUS and
Step
Action
Long Ranger Gels
1
Referring to the appropriate list of ingredients above, and your user’s manual,
for the
gather all the necessary equipment and ingredients.
ABI PRISM 377
2
Prepare all stock solutions per the appropriate list of ingredients above.
3
Clean the gel plates thoroughly and mount them in the gel pouring cassette (or
alternative device).
To prepare 5.0% Long Ranger, and 4.8 and 5.25% PAGE-PLUS gels:
Step
Action
1
Weigh out the urea and carefully transfer it to a stoppered, graduated cylinder.
2
Using a pipette, add the appropriate amount of gel stock solution and 10X TBE
buffer to the cylinder.
3
Adjust the volume to 45 mL by slowly adding deionized water, and tapping the
cylinder to release air bubbles trapped by the urea.
4
Stopper the cylinder and invert to dissolve the urea.
5
Allow the solution to warm to room temperature.
6
Add deionized water to make the final volume (50 mL).
7
Stopper the cylinder and mix the contents thoroughly.
8
Filter the solution through a 0.2-µm cellulose nitrate filter.
9
Degas for 2–5 minutes, and transfer the solution to a wide-mouthed container.
Note
Degas time for all gels should be constant to ensure a reproducible
polymerization rate for all gels.
Gel Preparation A-11
Adding the polymerizing reagents:
Step
Action
1
IMPORTANT
If the plates are not clean and mounted in the gel cassette or
other device, clean and mount them now before adding the polymerizing agents to
your solution.
2
Add freshly made 10% APS, and swirl carefully to mix without introducing air
bubbles.
Note
Be as accurate and reproducible as possible when making the 10% APS
solution. Significant variation in this reagent can produce changes in data quality.
3
Add TEMED, and swirl carefully to mix without introducing air bubbles.
4
Cast the gel using one of the methods described in the ABI PRISM 377 DNA
Sequencer User’s Manual.
5
Allow the gel to polymerize for 2 hours before using.
Ingredients and Run For 34-cm WTR Runs
Conditions for the
5.75% Long Ranger Gel, 8.3 M Urea
ABI 373
Ingredient
For 80 mL
urea
40.0 g
50% gel stock solution
9.2 mL
10X TBE
deionized water
Run Conditions
Use the standard run time of
14 hours.
8.0 mL
to 80 mL
10% APS
400 µL
TEMED
40 µL
5.75% PAGE-PLUS Gel, 8.3 M Urea
Ingredient
urea
40.0 g
40% gel stock solution
11.5 mL
10X TBE
8.0 mL
deionized water
A-12 Gel Preparation
For 80 mL
to 80 mL
10% APS
480 µL
TEMED
48 µL
Run Conditions
Use the standard run time of
14 hours.
For 48-cm WTR Runs
5.0% Long Ranger Gel, 8.3 M Urea
Ingredient
urea
For 100 mL
50 g
50% gel stock solution
10 mL
10X TBE
10 mL
deionized water
Run Conditions
Use the standard run time of
18 hours.
to 100 mL
10% APS
500 µL
TEMED
50 µL
5.25% PAGE-PLUS Gel, 8.3 M Urea
Ingredient
urea
For 100 mL
50 g
40% gel stock solution
13.2 mL
10X TBE
10.0 mL
deionized water
Run Conditions
Use the standard run time of
18 hours.
to 100 mL
10% APS
500 µL
TEMED
50 µL
Gel Preparation A-13
Preparing Preliminary gel preparation steps:
PAGE-PLUS and
Step
Action
Long Ranger Gels
1
Referring to the appropriate list of ingredients above, and your user’s manual,
for the ABI 373
gather all the necessary equipment and ingredients.
2
Prepare all stock solutions per the appropriate list of ingredients above.
3
Clean the gel plates thoroughly and prepare them for gel pouring.
To prepare 5.0% Long Ranger, and 4.8 and 5.25% PAGE-PLUS gels:
Step
Action
1
Weigh out the urea and carefully transfer it to a 250-mL, stoppered, graduated
cylinder.
2
Using a pipette, add the appropriate amount of gel stock solution and 10X TBE
buffer to the cylinder.
3
Adjust the volume to 75 mL by slowly adding deionized water. Tap the cylinder to
release air bubbles trapped by the urea.
4
Stopper the cylinder and invert to dissolve the urea.
5
Allow the solution to warm to room temperature.
6
Add deionized water to make the final volume:
♦
80 mL for 34-cm gels
♦
100 mL for 48-cm gels
7
Stopper the cylinder and mix the contents thoroughly.
8
Filter the solution through a 0.2-µm cellulose nitrate filter.
9
Degas for 2–5 minutes, and transfer the solution to a wide-mouthed container.
Note
Degas time for all gels should be constant to ensure a reproducible
polymerization rate for all gels.
Adding the polymerizing reagents:
Step
Action
1
IMPORTANT
If the plates are not clean and ready for gel pouring, prepare
them before adding the polymerizing agents to your solution.
2
Add freshly made 10% APS, and swirl carefully to mix without introducing air
bubbles.
Note
Be as accurate and reproducible as possible when making the 10% APS
solution. Significant variation in this reagent can produce changes in data quality.
A-14 Gel Preparation
3
Add TEMED, and swirl carefully to mix without introducing air bubbles.
4
Cast the gel using one of the methods described in the 373 DNA Sequencing
System User’s Manual.
5
Allow the gel to polymerize for 2 hours before using.
Preparing APS, TBE Buffer, and Deionized Formamide
10% Ammonium IMPORTANT
Persulfate dissolve.
Step
1
Use fresh ammonium persulfate. The crystals should crackle as they
Action
Weigh out 0.50 ± 0.005 g of ammonium persulfate (APS) into a 15-mL
polypropylene tube.
! WARNING ! CHEMICAL HAZARD. Always wear appropriate safety
attire (full-length laboratory coat, protective glasses, gloves, etc.) when
handling and mixing hazardous chemicals. Always work under a chemical
fume hood when handling and mixing hazardous chemicals. The room in
which you work must have proper ventilation and a waste-collection system.
2
Using a P-5000 Pipetman or equivalent, add 5 mL of deionized water to the tube.
3
Vortex until all crystals dissolve.
Note
Optimally, APS should be prepared fresh daily. At the very least, store 10% stocks at
–15 to –25 °C and replace every week. Listen for a crackling sound when adding water to dry
APS. Material that has broken down due to high humidity or liquid contamination will no longer
crackle when moistened.
10X TBE To make 500 mL of 10X TBE:
Step
1
Action
To a 500-mL container, add the following:
♦
54 g Tris
♦
28 g Boric acid
♦
4 g Ethylenediaminetetraacetic acid, disodium salt (Na2EDTA)
♦
Distilled, deionized water to 500 mL
IMPORTANT
Use Tris base (see page 4-2). Use disodium EDTA to make
10X TBE stock. Some major laboratory suppliers provide monosodium EDTA or
tetrasodium EDTA.
2
Mix ingredients thoroughly by vortexing.
3
Verify that the pH is 8.2–8.3.
Note
10X TBE stored at room temperature should be used within 1 month. Do not use if a
precipitate is present.
Gel Preparation A-15
Deionized IMPORTANT
Always use deionized formamide to prepare loading buffers. Over time,
formamide
hydrolyzes
to formic acid and formate. Deionized formamide stock lasts for 3 months
Formamide
at –15 to –25 °C
Step
1
Action
Mix 50 mL of formamide and 5 g of AG501 X8 ion-exchange resin.
! WARNING ! CHEMICAL HAZARD. Formamide is a teratogen and is
harmful by inhalation, skin contact, and ingestion. Use in a well-ventilated
area. Use chemical-resistant gloves and safety glasses when handling.
2
Stir for 30 minutes at room temperature.
3
Check that the pH is greater than 7.0 using pH paper.
If the pH is not greater than 7.0, decant the formamide into a beaker containing
another 5 g of ion-exchange resin and repeat 30-minute stirring at room
temperature.
A-16 Gel Preparation
4
When the pH is greater than 7.0, allow the beads to settle to the bottom of the
beaker. Remove the supernatant (formamide), taking care not to disturb the beads.
5
Dispense the deionized formamide into aliquots of 500 µL and store for up to
3 months at –15 to –25 °C.
6
Use one aliquot per set of samples. Discard any unused deionized formamide.
IUB Codes
B
B
IUB Codes
Complements
A = adenosine
S = G or C (Strong—3 H bonds)
A
T, U
R
Y
C = cytidine
W = A or T (Weak—2 H bonds)
C
G
Y
R
G = guanosine
Y = C or T (pYrimidine)
G
C
K
M
T = thymidine
B = C, G, or T
T
A
M
K
U = uracil
D = A, G, or T
U
A
S
W
K = G or T (Keto)
H = A, C, or T
D
H
W
S
M = A or C (aMino)
V = A, C, or G
H
D
B
V
R = A or G (puRine)
N = aNy base
N
N
V
B
IUB Codes B-1
References
C
C
Literature Ausubel, F.M., Brent, R., Kingstin, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and
References Struhl, K., eds. 1998. Current Protocols in Molecular Biology. John Wiley and Sons:
New York, NY, p. A.3.0.2.
Barr, P.J., Thayer, R.M., Laybourn, P., Najarian, R.C., Seela, F., and Tolan, D.R. 1986.
7-deaza-2´-deoxyguanosine-5´-triphosphate: enhanced resolution in M13 dideoxy
sequencing. BioTechniques 4: 428–432.
Baskaran, N., Kandpal, R.P., Bhargava, A.K., Glynn, M.W., Bale, A., and Weissman,
S.M. 1996. Uniform amplification of a mixture of deoxyribonucleic acids with varying
GC content. Genome Res. 6: 633–638.
Burgett, S.G., and Rosteck, P.R., Jr. 1994. Use of dimethyl sulfoxide to improve
fluorescent, Taq cycle sequencing. In Automated DNA Sequencing and Analysis, ed.
Adams, M.D., Fields, C., and Venter, J.C. Academic Press: San Diego, CA,
pp. 211–215.
Devine, S.E., and Boeke, J.D. 1994. Efficient integration of artificial transposons into
plasmid targets in vitro: a useful tool for DNA mapping, sequencing, and functional
analysis. Nucleic Acids Res. 22: 3765–3772.
Devine, S.E., Chissoe, S.L., Eby, Y., Wilson, R.K., and Boeke, J.D. 1997. A
transposon-based strategy for sequencing repetitive DNA in eukaryotic genomes.
Genome Res. 7: 551–563.
Fernandez-Rachubinski, F., Eng, B., Murray, W.W., Blajchman, M.A., and Rachubinski,
R.A. 1990. Incorporation of 7-deaza dGTP during the amplification step in the
polymerase chain reaction procedure improves subsequent DNA sequencing. DNA
Seq. 1: 137–140.
Henke, W., Herdel, K., Jung, K., Schnorr, D., and Loening, S.A. 1997. Betaine
improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res.
25: 3957–3958.
Hoefer, Inc. 1993. Hoefer TKO 100 Mini-fluorometer Operator’s Manual:
20788/Rev C/3-05-93.
Innis, M.A., and Gelfand, D.H. 1990. Optimization of PCRs. In PCR Protocols: A Guide
to Methods and Applications, ed. Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White,
T.J. Academic Press: San Diego, CA, pp. 3–12.
Innis, M.A. 1990. PCR with 7-deaza-2´-deoxyguanosine triphosphate. In PCR
Protocols: A Guide to Methods and Applications, ed. Innis, M.A., Gelfand, D.H.,
Sninsky, J.J., and White, T.J. Academic Press: San Diego, CA, pp. 54–59.
References C-1
Khan, A.S., Wilcox, A.S. , Hopkins, J.A., and Sikela, J.M. 1991. Efficient double
stranded sequencing of cDNA clones containing long poly(A) tails using anchored
poly(dT) primers. Nucleic Acids Res. 19: 1715.
Kornberg, A., and Baker, T. 1992. DNA Replication, 2nd. ed. W.H. Freeman and
Company, New York, NY, pp. 132–133.
Landre, P.A., Gelfand, D.H., and Watson, R.M. 1995. The use of cosolvents to
enhance amplification by the polymerase chain reaction. In PCR Strategies, ed. Innis,
M.A., Gelfand, D.H., and Sninsky, J.J. Academic Press: San Diego, CA, pp. 3–16.
Lee, L.G., Spurgeon, S.L., Heiner, C.R., Benson, S.C., Rosenblum, B.B., Menchen,
S.M., Graham, R.J., Constantinescu, A., Upadhya, K.G., and Cassel, J.M. 1997. New
energy transfer dyes for DNA sequencing. Nucleic Acids Res. 25: 2816–2822.
Lobet, Y., Peacock, M.G., and Cieplak, W., Jr. 1989. Frame-shift mutation in the lacZ
gene of certain commercially available pUC18 plasmids. Nucleic Acids Res. 17: 4897.
Marra, M., Weinstock, L.A., and Mardis, E.R. 1996. End sequence determination from
large insert cloning using energy transfer fluorescent primers. Genome Res.
6: 1118–1122.
McMurray, A.A., Sulston, J.E., and Quail, M.A. 1998. Short-insert libraries as a
method of problem solving in genome sequencing. Genome Res. 8: 562–566.
Mills, D.R., and Kramer, F.R. 1979. Structure-independent nucleotide sequence
analysis. Proc. Natl. Acad. Sci (USA) 76: 2232–2235.
Mizusawa, S., Nishimura, S., and Seela, F. 1986. Improvement of the dideoxy chain
termination method of DNA sequencing by use of deoxy-7-deazaguanosine
triphosphate in place of dGTP. Nucleic Acids Res. 14: 1319–1324.
Molecular Probes, Inc. 1995. User Bulletin #MP-7581/4-01-95: Picogreen dsDNA
Quantitation Reagent (P/N P-7581).
Rosenblum, B.B., Lee, L.G., Spurgeon, S.L., Khan, S.H., Menchen, S.M., Heiner,
C.R., and Chen, S.M. 1997. New dye-labeled terminators for improved DNA
sequencing patterns. Nucleic Acids Res. 25: 4500–4504.
Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci (USA) 74: 5463–5467.
Tabor, S., and Richardson, C.C. 1990. DNA sequence analysis with a modified
bacteriophage T7 DNA polymerase: effect of pyrophosphorolysis and metal ions. J.
Biol. Chem. 265: 8322–8328.
Tabor, S., and Richardson, C.C. 1995. A single residue in DNA polymerases of
Escherichia coli DNA polymerase I family is critical for distinguishing between deoxyand dideoxynucleotides. Proc. Natl. Acad. Sci (USA) 92: 6339–6343.
Thomas, M.G., Hesse, S.A., McKie, A.T., and Farzaneh, F. 1993. Sequencing of cDNA
using anchored oligo dT primers. Nucleic Acids Res. 16: 3915–3916.
Thweatt, R., Goldstein, S., and Shmookler Reis, R.J. 1990. A universal primer mixture
for sequence determination at the 3´ ends of cDNAs. Anal. Biochem. 190: 314–316.
Watson, J.D., Hopkins, N.H., Roberts, J.W., Steitz, J.A., and Weiner, A.M. 1987.
Molecular Biology of the Gene, 4th ed., Benjamin-Cummings: Menlo Park, CA.
C-2 References
Werle, E., Schneider, C., Renner, M., Volker, M., and Fiehn, W. 1994. Convenient
single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids
Res. 22: 4354–4355.
WWW Sites ♦
Bio-Rad Laboratories:
http://www.bio-rad.com/cgi-bin/Tango.cgi/ApplicationBits/literature.qry
♦
Centre National de Séquençage (CNS, or Génoscope): http://www.cns.fr
♦
Ligochem: http://www.ligochem.com
♦
Applied Biosystems: www.appliedbiosystems.com/techsupport
(Genetic Analysis page: www.appliedbiosystems.com/techsupport)
♦
QIAGEN: http://www.qiagen.com
♦
Sanger Centre: http://www.sanger.ac.uk
♦
The Institute for Genome Research (TIGR): http://www.tigr.org
♦
University of Oklahoma Advanced Center for Genome Technology (ACGT):
http://www.genome.ou.edu
♦
University of Washington Genome Center:
http://www.genome.washington.edu/uwgc
♦
Washington University School of Medicine Genome Sequencing Center (GSC):
http://genome.wustl.edu/gsc
References C-3
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D
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ABI PRISM ® 3700 DNA Analyzer
ABI PRISM ® 3100 Genetic Analyzer
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Technical Support D-1
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Chemiluminescence
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LC/MS
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Synthesis
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Technical Support D-3
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Technical Support D-5
Part Numbers
E
E
ABI PRISM DNA Sequencing Kits and Reagents
Kits Ready Reaction formulations contain all necessary reagents in one stable premix.
Core Kit configurations contain all essential reagents packaged in separate tubes.
ABI PRISM™ dRhodamine Terminator Cycle Sequencing Kits with AmpliTaq® DNA
Polymerase, FS
P/N
Kit
Reactions
403044
Ready Reaction
100
403045
Ready Reaction
1000
4303143
Ready Reaction
5000
ABI PRISM BigDye™ Terminator Cycle Sequencing Kits with AmpliTaq DNA
Polymerase, FS
P/N
Kit
Reactions
4303573
Ready Reaction
24
4303149
Ready Reaction
100
4303150
Ready Reaction
1000
4303151
Ready Reaction
5000
ABI PRISM BigDye™ Primer Cycle Sequencing Ready Reaction Kits with AmpliTaq DNA
Polymerase, FS
P/N
Primer
Reactions
403051
–21 M13
100
403049
–21 M13
5000
403052
M13 Reverse
100
403050
M13 Reverse
5000
Part Numbers E-1
ABI PRISM Dye Terminator Cycle Sequencing Kits with AmpliTaq DNA Polymerase, FS
P/N
Kit
Reactions
402080
Ready Reaction
100
402119
Ready Reaction
1000
402118
Core Kit
100
ABI PRISM Dye Primer Cycle Sequencing Ready Reaction Kits with AmpliTaq DNA
Polymerase, FS
P/N
Primer
Reactions
402111
–21 M13
100
402109
M13 Reverse
100
ABI PRISM Dye Primer Cycle Sequencing Core Kits with AmpliTaq DNA Polymerase, FS
P/N
E-2 Part Numbers
Primer
Reactions
402071
–21 M13
100
402072
M13 Reverse
100
402073
–21 M13/M13 Reverse
100
402126
T7
100
402127
T3
100
402128
SP6
100
402129
T7/SP6
100
402130
T3/T7
100
402125
Kit reagents only (primerless)
100
Dye-Labeled Includes 20 pmol of FAM- and JOE-labeled primer, 40 pmol of TAMRA- and
Primers ROX-labeled primer, and a control template. Enough for 50 ss- or dsDNA sequencing
reactions.
P/N
Matrix and
Sequencing
Standards
Sample Preparation
Reagents
Primers
401131
–21 M13 Dye Primers (4 x 50), 5´ TGT AAA ACG ACG GCC AGT 3´
401130
M13 Reverse Dye Primers (4 x 50), 5´ CAG GAA ACA GCT ATG ACC 3´
401127
T7 Dye Primers (4 x 50), 5´ TAA TAC GAC TCA CTA TAG GG 3´
401128
T3 Dye Primers (4 x 50), 5´ ATT AAC CCT CAC TAA AGG GA 3´
401129
SP6 Dye Primers (4 x 50), 5´ ATT TAG GTG ACA CTA TAG 3´
403013
PI (+) Dye Primers (4 x 50), 5´ CAG GAC ATT GGA TGC TGA GAA TTC G 3´
403014
PI (–) Dye Primers (4 x 50), 5´ CAG GAG CCG TCT ATC CTG CTT GC 3´
P/N
Standard
403047
dRhodamine Matrix Standards Kit
401071
Dye Terminator Matrix Standards Kit
401114
Dye Primer Matrix Standards Kit
4303120
dRhodamine Terminator Cycle Sequencing Standard
4304154
BigDye Terminator Cycle Sequencing Standard
402830
Dye Terminator Cycle Sequencing Standard
401920
Dye Primer Cycle Sequencing Standard
P/N
Standard
402790
ABI PRISM Plasmid Miniprep Kit (100 purifications)
402791
ABI PRISM Plasmid Miniprep Kit (500 purifications)
4305605
5X Sequencing Buffer (600 reactions)
4305603
5X Sequencing Buffer (5400 reactions)
Part Numbers E-3
Reagent Kit
Protocols
E-4 Part Numbers
P/N
Protocol
403041
ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit
Protocol
4303237
ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit Protocol
403057
ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit Protocol
402078
ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit Protocol
402116
ABI PRISM Dye Terminator Cycle Sequencing Core Kit Protocol
402113
ABI PRISM Dye Primer Cycle Sequencing Ready Reaction Kit Protocol
402114
ABI PRISM Dye Primer Cycle Sequencing Core Kit Protocol
402920
Primer Island Transposition Kit Protocol
ABI PRISM 310 Genetic Analyzer
Autosampler
Tray Kits
P/N
402867
Kit
48-Tube Sample Tray Kit
Includes: 48-Tube Sample Trays (2), 0.5-mL Tube Septa (500), 0.5-mL Sample
Tubes (500). (Individual Part Numbers: One 48-Tube Sample Tray, P/N 005572;
0.5-mL Tube Septa, P/N 401956; 0.5-mL Sample Tubes, P/N 401957)
402868
96-Tube Sample Tray Kit
Includes: 96-Tube Septa Clips (4), 0.2-mL Tube Septa Strips (24 strips,
480 septa), 0.2-mL Sample Tubes (1000), MicroAmp® Tray and Retainer
(10 sets), MicroAmp® Base (10). (Individual Part Numbers: Septa Clips,
P/N 402866; 0.2-mL Tube Septa Strips, P/N 402059; 0.2 mL MicroAmp®
Tubes, 8/strip, P/N N801-0580; MicroAmp Tray and Retainer, P/N 403081;
MicroAmp Base, P/N N801-0531; 96-Well Tray Adapter, P/N 4305051)
Polymers and
Polymers and Consumables for the ABI PRISM® 310 Genetic Analyzer
Consumables
P/N
402837
Item
Performance Optimized Polymer 6
Size/Amt
(POP-6™)
3 mL
Generally used for sequencing
No template suppression reagent included
200 sample run
402844
Performance Optimized Polymer 6 (POP-6) with TSR
3 mL
Includes two 4-mL vials of Template Suppression Reagent
200 sample runs
403076
POP-6 polymer with TSR for Shared Instruments
3 mL
Includes eight 4-mL vials of Template Suppression Reagent
200 sample runs
402824
10X Genetic Analyzer Buffer with EDTA
25 mL
Used with POP-6 polymer
402839
ABI PRISM 310 Capillaries, 47-cm x 50-µm (internally uncoated)
5/pkg
Used with POP-6 polymer for rapid sequencing
500 sample runs (100 runs/capillary)
402840
ABI PRISM 310 Capillaries, 61-cm x 50-µm (internally uncoated)
2/pkg
Used with POP-6 polymer for long-read sequencing
200 sample runs (100 runs/capillary)
401957
Genetic Analyzer Sample Tubes (0.5-mL)
500/pkg
401956
Genetic Analyzer Septa for 0.5-mL Sample Tubes
500/pkg
For 48-Tube Tray
402059
Genetic Analyzer Septa Strips (0.2-mL tube)
For 96-Tube Tray
485/pkg
(24 strips)
Part Numbers E-5
Polymers and Consumables for the ABI PRISM® 310 Genetic Analyzer
P/N
402866
Item
Genetic Analyzer Retainer Clips
(continued)
Size/Amt
4/pkg
(96-Tube Tray Septa Clips)
N801-0580
403081
MicroAmp 0.2-mL Sample Tubes, 8/strip
1000/pkg
MicroAmp Tray and Retainer
10 sets
MicroAmp Base
10/pkg
4305051
96-Well Tray Adapter
1 each
401958
Genetic Analyzer Capillary Cutters
2 each
401955
Genetic Analyzer Buffer Vials (4.0-mL)
50/pkg
N801-0531
Includes cap adapters
005914
Platinum cathode electrode
1 each
604418
1.0-mL Glass Syringe
1 each
Used for GeneScan® and sequencing applications
Contains syringe O-rings and ferrule
604042
GeneScan Glass Syringe (2.5-mL)
1 each
Contains syringe O-rings and ferrule
603803
DNA Sequencing Glass Syringe (250-µL)
1 each
Contains syringe O-rings and ferrule
221102
Syringe O-rings
1 each
O-ring inside of glass syringe assembly
005401
Syringe ferrule
1 each
Ferrule inside of glass syringe assembly
005404
Capillary Fitting
1 each
Screw fitting used to hold the capillary in the pump block
005572
0.5-mL Sample Tray
1 each
Holds 48 0.5-mL sample tubes
603796
Waste vial
1 each
Vial attaches to the gel pump block, collects waste generated
during gel pump priming with Sequence Polymer
005402
Anode buffer jar
1 each
Buffer jar attaches to gel pump block, holds the anode buffer
604076
Valve, waste vial
1 each
Gel pump block manual valve, the waste vial attaches to the
fitting on this valve
604075
Valve, plastic syringe, Luer
1 each
Gel pump block manual valve, the DNA sequencing polymer
plastic syringe attaches to the fitting on this valve
310021
Thermal Tape
For affixing the capillary to the heat plate
E-6 Part Numbers
1 each
Chemical These kits are shipped with new instruments for the purpose of installation and
Installation Kits training.
P/N
402089
Kit
ABI PRISM 310 Basic Install Kit
Included with purchase of ABI PRISM 310 Genetic Analyzer
Includes: 310 Genetic Analyzer Buffer with EDTA, Leak Test Capillary,
Sensitivity Standard, Genetic Analyzer Buffer Vials, Genetic Analyzer Septa,
Genetic Analyzer Capillary Cutters, 5-mL Syringe
402090
ABI PRISM 310 DNA Sequencing and GeneScan Install Kit
Included when both the DNA Sequencing Analysis and GeneScan Modules
(677-30 and 672-30, respectively) are purchased
Includes: POP-6 polymer, TSR, 61-cm x 50-µm i.d. Capillaries, POP-4™
polymer, 47-cm x 50-µm i.d. Capillaries, 310 Genetic Analyzer Buffer with
EDTA, Fluorescent Genotyping Demonstration Kit B, GeneScan-500 [TAMRA]
Internal Lane Size Standard, formamide, Amberlite MB-1A, Dye Primer Matrix
Standards, Dye Terminator Matrix Standards, Fluorescent Amidite Matrix
Standards, NED Matrix Standard, dRhodamine Matrix Standards, BigDye
Terminator Cycle Sequencing Standard
401820
ABI PRISM 310 DNA Sequencing Install Kit
Included with purchase of the DNA Sequencing Analysis Module (677-30)
Includes: POP-6 polymer, TSR, 61-cm x 50-µm i.d. Capillaries, dRhodamine
Matrix Standards, BigDye Terminator Cycle Sequencing Standard
Part Numbers E-7
ABI PRISM 377 DNA Sequencer
Plates and Spacers
P/N
401878
Item
48-cm Glass Plates/Spacers Kit
Includes: two sets of 48-cm Well-to-Read Glass Plates and Gel Spacers
4305810
48-cm Glass Plates/Spacers Kit for the for the ABI PRISM® 377 with 96-Lane
Upgrade
Includes: one set of 48-cm Well-to-Read Glass Plates and Gel Spacers
401876
36-cm Glass Plates/Spacers Kit
Includes: two sets of 36-cm Well-to-Read Glass Plates and Gel Spacers
4305693
36-cm Glass Plates/Spacers Kit for the ABI PRISM 377 with 96-Lane Upgrade
Includes: one set of 36-cm Well-to-Read Glass Plates and Gel Spacers
401835
48-cm Rear Glass Plate
401838
48-cm Front Glass Plate
4305387
48-cm 0.4-mm Stepped Front Glass Plate for the ABI PRISM 377 with 96-Lane
Upgrade
401837
Two 48-cm Gel Spacers, 0.2 mm thick
401839
36-cm Rear Glass Plate
401840
36-cm Front Glass Plate
4305384
36-cm 0.4-mm Stepped Front Glass Plate for the ABI PRISM 377 with 96-Lane
Upgrade
401836
Two 36-cm Gel Spacers, 0.2 mm thick
Combs
P/N
E-8 Part Numbers
Item
402168
18-well Sharktooth Comb, 0.2 mm thick
401827
24-well Sharktooth Comb, 0.2 mm thick
401922
32-well Sharktooth Comb, 0.2 mm thick
401828
36-well Sharktooth Comb, 0.2 mm thick
402177
48-well Sharktooth Comb, 0.2 mm thick
402180
64-well Sharktooth Comb, 0.2 mm thick
4305385
100-well Sharktooth Comb, 0.4 mm thick for the ABI PRISM 377 with 96-Lane
Upgrade
Cassette, Buffer
Chambers, and Heat
Plate
P/N
Item
604297
Gel Cassette
401991
Gel Pouring Fixture Kit (contains top and bottom fixtures, clamps, and syringe)
4304406
Upper Buffer Chamber- new
4304409
Gasket Replacement Kit for Upper Buffer Chamber P/N 4304406
604078
Upper Buffer Chamber- obsolete
604524
Gasket Replacement Kit for Upper Buffer Chamber P/N 604078
603875
Lower Buffer Chamber
603822
Upper Buffer Electrode Assembly
603823
Lower Buffer Electrode Assembly
603833
Front 36-cm Well-to-Read Heat Plate
ABI 373 DNA Sequencer
Combs and Spacers
P/N
Item
401472
24-well Sharktooth Comb, 0.4 mm thick
401580
32-well Sharktooth Comb, 0.4 mm thick
401473
36-well Sharktooth Comb, 0.4 mm thick
402179
48-well Sharktooth Comb, 0.4 mm thick
402182
64-well Sharktooth Comb, 0.4 mm thick
402214
Casting Comb for Sharktooth, 0.4 mm thick
402048
Uni-length Glass Plate Spacer, 0.4 mm thick
401582
24-well Sharktooth Comb, 0.3 mm thick
401581
32-well Sharktooth Comb, 0.3 mm thick
401583
36-well Sharktooth Comb, 0.3 mm thick
402178
48-well Sharktooth Comb, 0.3 mm thick
402181
64-well Sharktooth Comb, 0.3 mm thick
402215
Casting Comb for Sharktooth, 0.3 mm thick
402047
Uni-length Glass Plate Spacer, 0.3 mm thick
Plates
P/N
Plate
401617
48-cm Notched Glass Plate (Stretch Configuration)
401618
48-cm Plain Glass Plate (Stretch Configuration)
401069
24/34-cm Notched Glass Plate (Stretch Configuration)
401498
24/34-cm Plain Glass Plate (Stretch Configuration)
Part Numbers E-9
Buffer Chambers
P/N
Item
603750
Small Upper Buffer Assembly
603751
Large Upper Buffer Assembly
603410
Lower Buffer Assembly
200576
Gasket (Silicone Foam Cord) for Upper Buffer Chamber Seal
201410
Gasket Adhesive (for use with P/N 200576)
Documentation and Software
User’s Manuals and
Other Documents
P/N
Item
903565
ABI PRISM 310 Genetic Analyzer User’s Manual
902376
373 DNA Sequencing System User’s Manual
904258
373 DNA Sequencer With XL Upgrade User’s Manual
4304367
Using the ABI 373 BigDye Filter Wheel User Bulletin
903433
ABI PRISM 377 DNA Sequencer User’s Manual
904210
ABI PRISM 377-18 DNA Sequencer User’s Manual
904412
ABI PRISM 377 DNA Sequencer XL Upgrade User’s Manual
4305423
ABI PRISM 377 DNA Sequencer 96-Lane Upgrade User’s Manual
903939
CATALYST 800 Molecular Biology LabStation User’s Manual
904414
ABI PRISM 877 Integrated Thermal Cycler User’s Manual
904532
ABI PRISM DNA Sequencing Analysis Software (Version 3.0) User’s Manual
4304075
ABI PRISM DNA Sequencing Analysis Software (Version 3.2) User’s Manual
770901
Comparative PCR Sequencing, A Guide to Sequencing-Based Mutation
Detection
770905
Guide to PCR Enzymes
4304655
Precipitation Methods to Remove Residual Dye Terminators from Sequencing
Reactions User Bulletin
Software
P/N
E-10 Part Numbers
Item
4303032
ABI PRISM 310 Training CD
402089
Primer Express™ Software
Index
with BigDye Filter Wheel, choosing
chemistry 2-15 to 2-16
ABI 373 with XL Upgrade
description of 1-7
run modules 1-12, 6-2
See Also ABI 373
ABI PRISM 310
choosing a sequencing
chemistry 2-15 to 2-16
dye set/primer (mobility) files 6-5
instrument description 1-10
part numbers E-5 to E-7
run modules 1-12 to 1-13, 6-2
troubleshooting capillary
electrophoresis 7-55 to
A
7-61
A260, converting to
capillary failure 7-55
concentration 3-17
current decreases over
ABI 373
run 7-58
19:1 polyacrylamide gels A-3
current too high 7-58
29:1 polyacrylamide gels A-7 to
data not automatically
A-8
analyzed 7-57
choosing a sequencing
extra peaks under strong
chemistry 2-16
peak 7-60
dye set/primer (mobility) files 6-5
extraneous peaks 7-60
filter sets 1-8
fluctuating current 7-58
Long Ranger gels A-12 to A-14
fragments migrate
PAGE-PLUS gels A-12 to A-14
slowly 7-61
part numbers E-9 to E-10
high baseline 7-59
troubleshooting gel
inconsistent peak
electrophoresis 7-44 to
mobilities 7-60
7-54
low current 7-58
buffer leaks 7-47 to 7-48
no current 7-57 to 7-58
excess salt 7-45
no signal 7-58 to 7-59
fluorescent
noisy baseline 7-60
contaminants 7-46
poor base spacing 7-60
gel extrusion 7-50
poor resolution 7-61
gel runs too quickly 7-53
runs get faster 7-61
gel runs too slowly 7-53
runs get slower 7-61
green streak in lane 7-54
signal too high 7-59
greenish-yellow haze 7-54
signal too low 7-59
lanes appear smeared 7-54
spikes 7-55 to 7-56
polymerization too slow 7-54
spikes in baseline 7-60
poor resolution caused by
stop peak 7-60
gel 7-53
virtual filter sets 1-11
poor-quality acrylamide 7-44 ABI PRISM 377
poor-quality gel plates 7-52
19:1 polyacrylamide gels A-2
red streaks, vertical 7-49
29:1 polyacrylamide gels A-6 to
swirls in gel 7-53
A-7
temporary loss of
choosing a sequencing
signal 7-51
chemistry 2-15 to 2-16
dye set/primer (mobility) files 6-5
Numerics
310. See ABI PRISM 310
36-lane gels, express load
option 3-49
373 and 373XL. See ABI 373 and ABI
373 with XL Upgrade
377 and 377XL. See ABI PRISM 377
and ABI PRISM 377 with XL
Upgrade
377-18, description of 1-9
96-Lane Upgrade, description of 1-9
96-well plate purification
protocol 3-35
instrument description 1-8 to 1-9
Long Ranger gels A-10 to A-12
PAGE-PLUS gels A-10 to A-12
part numbers E-8 to E-9
run modules 1-12 to 1-13, 6-2
troubleshooting gel
electrophoresis 7-44 to
7-54
buffer leaks 7-47 to 7-48
excess salt 7-45
fluorescent
contaminants 7-46
gel extrusion 7-50
gel runs too quickly 7-53
gel runs too slowly 7-53
green streak in lane 7-54
greenish-yellow haze 7-54
lanes appear smeared 7-54
polymerization too slow 7-54
poor resolution caused by
gel 7-53
poor-quality acrylamide 7-44
poor-quality gel plates 7-52
red streaks, vertical 7-49
swirls in gel 7-53
temporary loss of
signal 7-51
virtual filter sets 1-11
ABI PRISM 377 with XL Upgrade
description of 1-9
See Also ABI PRISM 377
ABI PRISM 377-18, description of 1-9
ABI PRISM 877 ITC, cycle
sequencing 3-32
ABI PRISM DNA sequencing kits and
reagents E-1 to E-4
dye-labeled primers E-3
kits E-1 to E-2
matrix and sequencing
standards E-3
reagent kit protocols, part
numbers E-4
acrylamide, gel electrophoresis 4-2
ammonium persulfate (APS) 4-2
preparing A-15
AmpliTaq DNA Polymerase, FS
description of 1-5
B
background fluorescence, problem with
gel 4-4
Index-1
bacterial artificial chromosome (BAC)
DNA templates
cycle sequencing
using BigDye primers 3-30
using BigDye
terminators 3-28
preparing 3-9
base spacing, default value 7-8
basecaller, choosing the correct
basecaller 6-6
Beer’s Law, converting A260 to
concentration 3-17
BigDye Filter Wheel
choosing chemistry 2-15 to 2-16
to use new chemistries 1-8
BigDye primers
chemistry, description of 2-9 to
2-11
cycle sequencing 3-29 to 3-30
dye/base relationships 2-14
ethanol precipitation
method 3-47
preparing sequencing
reactions 3-25 to 3-26
BigDye terminators
chemistry, description of 2-5 to
2-6
cycle sequencing 3-27 to 3-29
dye/base relationships 2-14
ethanol precipitation
method 3-38 to 3-40
isopropanol precipitation
method 3-36 to 3-38
preparing sequencing
reactions 3-22 to 3-24
troubleshooting, GT-rich
template 7-34 to 7-35
bleedthrough, troubleshooting
sequencing data 7-43
C
capillary electrophoresis
optimizing 5-1 to 5-8
consumables 5-2 to 5-3
effect of salt
contamination 5-4
electrokinetic injection 5-4 to
5-6
electrophoresis
conditions 5-7
run parameters for
sequencing
chemistries 5-8
preparing and loading
samples 3-53 to 3-54
loading the sample 3-54
preparing reaction
mixture 3-53
preparing the sample 3-53
sample volume 3-53
Index-2
troubleshooting 7-55 to 7-61
table 7-57 to 7-61
CATALYST 800, cycle
sequencing 3-31
chemistries
AmpliTaq DNA Polymerase,
FS 1-5
chemistry-specific mobility
information 6-4
choosing a sequencing
chemistry 2-15 to 2-16
compatibility with filter sets 2-13
compatibility with
instruments 2-13
dye primer cycle sequencing
kits 2-8 to 2-11
BigDye primers 2-9 to 2-11
fluorescein/rhodamine dye
primers 2-8 to 2-9
dye spectra 2-12
dye terminator cycle sequencing
kits 2-2 to 2-7
BigDye terminators 2-5 to
2-6
dRhodamine terminators 2-3
to 2-5
rhodamine dye
terminators 2-2 to 2-3
dye/base relationships 2-14
dye-labeled primers, description
of 1-6
dye-labeled terminators,
description of 1-5
run parameters for capillary
electrophoresis 5-8
chemistry guide, what’s new in this
guide 1-1
cleaning dirty templates 3-16
compressions
troubleshooting sequencing
data 7-31 to 7-32, 7-43
consumables
capillary electrophoresis 5-2 to
5-3
contamination
affecting quality of DNA
template 3-15
avoiding problems with
sequencing gels 4-4
control DNA, effect on template
quality 3-15
Custom Oligonucleotide Synthesis
Service 3-19
cycle sequencing
advantages of 1-4
description of 1-4
performing DNA sequencing
reactions 3-27 to 3-32
dye primer chemistries 3-29
to 3-32
dye terminator
chemistries 3-27 to
3-29, 3-31, 3-32
preparing sequencing
reactions 3-21 to 3-26
dye primer chemistries 3-24
to 3-26
dye terminator
chemistries 3-21 to
3-24
D
data analysis, setting range 6-15 to
6-23
Peak 1 Location for dye primer
chemistries 6-16 to 6-18
Peak 1 Location for dye terminator
chemistries 6-18 to 6-20
Stop Point 6-21 to 6-23
Data Utility software
using to make matrix file 6-7
deionized formamide 3-50
preparing A-16
DNA quality, determining 3-16
DNA sequence troubleshooting 7-30
to 7-38
compressions 7-31 to 7-32
false stops in dye primer
chemistry 7-30 to 7-31
GC-rich templates 7-32 to 7-33
GT-rich template with BigDye
terminators 7-34 to 7-35
homopolymer regions 7-35 to
7-37
using anchored primers 7-36
repetitive DNA 7-38
secondary structure 7-33
DNA Sequencing Polymer (DSP)
capillary electrophoresis 5-2
DNA sequencing, performing
capillary electrophoresis,
preparing and loading
samples 3-53 to 3-54
loading the sample 3-54
preparing reaction
mixture 3-53
preparing the sample 3-53
sample volume 3-53
choosing a sequencing
chemistry 2-15 to 2-16
cycle sequencing 3-27 to 3-32
dye primer chemistries 3-29
to 3-32
dye terminator
chemistries 3-27 to
3-29, 3-31, 3-32
electrophoresis, preparing
for 3-33 to 3-49
96-well plate purification
protocol 3-35
dye primer chemistries 3-33
procedures 3-46 to 3-49
dye terminator
chemistries 3-33
procedures 3-34 to 3-45
ethanol precipitation
BigDye primers 3-47
BigDye
terminators 3-38
to 3-40
fluorescein/rhodamine
dye primers 3-46
ethanol/MgCl2
precipitation 3-43 to
3-45
ethanol/sodium acetate
precipitation 3-41 to
3-42
express load for
36-lane 3-49
isopropanol
precipitation 3-36 to
3-38
methods, table of 3-33
shrimp alkaline phosphatase
digestion 3-45
spin column purification 3-34
to 3-35
gel electrophoresis, preparing and
loading samples 3-50 to
3-52
loading
recommendations 350
loading samples 3-52
preparing loading
buffer 3-50
sample loading
volumes 3-51
preparing cycle sequencing
reactions 3-21 to 3-26
dye primer chemistries 3-24
to 3-26
dye terminator
chemistries 3-21 to
3-24
primer design and
quantitation 3-18 to 3-19
DNA templates
determining quality of
templates 3-15 to 3-16
DNA template quantity 3-17
preparing
BAC DNA templates 3-9
performing DNA
reactions 3-2 to 3-9
plasmid DNA templates 3-6
to 3-8
single-stranded DNA
templates 3-2 to 3-6
sequencing PCR templates 3-10
to 3-14
troubleshooting, poor quality
template 7-16
Documents-on-Demand D-2
dRhodamine terminators
chemistry description 2-3 to 2-5
cycle sequencing 3-27 to 3-29
dye spectra 2-12
dye/base relationships 2-14
preparing sequencing
reactions 3-21
dye peaks, excess dye peaks at the
beginning of the
sequence 7-42
dye primer chemistries
cycle sequencing 3-29 to 3-30
cycle sequencing kits 2-8 to 2-11
BigDye primers 2-9 to 2-11
fluorescein/rhodamine dye
primers 2-8 to 2-9
dye spectra 2-12
dye/base relationships 2-14
false stops in dye primer
chemistry 7-30 to 7-31
ways to obtain the
sequence 7-31
Peak 1 Location for data
analysis 6-16 to 6-18
preparing sequencing
reactions 3-24 to 3-26
secondary structure 7-33
troubleshooting, stop peaks in dye
primer chemistry 7-43
dye set/primer files
choosing 6-3 to 6-5
chemistry-specific mobility
information 6-4
using the wrong file 6-3
mobility correction,
described 1-13
troubleshooting software
settings 7-62
dye spectra 2-12
dye terminator chemistries
cycle sequencing 3-27 to 3-29
cycle sequencing kits 2-2 to 2-7
BigDye terminators 2-5 to
2-6
dRhodamine terminators 2-3
to 2-5
rhodamine dye
terminators 2-2 to 2-3
dye spectra 2-12
dye/base relationships, table
of 2-14
Peak 1 Location for data
analysis 6-18 to 6-20
preparing sequencing
reactions 3-21 to 3-24
dye/base relationships 2-14
dye-labeled primers
description of 1-6
part numbers E-3
dye-labeled terminators, description
of 1-5
E
electrokinetic injection, optimizing 5-4
to 5-6
description of 5-4
modifying injection time 5-4 to
5-5
modifying injection voltage 5-6
setting electrokinetic injection
values 5-6
electrophoresis
preparing extension
products 3-33 to 3-49
96-well plate purification
protocol 3-35
dye primer chemistries 3-33
procedures 3-46 to 3-49
dye terminator
chemistries 3-33
procedures 3-34 to 3-45
ethanol precipitation
BigDye primers 3-47
BigDye
terminators 3-38
to 3-40
fluorescein/rhodamine
dye primers 3-46
ethanol/MgCl2
precipitation 3-43 to
3-45
ethanol/sodium acetate
precipitation 3-41 to
3-42
express load for 36-lane
gels 3-49
isopropanol
precipitation 3-36 to
3-38
methods, table of 3-33
shrimp alkaline phosphatase
digestion 3-45
spin column purification 3-34
to 3-35
See also capillary electrophoresis,
gel electrophoresis
e-mail address, technical support D-3
evaluating data 7-2 to 7-15
gel files 7-2 to 7-4
practical examples 7-10 to 7-15
early signal loss 7-14 to 7-15
no usable sequence 7-10
noise 7-11 to 7-13
poor mobility correction 7-13
to 7-14
Index-3
preparing A-1 to A-16
matrix files
19:1 polyacrylamide
and sequencing standards, part
gels A-2 to A-5
numbers E-3
29:1 polyacrylamide
creating 6-7 to 6-13
gels A-6 to A-9
Data Utility software, using to
APS, TBE buffer, and
make file 6-7
deionized
making Filter Set E 6-8 to
formamide A-15 to
6-13
A-16
making instrument file from
gel formulations, table of A-1
sample file 6-14
Long
Ranger
and
overview
6-7
F
PAGE-PLUS
when to remake file 6-7
files, instrument files 1-14 to 1-16
gels A-10 to A-14
noise caused by incorrect or poor
matrix files, what’s in the file 1-16
overview A-1
file 7-13
multicomponent analysis 1-14 to
Genetic Analyzer Buffer, description
troubleshooting software
1-15
of 5-2
settings 7-63 to 7-64
Filter Set E, making 6-8 to 6-13
genomic DNA templates
what’s in the file 1-16
filter sets
cycle sequencing using BigDye
melting temperature (Tm),
ABI 373 1-8
terminators 3-29
estimating 3-18
compatibilities with
mobility correction, described 1-13
chemistries 2-13
mobility files
H
filter wheel, ABI PRISM BigDye Filter
See dye set/primer files
Wheel 1-8
help D-1 to D-5
molecular weights,
fluorescein/rhodamine dye primers.
Documents-on-Demand D-2
oligonucleotides 3-19
See dye primer chemistries
e-mail address D-3
multicomponent analysis,
fluorescent sequencing, description
fax, phone and hours D-1
described 1-14 to 1-15
of 1-3
Internet (WWW) address D-1
regional offices D-3 to D-5
N
homopolymer
regions, poor data
G
following 7-43
no recognizable sequence
GC-rich templates,
humidity, capillary electrophoresis,
troubleshooting 7-39
troubleshooting 7-32 to 7-33
optimizing 5-7
no usable sequence
gel electrophoresis
practical example 7-10
optimizing 4-1 to 4-9
noise
I
avoiding problems with
caused by incorrect or poor matrix
sequencing gels 4-4
initiator concentrations
file 7-13
to 4-9
effect on gel 4-3
throughout the sequence
polyacrylamide gels, theory
injection time, modifying 5-4 to 5-5
practical example 7-11 to
of 4-1
injection voltage, modifying 5-6
7-12
reagents 4-2 to 4-3
instrument files 1-14 to 1-16
sequencing data 7-40
preparing and loading
matrix files, what’s in the file 1-16
up to or after a point in sequence
samples 3-50 to 3-52
multicomponent analysis 1-14 to
practical example 7-12
loading
1-15
sequencing data 7-41
recommendations 3- Internet (WWW) address D-1
50
Documents-on-Demand D-2
O
loading samples 3-52
IUB codes B-1
preparing loading
oxygen, avoiding gel problems 4-5
buffer 3-50
L
sample loading
P
laboratory temperature, capillary
volumes 3-51
electrophoresis, optimizing 5-7 PAGE-PLUS gels, protocol and run
troubleshooting 7-44 to 7-54
literature references C-1 to C-3
conditions A-10 to A-14
table 7-53 to 7-54
Long Ranger gels, protocol and run
for the ABI 373 A-12 to A-14
gels
conditions A-10 to A-14
for the ABI PRISM 377 A-10 to
avoid problems with 4-4 to 4-9
for the ABI 373 A-12 to A-14
A-12
cleaning gel plates 4-6 to 4-9
for the ABI PRISM 377 A-10 to
part numbers
contaminants 4-4
ABI 373 E-9 to E-10
A-12
gel plate quality 4-6
ABI PRISM 310 E-5 to E-7
polymerization 4-4 to 4-5
ABI PRISM 377 E-8 to E-9
M
red streaks 4-5
ABI PRISM DNA sequencing kits
material safety data sheets (MSDS),
using fresh gels 4-5
and reagents E-1 to E-4
how to obtain D-2
evaluating 7-2 to 7-4
dye-labeled primers E-3
sample files 7-5 to 7-9
using the Annotation
View 7-8 to 7-9
using the Electropherogram
View 7-5 to 7-6
using the Raw Data
View 7-6 to 7-8
express load option, 36-lane
gels 3-49
Index-4
kits E-1 to E-2
matrix and sequencing
standards E-3
reagent kit protocols E-4
user’s manuals and
software E-10
PCR sequencing, setting Stop
Point 6-21 to 6-23
Performance Optimized Polymer 6
(POP-6), description of 5-2
plasmid DNA templates,
preparing 3-6 to 3-8
polyacrylamide gels
19:1, protocol and run
conditions A-2 to A-5
for the ABI 373 A-3
for the ABI PRISM 377 A-2
preparing 40% acrylamide
stock A-2
29:1, protocol and run
conditions A-6 to A-9
for the ABI 373 A-7 to A-8
for the ABI PRISM 377 A-6 to
A-7
preparing 40% acrylamide
stock A-6
theory of 4-1
polymers
capillary electrophoresis 5-2
poor mobility correction
sequencing data 7-41
POP-6, description of 5-2
primer design, effect on sequencing
data 3-18 to 3-19
pull-up peaks, troubleshooting
sequencing data 7-43
sequencing reactions 7-22 to
7-23
Q
quantitation, converting A260 to
concentration 3-17
R
reagents
gel electrophoresis 4-2 to 4-3
handling and storage 3-20
kit protocols, part numbers E-4
red rain (streaks)
avoiding problems with gel 4-5
gel electrophoresis,
troubleshooting 7-49
references
literature C-1 to C-3
WWW sites C-3
rhodamine dye terminators. See dye
terminator chemistries
run modules
choosing a run module 6-2
introduction to 1-12 to 1-13
run parameters, for capillary
electrophoresis 5-8
run temperature, capillary
electrophoresis, optimizing 5-7
run time, capillary electrophoresis,
optimizing 5-7
S
salt contamination, from template
preparation 7-25 to 7-26
sample files, evaluating 7-5 to 7-9
using the Annotation View 7-8 to
7-9
using the Electropherogram
View 7-5 to 7-6
using the Raw Data View 7-6 to
7-8
Sanger Dideoxy sequencing,
description of 1-2
sequencing data
troubleshooting table 7-39 to
7-43
sequencing instruments. See specific
instrument
sequencing PCR templates 3-10 to
3-14
sequencing reactions
factors affecting data quality 3-1
preparing 3-21 to 3-26
BigDye primers 3-25 to 3-26
BigDye terminators 3-22 to
3-24
dRhodamine
terminators 3-21
fluorescein/rhodamine dye
primers 3-24
reaction tubes, type of 3-20
reagent handling and reaction
storage 3-20
rhodamine dye
terminators 3-21
thermal cyclers, type of 3-20
signal
early signal loss
practical example 7-14 to
7-15
troubleshooting sequencing
data 7-41 to 7-42
preventing loss of 4-9
temporary loss of, gel
electrophoresis 7-51
single-stranded DNA templates,
preparing 3-2 to 3-6
software
optimizing settings 6-2 to 6-23
choosing a dye set/primer
file 6-3 to 6-5
choosing a run module 6-2
choosing the correct
basecaller 6-6
creating instrument (matrix)
files 6-7 to 6-13
dye set/primer files, table
of 6-5
setting the data analysis
range 6-15 to 6-23
Sequencing Analysis software,
described 1-16
troubleshooting 7-62 to 7-65
incorrect dye set/primer
file 7-62
incorrect or poor-quality matrix
file 7-63 to 7-64
incorrect Peak 1
Location 7-64
incorrect run module 7-62
spin column purification 3-34 to 3-35
T
tables
choosing a sequencing
chemistry 2-15 to 2-16
DNA quantity, used in sequencing
reactions 3-17
dye set/primer (mobility) files 6-5
dye/base relationships 2-14
methods of preparing extension
products for
electrophoresis 3-33
run modules 6-2
sample loading volumes, gel
electrophoresis 3-51
troubleshooting capillary
electrophoresis 7-57 to
7-61
troubleshooting gel
electrophoresis 7-53 to
7-54
troubleshooting sequencing
data 7-39 to 7-43
TBE buffer 4-2
preparing A-15
technical support D-1 to D-5
Documents-on-Demand D-2
e-mail address, D-3
fax, phone and hours D-1
Internet (WWW) address D-1
regional offices D-3 to D-5
TEMED, gel electrophoresis 4-3
temperature
avoiding gel problems 4-4
run temperature, optimizing for
capillary
electrophoresis 5-7
Template Suppression Reagent (TSR)
capillary electrophoresis 5-2
templates
determining DNA template
quality 3-15 to 3-16
Index-5
DNA template quantity 3-17
amount used 3-17
quantitation 3-17
preparing DNA templates 3-2 to
3-9
BAC 3-9
plasmid 3-6 to 3-8
single-stranded 3-2 to 3-6
sequencing PCR templates 3-10
to 3-14
troubleshooting, poor quality
template 7-16
thermal cyclers, preparing sequencing
reactions 3-20
Tm, estimating 3-18
troubleshooting
avoiding problems with
sequencing gels 4-4 to
4-9
cleaning gel plates 4-6 to 4-9
contaminants 4-4
gel plate quality 4-6
polymerization 4-4 to 4-5
red streaks 4-5
using fresh gels 4-5
capillary electrophoresis 7-55 to
7-61
capillary failure 7-55
current decreases over
run 7-58
current too high 7-58
data not automatically
analyzed 7-57
extra peaks under strong
peak 7-60
extraneous peaks 7-60
fluctuating current 7-58
fragments migrate
slowly 7-61
high baseline 7-59
inconsistent peak
mobilities 7-60
low current 7-58
no current 7-57 to 7-58
no signal 7-58 to 7-59
noisy baseline 7-60
poor base spacing 7-60
poor resolution 7-61
runs get faster 7-61
runs get slower 7-61
signal too high 7-59
signal too low 7-59
spikes 7-55 to 7-56
spikes in baseline 7-60
stop peak 7-60
troubleshooting table 7-57 to
7-61
data evaluation, practical
examples 7-10 to 7-15
no usable sequence 7-10
Index-6
noise 7-11 to 7-13
determining quality of DNA
template 3-15 to 3-16
DNA sequence composition 7-30
to 7-38
compressions 7-31 to 7-32
false stops in dye primer
chemistry 7-30 to
7-31
GC-rich templates 7-32 to
7-33
GT-rich template with BigDye
terminators 7-34 to
7-35
homopolymer regions 7-35
to 7-37
using anchored
primers 7-36
repetitive DNA 7-38
secondary structure 7-33
gel electrophoresis 7-44 to 7-54
buffer leaks 7-47 to 7-48
excess salt 7-45
fluorescent
contaminants 7-46
gel extrusion 7-50
gel runs too quickly 7-53
gel runs too slowly 7-53
green streak in lane 7-54
greenish-yellow haze 7-54
lanes appears smeared 7-54
polymerization too slow 7-54
poor resolution caused by
gel 7-53
poor-quality acrylamide 7-44
poor-quality gel plates 7-52
red streaks, vertical 7-49
swirls in gel 7-53
temporary loss of
signal 7-51
troubleshooting table 7-53 to
7-54
poor template quality 3-15
primer problems and
causes 3-19
sequencing data 7-39 to 7-43
broad, red peak between base
200 and 350 7-43
compressions 7-43
early signal loss 7-41 to 7-42
excess dye peaks at the
beginning of the
sequence 7-42
no recognizable
sequence 7-39
noise up to or after point in
sequence 7-41
noisy data throughout
sequence 7-40
poor data following long
homopolymer
region 7-43
poor mobility correction 7-41
pull-up peaks
(bleedthrough) 7-43
stop peaks in dye primer
chemistry 7-43
troubleshooting table 7-39 to
7-43
sequencing reactions 7-16 to
7-29
excess dye peaks 7-27 to
7-29
poor quality template 7-16
primer-related
problems 7-19 to
7-22
pull-up peaks 7-22 to 7-23
salt contamination from
template
preparation 7-25 to
7-26
stop peaks in PCR
sequencing 7-24
software settings 7-62 to 7-65
incorrect dye set/primer
file 7-62
incorrect or poor quality matrix
file 7-63 to 7-64
incorrect Peak 1
Location 7-64
incorrect run module 7-62
TSR (Template Suppression Reagent)
capillary electrophoresis 5-2
U
urea, gel electrophoresis 4-2
user’s manuals, part numbers E-10
V
virtual filter sets, ABI PRISM 310 and
ABI PRISM 377 1-11
W
WWW address D-1
Documents-on-Demand
D-2
X
XL Upgrade
ABI 373
description of 1-7
run modules 1-12, 6-2
ABI PRISM 377
description of 1-9
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on six continents. For international office locations,
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