PowerPlex® 16 System - Projects at NFSTC.org

PowerPlex® 16 System - Projects at NFSTC.org
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 1
Technical Manual
PowerPlex® 16 System
INSTRUCTIONS FOR USE OF PRODUCTS DC6530 AND DC6531
This Technical Manual has been revised. Please read it
carefully.
• The PowerPlex® 16 System shipped with this Technical
Manual incorporates improved manufacturing processes.
• The PowerPlex® 16 Allelic Ladder Mix and PowerPlex® 16
10X Primer Pair Mix included in the kit are not compatible
with previous lots of the PowerPlex® 16 System.
• A new matrix must be generated using one of the new
matrices: PowerPlex® Matrix Standards 310 (Cat.# DG4640)
or PowerPlex® Matrix Standards 3100/3130 (Cat.# DG4650).
PRINTED IN USA
Revised 1/07
Part# TMD012
tmd012.0107.qxp
1/12/2007
2:33 PM
Page 1
PowerPlex® 16 System
All technical literature is available on the Internet at: www.promega.com/tbs/
Please visit the web site to verify that you are using the most current version of this Technical Manual.
Please contact Promega Technical Services if you have questions on use of this system.
E-mail: [email protected]
I.
Description..................................................................................................................................2
II.
Product Components and Storage Conditions ....................................................................4
III.
Before You Begin .......................................................................................................................5
A. Precautions ........................................................................................................................5
B. Matrix Standardization or Spectral Calibration ..........................................................6
IV.
Protocols for DNA Amplification Using the PowerPlex® 16 System .............................7
A. Amplification Setup.........................................................................................................7
B. Amplification Thermal Cycling .....................................................................................9
V.
Instrument Setup and Sample Preparation........................................................................11
A. Detection of Amplified Fragments Using the ABI PRISM®
3100 or 3100-Avant Genetic Analyzer with Data Collection
Software, Version 2.0, and the Applied Biosystems 3130 or
3130xl Genetic Analyzer................................................................................................11
B. Detection of Amplified Fragments Using the ABI PRISM® 3100
Genetic Analyzer with Data Collection Software, Version 1.0.1 or 1.1 .................13
C. Detection of Amplified Fragments Using the ABI PRISM® 310
Genetic Analyzer............................................................................................................16
D. Detection of Amplified Fragments Using the ABI PRISM® 377 DNA
Sequencer.........................................................................................................................18
VI.
Data Analysis ...........................................................................................................................22
A. PowerPlex® Panel and Bin Sets with GeneMapper® ID, Version 3.2.....................22
B. Creating a Casework Analysis Method with GeneMapper® ID Software............23
C. Creating a Databasing or Paternity Analysis Method
with GeneMapper® ID Software..................................................................................27
D. Sample Analysis Using the GeneScan® Software
and PC Operating Systems...........................................................................................29
E. Sample Analysis Using the GeneScan® Software
and Macintosh® Operating Systems ...........................................................................31
F.
Sample Analysis Using the Genotyper® Software
and PowerTyper™ 16 Macro .......................................................................................32
G. Controls ...........................................................................................................................35
H. Results..............................................................................................................................35
VII.
Troubleshooting.......................................................................................................................37
A. Amplification and Fragment Detection ......................................................................37
B. GeneMapper® ID Analysis Software...........................................................................40
C. PowerTyper™ 16 Macro ...............................................................................................42
VIII.
References .................................................................................................................................44
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 1
tmd012.0107.qxp
1/12/2007
12:33 PM
IX.
I.
Page 2
Appendix ...................................................................................................................................46
A. Advantages of STR Typing...........................................................................................46
B. Advantages of Using the Loci in the PowerPlex® 16 System..................................47
C. Power of Discrimination ...............................................................................................50
D. DNA Extraction and Quantitation Methods..............................................................52
E. The Internal Lane Standard 600...................................................................................52
F.
Preparing the PowerPlex® 16 System Master Mix ....................................................53
G. Agarose Gel Electrophoresis of Amplification Products (Optional)......................53
H. Composition of Buffers and Solutions........................................................................54
I.
Related Products ............................................................................................................55
Description
STR (short tandem repeat) loci consist of short, repetitive sequence elements 3–7 base
pairs in length (1–4). These repeats are well distributed throughout the human genome
and are a rich source of highly polymorphic markers, which may be detected using
the polymerase chain reaction (5–8). Alleles of STR loci are differentiated by the
number of copies of the repeat sequence contained within the amplified region and
are distinguished from one another using radioactive, silver stain or fluorescence
detection following electrophoretic separation.
The PowerPlex® 16 System(a–d) (9,10) allows the co-amplification and three-color
detection of sixteen loci (fifteen STR loci and Amelogenin), including Penta E,
D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, Amelogenin, Penta D,
CSF1PO, D16S539, D7S820, D13S317 and D5S818. One primer for each of the Penta E,
D18S51, D21S11, TH01 and D3S1358 loci is labeled with fluorescein (FL); one primer
for each of the FGA, TPOX, D8S1179, vWA and Amelogenin loci is labeled with
carboxy-tetramethylrhodamine (TMR); and one primer for each of the Penta D,
CSF1PO, D16S539, D7S820, D13S317 and D5S818 loci is labeled with 6-carboxy-4´,5´dichloro-2´,7´-dimethoxy-fluorescein (JOE). All sixteen loci are amplified
simultaneously in a single tube and analyzed in a single injection or gel lane.
The PowerPlex® 16 Monoplex System, Penta E (Fluorescein) (Cat.# DC6591) and
PowerPlex® 16 Monoplex System, Penta D (JOE) (Cat.# DC6651) are available to
amplify the Penta E and Penta D loci, respectively. Each monoplex system allows
amplification of a single locus to confirm results obtained with the PowerPlex® 16
System, PowerPlex® 16 BIO System or PowerPlex® 2.1 System. The monoplex
systems can also be used to re-amplify DNA samples when one or more of the loci
do not amplify initially due to nonoptimal amplification conditions or poor DNA
template quality.
The PowerPlex® 16 System is compatible with the ABI PRISM® 310, 3100 and 3100Avant Genetic Analyzers, Applied Biosystems 3130 and 3130xl Genetic Analyzers
and ABI PRISM® 377 DNA Sequencer. The protocols presented in this manual were
tested at Promega Corporation. Amplification and detection instrumentation may
vary. You may need to optimize protocols including cycle number and injection time
(or loading volume) for each laboratory instrument. In-house validation should be
performed.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 2
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 3
The PowerPlex® 16 System provides all of the materials necessary for amplification
of STR regions of purified genomic DNA except for AmpliTaq Gold® DNA
polymerase. This manual contains separate protocols for use of the PowerPlex® 16
System with the Perkin-Elmer model 480 and GeneAmp® PCR system 9600, 9700 and
2400 thermal cyclers in addition to protocols for separation of amplified products and
detection of separated material (Figure 1). Protocols for operation of the fluorescencedetection instruments should be obtained from the instrument manufacturer.
Information on Promega’s other fluorescent STR systems and detection of amplified
STR fragments using silver staining is available upon request from Promega or
online at: www.promega.com
Amplification Setup
Section IV.A
Thermal Cycling
Section IV.B
GeneAmp® PCR System 9700
GeneAmp® PCR System 9600
GeneAmp® PCR System 2400
Model 480 Thermal Cycler
Instrument Setup and Sample Preparation
Section V
Applied
Biosystems 3130
or 3130xl Genetic
Analyzer
Section V.A
ABI PRISM® 3100 or
3100-Avant Genetic
Analyzer with Data
Collection Software,
Version 2.0
ABI PRISM® 3100
Genetic Analyzer
with Data Collection
Software, Version
1.0.1 or 1.1
Section V.A
Section V.B
ABI PRISM® 310
Genetic Analyzer
ABI PRISM® 377
DNA Sequencer
Section V.C
Section V.D
5120MA
Data Analysis
Section VI
GeneMapper® ID Software, GeneScan® Software
Versions 3.1 and 3.2
and PC Operating
Systems
GeneScan® Software
and Macintosh® Operating
Systems
Figure 1. An overview of the PowerPlex® 16 System protocol.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 3
tmd012.0107.qxp
1/12/2007
12:33 PM
II.
Page 4
Product Components and Storage Conditions
Product
PowerPlex® 16 System
Size
100 reactions
Cat.#
DC6531
Not For Medical Diagnostic Use. Cat.# DC6531 contains sufficient reagents for 100 reactions
of 25µl each. Includes:
Pre-amplification Components Box (Blue Label)
1 × 300µl
Gold ST★R 10X Buffer
1 × 250µl
PowerPlex® 16 10X Primer Pair Mix
25µl
9947A DNA (10ng/µl)
Post-amplification Components Box (Beige Label)
1 × 25µl
PowerPlex® 16 Allelic Ladder Mix
1× 150µl
Internal Lane Standard (ILS) 600
1
Protocol
Product
PowerPlex® 16 System
Size
400 reactions
Cat.#
DC6530
Not For Medical Diagnostic Use. Cat.# DC6530 contains sufficient reagents for 400 reactions
of 25µl each. Includes:
Pre-amplification Components Box (Blue Label)
4 × 300µl
Gold ST★R 10X Buffer
4 × 250µl
PowerPlex® 16 10X Primer Pair Mix
25µl
9947A DNA (10ng/µl)
Post-amplification Components Box (Beige Label)
4 × 25µl
PowerPlex® 16 Allelic Ladder Mix
4 × 150µl
Internal Lane Standard (ILS) 600
1
Protocol
!
The PowerPlex® 16 Allelic Ladder Mix is provided in a separate, sealed bag for
shipping. This component should be moved to the post-amplification box after
opening.
Storage Conditions: Store all components at –20°C in a nonfrost-free freezer. The
PowerPlex® 16 10X Primer Pair Mix, PowerPlex® 16 Allelic Ladder Mix and the
Internal Lane Standard 600 are light-sensitive and must be stored in the dark. We
strongly recommend that pre-amplification and post-amplification reagents be stored
and used separately with different pipettes, tube racks, etc.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 4
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 5
Available Separately
Product
Blue Dextran Loading Solution*
PowerTyper™ Macros (Release 2.0)**
Size
3ml
1 CD-ROM
Cat.#
DV4351
DG3470
*For Laboratory Use.
**Not For Medical Diagnostic Use.
The PowerTyper™ Macros (Release 2.0), for use with Genotyper® software, are
available from Promega. This CD-ROM contains the file “PowerTyper™ 16 Macro
(Release 2.0)” for use with the PowerPlex® 16 System. The macros can also be
downloaded at: www.promega.com/geneticidtools/
The proper panel and bin files for use with GeneMapper® ID software can be obtained
from the Promega web site at: www.promega.com/geneticidtools/panels_bins/
Matrix standards are required for initial setup of the color separation matrix. The
matrix standards are sold separately and are available for the ABI PRISM® 310
Genetic Analyzer and 377 DNA Sequencer (PowerPlex® Matrix Standards, 310) and
the ABI PRISM® 3100 and 3100-Avant Genetic Analyzers and Applied Biosystems
3130 and 3130xl Genetic Analyzers (PowerPlex® Matrix Standards, 3100/3130). See
Section IX.I for ordering information.
III. Before You Begin
III.A. Precautions
The application of PCR-based typing for forensic or paternity casework requires
validation studies and quality-control measures that are not contained in this
manual (11,12). The quality of the purified DNA sample, as well as small changes
in buffers, ionic strength, primer concentrations, choice of thermal cycler and
thermal cycling conditions, can affect the success of a PCR amplification. We
suggest strict adherence to recommended procedures for amplification, as well
as electrophoresis and fluorescence detection.
PCR-based STR analysis is subject to contamination by very small amounts of
nontemplate human DNA. Extreme care should be taken to avoid crosscontamination when preparing sample DNA, handling primer pairs, assembling
amplification reactions and analyzing amplification products. Reagents and
materials used prior to amplification (Gold ST★R 10X Buffer and PowerPlex® 16
10X Primer Pair Mix) are provided in a separate box and should be stored
separately from those used following amplification (PowerPlex® 16 Allelic
Ladder Mix and Internal Lane Standard 600). Always include a negative control
reaction (i.e., no template) to detect reagent contamination. We highly
recommend the use of gloves and aerosol-resistant pipette tips (e.g., ART® tips,
Section IX.I).
Some of the reagents used in the analysis of STR products are potentially
hazardous and should be handled accordingly. Table 1 describes the potential
hazards associated with such reagents.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 5
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 6
Table 1. Hazardous Reagents.
Reagents for ABI PRISM® 310, 3100 and 3100-Avant
Genetic Analyzers and Applied Biosystems 3130 and
3130xl Genetic Analyzers
Hazard
formamide
irritant, teratogen
Reagents for ABI PRISM® 377 DNA Sequencer
acrylamide (Long Ranger ® gel solution)
ammonium persulfate
Hazard
suspected carcinogen,
toxic
oxidizer, corrosive
formamide (contained in the Blue Dextran Loading Solution)
irritant, teratogen
TEMED
corrosive, flammable
urea
irritant
III.B. Matrix Standardization or Spectral Calibration
Proper generation of a matrix file is critical to evaluate multicolor systems with
the ABI PRISM® 310, 3100 and 3100-Avant Genetic Analyzers and Applied
Biosystems 3130 and 3130xl Genetic Analyzers. A matrix must be generated for
each individual instrument.
The PowerPlex® Matrix Standards, 310 (Cat.# DG4640), is required for matrix
standardization for the ABI PRISM® 310 Genetic Analyzer and ABI PRISM® 377
DNA Sequencer. For best results, the PowerPlex® Matrix Standards, 3100/3130
(Cat.# DG4650), should not be used to generate a matrix on the ABI PRISM®
310 Genetic Analyzer.
The PowerPlex® Matrix Standards, 3100/3130 (Cat.# DG4650), is required for
spectral calibration on the ABI PRISM® 3100 and 3100-Avant Genetic Analyzers
and Applied Biosystems 3130 and 3130xl Genetic Analyzers. The PowerPlex®
Matrix Standards, 310 (Cat.# DG4640), cannot be used to generate a matrix on
these instruments.
For protocols and additional information on matrix standardization, see the
PowerPlex ® Matrix Standards, 310, Technical Bulletin #TBD021, which is supplied
with Cat.# DG4640. For protocols and additional information about spectral
calibration, see the PowerPlex ® Matrix Standards, 3100/3130, Technical Bulletin
#TBD022, which is supplied with Cat.# DG4650. These manuals are available
upon request from Promega or online at: www.promega.com/tbs/
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 6
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 7
IV. Protocols for DNA Amplification Using the PowerPlex® 16 System
Materials to Be Supplied by the User
• thermal cycler model 480 or GeneAmp® PCR System 9600, 9700 or 2400
(Applied Biosystems)
• microcentrifuge
• 0.5ml or 0.2ml thin-walled microcentrifuge tubes or MicroAmp® optical 96-well
reaction plate (Applied Biosystems)
• 1.5ml amber-colored microcentrifuge tubes (Fisher Cat.# 05-402-26)
• aerosol-resistant pipette tips (see Section IX.I)
• AmpliTaq Gold® DNA polymerase (Applied Biosystems)
• Nuclease-Free Water (Cat.# P1193)
• Mineral Oil (Cat.# DY1151, for use with the model 480 thermal cycler)
We routinely amplify 0.5–1ng of template DNA in a 25µl reaction volume using the
protocols detailed below. Preferential amplification of the smaller loci can occur.
Expect to see high peak heights in the smaller loci and relatively lower peak heights
in the larger loci if more than the recommended amount of template is used. Reduce
the amount of template DNA or the number of cycles to correct this.
The PowerPlex® 16 System is optimized for the GeneAmp® PCR System 9700 thermal
cycler. Amplification protocols for the GeneAmp® PCR Systems 9600 and 2400
thermal cyclers and the Perkin-Elmer model 480 thermal cycler are provided.
IV.A. Amplification Setup
The use of gloves and aerosol-resistant pipette tips is highly recommended to
prevent cross-contamination. Keep all pre-amplification and post-amplification
reagents in separate rooms. Prepare amplification reactions in a room dedicated
for reaction setup. Use equipment and supplies dedicated for amplification setup.
!
Meticulous care must be taken to ensure successful amplification. A guide to
amplification troubleshooting is provided in Section VII.A.
1.
Thaw the Gold ST★R 10X Buffer and PowerPlex® 16 10X Primer Pair Mix.
Notes:
2.
1.
Mix reagents by vortexing for 15 seconds before each use. Do not
centrifuge the 10X Primer Pair Mix, as this may cause the primers to
be concentrated at the bottom of the tube.
2.
A precipitate may form in the Gold ST★R 10X Buffer. If this occurs,
warm the solution briefly at 37°C, then vortex until the precipitate is
in solution.
Determine the number of reactions to be set up. This should include
positive and negative control reactions. Add 1 or 2 reactions to this number
to compensate for pipetting error. While this approach does waste a small
amount of each reagent, it ensures that you will have enough PCR master
mix for all samples. It also ensures that each reaction contains the same
master mix.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 7
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 8
IV.A. Amplification Setup (continued)
3.
Place one clean, 0.2ml or 0.5ml reaction tube for each reaction into a rack,
and label appropriately. Alternatively, use a MicroAmp® plate, and label
appropriately.
Note: If using the GeneAmp® PCR System 9600, 9700 or 2400 thermal
cyclers, use 0.2ml MicroAmp® 8-strip reaction tubes or MicroAmp® plate.
For the Perkin-Elmer model 480 thermal cycler, we recommend the 0.5ml
GeneAmp® thin-walled reaction tubes.
4.
Add the final volume of each reagent listed in Table 2 into a sterile, 1.5ml
amber-colored tube. Mix gently.
Table 2 shows the component volumes per reaction. A worksheet to
calculate the required amount of each component of the PCR master mix is
provided in Section IX.F (Table 10).
Table 2. PCR Master Mix for the PowerPlex® 16 System.
PCR Master Mix Component1
nuclease-free water
Gold ST★R 10X Buffer
2.5µl
PowerPlex® 16 10X Primer Pair Mix
AmpliTaq
Gold®
DNA
Volume Per Reaction
to a final volume of 25.0µl
polymerase2
template DNA (0.5–1ng) 3
2.5µl
0.8µl (4u)
up to 19.2µl
total reaction volume
25µl
1Add nuclease-free water to the PCR master mix first, then add Gold ST★R 10X
Buffer, PowerPlex® 16 10X Primer Pair Mix and AmpliTaq Gold® DNA polymerase.
The template DNA will be added at Step 6.
2Assumes the AmpliTaq Gold® DNA polymerase is at 5u/µl. If the enzyme
concentration is different, the volume of enzyme must be adjusted accordingly.
3Store
DNA templates in nuclease-free water or TE-4 buffer (10mM Tris HCl [pH 8.0],
0.1mM EDTA). If the DNA template is stored in TE buffer that is not pH 8.0 or
contains a higher EDTA concentration, the volume of the DNA sample added
should not exceed 20% of the final reaction volume. PCR amplification efficiency
and quality can be greatly altered by changes in pH (due to added Tris-HCl),
available magnesium concentration (due to chelation by EDTA) or other PCR
inhibitors, which may be present at low concentrations depending on the source of
the template DNA and the extraction procedure used.
!
Amplification of >1ng of DNA template results in an imbalance in peak
heights from locus to locus. The smaller loci show greater amplification
yield than the larger loci. Reducing the number of cycles in the
amplification program by 2 to 4 cycles (i.e., 10/20 or 10/18 cycling) can
improve locus-to-locus balance.
5.
Pipet PCR master mix into each reaction tube.
6.
Pipet the template DNA (0.5–1ng) for each sample into the respective tube
containing PCR master mix.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 8
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 9
7.
For the positive amplification control, dilute the 9947A DNA positive
control to 0.5ng in the desired template DNA volume. Pipet 0.5ng of the
diluted DNA into a reaction tube containing the PCR master mix.
8.
For the negative amplification control, pipet nuclease-free water (instead
of template DNA) into a reaction tube containing the PCR master mix.
9.
If using the GeneAmp® PCR System 9600, 9700 or 2400 thermal cycler and
MicroAmp® reaction tubes or plates, no addition of mineral oil to the
reaction tubes is required. However, if using the model 480 thermal cycler
and GeneAmp® reaction tubes, add one drop of mineral oil to each tube
before closing.
Note: Allow the mineral oil to flow down the side of the tube and form an
overlay to limit sample loss or cross-contamination due to splattering.
IV.B. Amplification Thermal Cycling
This manual contains protocols for use of the PowerPlex® 16 System with the
Perkin-Elmer model 480 and GeneAmp® PCR system 9600, 9700 and 2400
thermal cyclers. For information about other thermal cyclers, please contact
Promega Technical Services by e-mail: [email protected]
Amplification and detection instrumentation may vary. You may need to
optimize protocols including cycle number and injection time (or loading
volume) for each laboratory instrument. Testing at Promega Corporation shows
that 10/22 cycles work well for 0.5–1ng of purified DNA templates. For higher
amounts of input DNA (i.e., FTA® paper) or to decrease sensitivity, fewer
cycles, such as 10/16, 10/18 or 10/20, should be evaluated. In-house validation
should be performed.
1.
Place the tubes or MicroAmp® plate in the thermal cycler.
2.
Select and run a recommended protocol. The preferred protocols for use
with the GeneAmp® PCR System 9600, 9700 and 2400 thermal cyclers and
the Perkin-Elmer model 480 thermal cycler are provided below.
3.
After completion of the thermal cycling protocol, store the samples at
–20°C in a light-protected box.
Note: Storage of amplified samples at 4°C or higher may produce
degradation products.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 9
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 10
Protocol for the GeneAmp® PCR
System 9700 Thermal Cycler1
Protocol for the GeneAmp® PCR
System 2400 Thermal Cycler
95°C for 11 minutes, then:
95°C for 11 minutes, then:
96°C for 1 minute, then:
96°C for 1 minute, then:
ramp 100% to 94°C for 30 seconds
ramp 29% to 60°C for 30 seconds
ramp 23% to 70°C for 45 seconds
for 10 cycles, then:
ramp 100% to 94°C for 30 seconds
ramp 100% to 60°C for 30 seconds
ramp 23% to 70°C for 45 seconds
for 10 cycles, then:
ramp 100% to 90°C for 30 seconds
ramp 29% to 60°C for 30 seconds
ramp 23% to 70°C for 45 seconds
for 22 cycles, then:
ramp 100% to 90°C for 30 seconds
ramp 100% to 60°C for 30 seconds
ramp 23% to 70°C for 45 seconds
for 22 cycles, then:
60°C for 30 minutes
60°C for 30 minutes
4°C soak
4°C soak
Protocol for the GeneAmp® PCR
System 9600 Thermal Cycler
Protocol for the Perkin-Elmer
Model 480 Thermal Cycler
95°C for 11 minutes, then:
95°C for 11 minutes, then:
96°C for 1 minute, then:
96°C for 2 minutes, then:
94°C for 30 seconds
ramp 68 seconds to 60°C (hold for 30 seconds)
ramp 50 seconds to 70°C (hold for 45 seconds)
for 10 cycles, then:
94°C for 1 minute
60°C for 1 minute
70°C for 1.5 minutes
for 10 cycles, then:
90°C for 30 seconds
ramp 60 seconds to 60°C (hold for 30 seconds)
ramp 50 seconds to 70°C (hold for 45 seconds)
for 22 cycles, then:
90°C for 1 minute
60°C for 1 minute
70°C for 1.5 minutes
for 22 cycles, then:
60°C for 30 minutes
60°C for 30 minutes
4°C soak
4°C soak
1When running the GeneAmp® PCR System 9700 thermal cycler, use the ramp rate for the
GeneAmp® PCR System 9600 thermal cycler.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 10
Printed in USA.
Revised 1/07
tmd012.0107.qxp
V.
1/12/2007
12:33 PM
Page 11
Instrument Setup and Sample Preparation
V.A. Detection of Amplified Fragments Using the ABI PRISM® 3100 or 3100-Avant
Genetic Analyzer with Data Collection Software, Version 2.0, and the Applied
Biosystems 3130 or 3130xl Genetic Analyzer
Materials to Be Supplied by the User
• 95°C dry heating block, water bath or thermal cycler
• crushed ice or ice-water bath
• aerosol-resistant pipette tips
• 3100 or 3130 capillary array, 36cm
• performance optimized polymer 4 (POP-4™) for the 3100 or 3130
• 10X genetic analyzer buffer with EDTA
• MicroAmp® optical 96-well plate and septa
• Hi-Di™ formamide (Applied Biosystems Cat.# 4311320)
• PowerPlex® Matrix Standards, 3100/3130 (Cat.# DG4650)
!
The quality of the formamide is critical. Use Hi-Di™ formamide with a
conductivity less than 100µS/cm. Freeze formamide in aliquots at –20°C. Multiple
freeze-thaw cycles or long-term storage at 4°C may cause a breakdown of the
formamide. Formamide with a conductivity greater than 100µS/cm may contain
ions that compete with DNA during injection. This results in lower peak heights
and reduced sensitivity. A longer injection time may not increase the signal.
!
Caution: Formamide is an irritant and a teratogen; avoid inhalation and contact
with skin. Read the warning label, and take appropriate precautions when
handling this substance. Always wear gloves and safety glasses when working
with formamide.
Sample Preparation
1.
Prepare a loading cocktail by combining and mixing the internal lane
standard and Hi-Di™ formamide as follows:
[(0.5µl ILS 600) × (# injections)] + [(9.5µl Hi-Di™ formamide) × (# injections)]
Note: The volume of internal lane standard used in the loading cocktail can
also be increased or decreased to adjust the intensity of the size standard
peaks. The optimal peak height for the 100-base fragment of the internal
lane standard is 500–1,000RFU. If the peak heights are too low, we
recommend altering the formamide/internal lane standard mix to contain
1.0µl of ILS 600 and 9.0µl of Hi-Di™ formamide. If the peak heights are
too high, we recommend altering the loading cocktail to contain 0.25µl of
ILS 600 and 9.75µl of formamide.
2.
Mix for 10–15 seconds using a vortex mixer.
3.
Pipet 10µl of formamide/internal lane standard mix into each well.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 11
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 12
V.A. Detection of Amplified Fragments Using the ABI PRISM® 3100 or 3100-Avant
Genetic Analyzer with Data Collection Software, Version 2.0, and the Applied
Biosystems 3130 or 3130xl Genetic Analyzer (continued)
4.
Add 1µl of amplified sample (or 1µl of the allelic ladder mix). Cover wells
with appropriate septa.
Note: Instrument detection limits vary; therefore, injection time or the
amount of product mixed with loading cocktail may need to be increased
or decreased. Use the “Module Manager” in the data collection software to
modify the injection time or voltage in the run module. If the peak heights
are higher than desired, the samples can be diluted in Gold ST★R 1X Buffer
before mixing with loading cocktail. This may result in uneven allele peak
heights across loci. For best results, use less DNA template in the
amplification reactions or reduce the number of cycles in the amplification
program by 2–4 cycles to achieve the desired signal intensity.
5.
Centrifuge plate briefly to remove air bubbles from the wells if necessary.
6.
Denature samples at 95°C for 3 minutes, then immediately chill on crushed
ice or in an ice-water bath for 3 minutes. Denature the samples just prior to
loading the instrument.
Instrument Preparation
Refer to the instrument users’ manual for instructions on cleaning, installing the
capillary array, performing a spatial calibration and adding polymer.
Analyze the samples as described in the user’s manual for the ABI PRISM® 3100
or 3100-Avant Genetic Analyzer with data collection software, version 2.0, and
the Applied Biosystems 3130 or 3130xl Genetic Analyzer with the following
exceptions.
1.
In the Module Manager, select “New”. Select “Regular” in the Type dropdown list, and select “HIDFragmentAnalysis36_POP4” in the Template
drop-down list. Confirm that the injection time is 5 seconds and the
injection voltage is 3kV. Lengthen the run time to 2,000 seconds. Give a
descriptive name to your run module, and select “OK”.
Note: Sensitivities of instruments may vary. The injection time and voltage
may be adjusted in the Module Manager. A suggested range for the
injection time is 3–22 seconds and for the injection voltage is 1–3kV.
2.
In the Protocol Manager, select “New”. Type a name for your protocol.
Select “Regular” in the Type drop-down list, and select the run module
you created in the previous step in the Run Module drop-down list.
Lastly, select “F” in the Dye-Set drop-down list. Select “OK.
3.
In the Plate Manager, create a new plate record as described in the
instrument user’s manual. In the dialog box that appears, select
“GeneMapper—Generic” in the Application drop-down list, and select the
appropriate plate type (96-well). Add entries in the owner and operator
windows, and select “OK”.
Note: If autoanalysis of sample data is desired, refer to the instrument
user’s manual for instructions.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 12
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
4.
12:33 PM
Page 13
In the GeneMapper® plate record, enter sample names in the appropriate
cells. Scroll to the right. In the “Results group 1” column, select the desired
results group. In the “Instrument Protocol 1” column, select the protocol
you created in Step 2. Be sure this information is present for each row that
contains a sample name. Select “OK”.
Note: To create a new results group, select “New” in the drop-down menu
in the results group column. Select the General tab, and enter a name.
Select the Analysis tab, and select “GeneMapper—Generic” in the
Analysis type drop-down list.
5.
Place samples in the instrument, and close the instrument doors.
6.
In the spectral viewer, confirm that dye set F is active, and set the correct
active calibration for dye set F.
7.
In the run scheduler, locate the plate record that you just created in Steps 3
and 4, and click once on the name to highlight it.
8.
Once the plate record is highlighted, click the plate graphic that corresponds
to the plate on the autosampler that contains your amplified samples.
9.
When the plate record is linked to the plate, the plate graphic will change
from yellow to green, and the green Run Instrument arrow becomes
enabled.
10. Click on the green Run Instrument arrow on the toolbar to start the
sample run.
11. Monitor electrophoresis by observing the run, view, array or capillaries
viewer window in the data collection software. Each injection will take
approximately 45 minutes.
V.B. Detection of Amplified Fragments Using the ABI PRISM® 3100 Genetic
Analyzer with Data Collection Software, Version 1.0.1 or 1.1
Materials to Be Supplied by the User
• 95°C dry heating block, water bath or thermal cycler
• crushed ice or ice-water bath
• aerosol-resistant pipette tips
• 3100 capillary array, 36cm
• performance optimized polymer 4 (POP-4™) for the 3100
• 10X genetic analyzer buffer with EDTA
• MicroAmp® optical 96-well plate and septa for the 3100
• Hi-Di™ formamide (Applied Biosystems Cat.# 4311320)
• PowerPlex® Matrix Standards, 3100/3130 (Cat.# DG4650)
!
The quality of the formamide is critical. Use Hi-Di™ formamide with a
conductivity less than 100µS/cm. Freeze formamide in aliquots at –20°C. Multiple
freeze-thaw cycles or long-term storage at 4°C may cause a breakdown of the
formamide. Formamide with a conductivity greater than 100µS/cm may contain
ions that compete with DNA during injection. This results in lower peak heights
and reduced sensitivity. A longer injection time may not increase the signal.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 13
tmd012.0107.qxp
1/12/2007
12:33 PM
!
Page 14
Caution: Formamide is an irritant and a teratogen; avoid inhalation and contact
with skin. Read the warning label, and take appropriate precautions when
handling this substance. Always wear gloves and safety glasses when working
with formamide.
Sample Preparation
1.
Prepare a loading cocktail by combining and mixing the internal lane
standard and Hi-Di™ formamide as follows:
[(0.5µl ILS 600) × (# injections)] + [(9.5µl Hi-Di™ formamide) × (# injections)]
Note: The volume of internal lane standard used in the loading cocktail can
also be increased or decreased to adjust the intensity of the size standard
peaks. The optimal peak height for the 100-base fragment of the internal
lane standard is 500–1,000RFU. If the peak heights are too low, we
recommend altering the formamide/internal lane standard mix to contain
1.0µl of ILS 600 and 9.0µl of Hi-Di™ formamide. If the peak heights are
too high, we recommend altering the loading cocktail to contain 0.25µl of
ILS 600 and 9.75µl of formamide.
2.
Mix for 10–15 seconds using a vortex mixer.
3.
Pipet 10µl of formamide/internal lane standard mix into each well.
4.
Add 1µl of amplified sample (or 1µl of the allelic ladder mix). Cover wells
with appropriate septa.
Note: Instrument detection limits vary; therefore, injection time or the
amount of product mixed with loading cocktail may need to be increased
or decreased. Use the Module Editor under “Tools” to modify the injection
time in the run module. If the peak heights are higher than desired, the
samples can be diluted in Gold ST★R 1X Buffer before mixing with
loading cocktail. The use of too much template DNA may result in uneven
allele peak heights across loci. For best results, use less DNA template in
the amplification reactions or reduce the number of cycles in the
amplification program by 2–4 cycles to achieve the desired signal intensity.
5.
Centrifuge plate briefly to remove air bubbles from the wells if necessary.
6.
Denature samples at 95°C for 3 minutes, then immediately chill on
crushed ice or in an ice-water bath for 3 minutes. Denature the samples
just prior to loading the instrument.
Instrument Preparation
Refer to the ABI PRISM® 3100 Genetic Analyzer User’s Manual for instructions on
cleaning the blocks, installing the capillary array, performing a spatial calibration
and adding polymer to the reserve syringe.
1.
Open the ABI PRISM® 3100 data collection software.
2.
Change the “GeneScan36_POP4DefaultModule” module run time to
2,000 seconds.
3.
Change the injection voltage to 3kV.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 14
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
4.
12:33 PM
Page 15
Change the injection time to 11 seconds.
Note: Sensitivities of instruments may vary. The injection time and voltage
may be adjusted in the Module Manager. A suggested range for the
injection time is 3–22 seconds and for the injection voltage is 1–3kV.
5.
Save the module with a new name (e.g.,
GeneScan36_POP4PowerPlex16_3kV_11secs_2000). Use this as the initial
run module for all runs.
6.
Open a new plate record. Name the plate, and select “GeneScan”. Select
the plate size (96-well). Select “Finish”.
7.
Complete the plate record spreadsheet for the wells you have loaded. Enter
appropriate information into the “sample name” and “color info” columns.
For allelic ladder samples, insert the word “ladder” into the “color info”
column for the blue, yellow and green dye colors. This information must
be entered to successfully analyze data with the PowerTyper™ 16 Macro
(Release 2.0).
8.
In the “BioLIMS Project” column, select “3100_Project1” from the pull-down
menu.
9.
In the “Dye Set” column, select “Z” from the pull-down menu.
10. When using the ABI PRISM® 3100 data collection software version 1.0.1 or
1.1, select “GeneScan36_POP4PowerPlex16_3kV_11secs_2000” from the
pull-down menu in the “Run Module 1” column.
11. To collect the data without autoanalyzing, select “No Selection” in the
“Analysis Module 1” column. Analysis parameters can be applied after data
collection and during data analysis using the GeneScan® analysis software.
12. Select “OK”. This new plate record will appear in the pending plate
records table on the plate setup page of the collection software.
13. Place samples in the instrument, and close the instrument doors.
14. Locate the pending plate record that you just created, and click once on the
name.
15. Once the pending plate record is highlighted, click on the plate graphic
that corresponds to the plate on the autosampler that contains your
amplified samples to link the plate to the plate record.
16. When the plate record is linked to the plate, the plate graphic will change
from yellow to green, the plate record moves from the pending plate
records table to the linked plate records table, and the Run Instrument
button becomes enabled.
17. Select “Run Instrument” on the toolbar to start the sample run.
18. Monitor electrophoresis by observing the run, status, array and capillary
views windows in the collection software. Each injection will take
approximately 45 minutes.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 15
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 16
V.C. Detection of Amplified Fragments Using the ABI PRISM® 310 Genetic Analyzer
Materials to Be Supplied by the User
• 95°C dry heating block, water bath or thermal cycler
• 310 capillaries, 47cm × 50µm
• performance optimized polymer 4 (POP-4™)
• glass syringe (1ml)
• 10X genetic analyzer buffer with EDTA
• sample tubes and septa
• aerosol-resistant pipette tips
• Hi-Di™ formamide (Applied Biosystems Cat.# 4311320)
• PowerPlex® Matrix Standards, 310 (Cat.# DG4640)
• crushed ice or ice-water bath
!
The quality of the formamide is critical. Use Hi-Di™ formamide with a
conductivity less than 100µS/cm. Freeze formamide in aliquots at –20°C. Multiple
freeze-thaw cycles or long-term storage at 4°C may cause a breakdown of the
formamide. Formamide with a conductivity greater than 100µS/cm may contain
ions that compete with DNA during injection. This results in lower peak heights
and reduced sensitivity. A longer injection time may not increase the signal.
!
Caution: Formamide is an irritant and a teratogen; avoid inhalation and contact
with skin. Read the warning label, and take appropriate precautions when
handling this substance. Always wear gloves and safety glasses when working
with formamide.
Sample Preparation
1.
Prepare a loading cocktail by combining the Internal Lane Standard 600
(ILS 600) and Hi-Di™ formamide as follows:
[(1.0µl ILS 600) × (# injections)] + [(24.0µl Hi-Di™ formamide) × (# injections)]
Note: The volume of internal lane standard used in the loading cocktail can
also be increased or decreased to adjust the intensity of the size standard
peaks. The optimal peak height for the 100-base fragment of the internal
lane standard is 500–1,000RFU. If the peak heights are too high, we
recommend altering the loading cocktail to contain 0.5µl of ILS 600 and
24.5µl of Hi-Di™ formamide.
2.
Mix for 10–15 seconds using a vortex mixer.
3.
Combine 25.0µl of the prepared loading cocktail and 1.0µl of amplified
sample.
Note: Instrument detection limits vary; therefore, injection time or the
amount of product mixed with loading cocktail may need to be increased
or decreased. If the peak heights are higher than desired, the samples can
be diluted in Gold ST★R 1X Buffer before mixing with loading cocktail.
This may result in uneven allele peak heights across loci. For best results,
use less template DNA in the amplification reactions or reduce the number
of cycles in the amplification program by 2–4 cycles (i.e., 10/18 or 10/20
cycling).
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 16
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 17
4.
Combine 25.0µl of the prepared loading cocktail and 1.0µl of the
PowerPlex® 16 Allelic Ladder Mix.
5.
Centrifuge the sample tubes briefly to remove air bubbles from the wells if
necessary.
6.
Denature the samples and ladder by heating at 95°C for 3 minutes, and
immediately chill on crushed ice or in an ice-water bath for 3 minutes.
Denature the samples just prior to loading.
7.
Assemble the tubes in the appropriate autosampler tray (48- or 96-tube).
8.
Place the autosampler tray in the instrument, and close the instrument
doors.
Instrument Preparation
Refer to the instrument users’ manual for instructions on cleaning the pump
block, installing the capillary, calibrating the autosampler and adding polymer
to the syringe.
1.
Open the ABI PRISM® 310 data collection software.
2.
Prepare a GeneScan® sample sheet as described in the ABI PRISM ® 310
Genetic Analyzer User’s Manual. Enter the appropriate sample information
in the “sample info” column.
For rows containing the PowerPlex® 16 Allelic Ladder Mix, insert the word
“ladder” in the “sample info” column for the blue dye color, yellow dye
color and green dye color. This information must be entered to successfully
analyze your data using the PowerTyper™ 16 Macro (Release 2.0).
3.
Create a new GeneScan® injection list. Select the appropriate sample sheet
by using the pull-down menu.
4.
Select the “GS STR POP4 (1ml) A” Module using the pull-down menu.
Change the injection time to 3 seconds and the run time to 30 minutes.
Keep the settings for the remaining parameters as shown below:
Inj. Secs:
Inj. kV:
Run kV:
Run °C:
Run Time:
!
3
15.0
15.0
60
30
You may need to optimize the injection time for individual instruments.
Injection times of 2–5 seconds are suggested for samples that contain 1ng
of template DNA.
Note: Migration of fragments may vary slightly over the course of a long
ABI PRISM® 310 Genetic Analyzer run. This may be due to changes in
temperature or changes in the column. When analyzing many samples,
injections of the allelic ladder at different times throughout the run can aid
in accurately genotyping the samples.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 17
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 18
V.C. Detection of Amplified Fragments Using the ABI PRISM® 310 Genetic Analyzer
(continued)
5.
Select the appropriate matrix file (Section III.B).
6.
To analyze the data automatically, select the auto analyze checkbox and the
appropriate analysis parameters and size standard. Refer to the ABI PRISM ®
310 Genetic Analyzer User’s Manual for specific information on these options.
7.
After loading the sample tray and closing the doors, select “Run” to start
the capillary electrophoresis system.
8.
Monitor the electrophoresis by observing the raw data and status
windows. Each sample will take approximately 40 minutes for syringe
pumping, sample injection and sample electrophoresis.
V.D. Detection of Amplified Fragments Using the ABI PRISM® 377 DNA
Sequencer
Materials to Be Supplied by the User
(Solution compositions are provided in Section IX.H.)
• Long Ranger ® gel solution (Cambrex Cat.# 50611) or Long Ranger Singel®
pack for ABI 377-36cm (Cambrex Cat.# 50691)
• 10% Ammonium Persulfate (Cat.# V3131)
• TEMED
• Urea (Cat.# V3171)
• TBE 10X buffer
• Nalgene® tissue culture filter (0.2 micron)
• 36cm front and rear glass plates
• 36cm gel spacers (0.2mm thick)
• 36-well sharkstooth comb or 34-well square-tooth comb (0.2mm thick)
• clamps (e.g., large office binder clamps)
• gel-loading pipette tips
• aerosol-resistant pipette tips (Section IX.I.)
• Liqui-Nox® or other detergent
• PowerPlex® Matrix Standards, 310 (Cat.# DG4640)
• Blue Dextran Loading Solution (Cat.# DV4351)
• crushed ice or ice-water bath
• 95°C dry heating block, water bath or thermal cycler
!
Caution: Acrylamide (Long Ranger® gel solution) is a neurotoxin and suspected
carcinogen; avoid inhalation and contact with skin. Read the warning label, and
take appropriate precautions when handling this substance. Always wear
gloves and safety glasses when working with acrylamide solutions.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 18
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 19
Polyacrylamide Gel Preparation
The following protocol is for preparation of a 36cm denaturing polyacrylamide
gel for use with the ABI PRISM® 377 DNA sequencer. Low-fluorescence glass
plates are recommended and may be obtained from the instrument manufacturer.
1.
Thoroughly clean the glass plates with hot water and a 1% Liqui-Nox®
solution. Rinse extremely well using deionized water. Allow the glass
plates to air-dry in a dust-free environment.
2.
Assemble the glass plates by placing 0.2mm side gel spacers between the
front and rear glass plates. Hold the plates together using binder clamps
(4 clamps on each side). Place the assembly horizontally on a test tube rack
or similar support.
3.
Prepare a 5% Long Ranger ® acrylamide gel (total of 50ml) by combining the
ingredients listed in Table 3. Stir the solution until the urea has dissolved.
Table 3. Preparation of a 5% Long Ranger ® Polyacrylamide Gel.
Component
5% Gel
Final Concentration
urea
18g
6M
deionized water
26ml
—
10X TBE
5ml
1X
50% Long Ranger ® gel solution
5ml
5%
total volume
50ml
Note: Long Ranger
Singel®
Packs may be used.
4.
Filter the acrylamide solution through a 0.2 micron filter (e.g., Nalgene®
tissue culture filter), and degas for 5 minutes.
5.
Add 35µl of TEMED and 250µl of fresh 10% ammonium persulfate to 50ml
of acrylamide solution, and mix gently.
6.
Using a disposable 60cc syringe, pour the gel by starting at the well end of
the plates and carefully injecting the acrylamide between the horizontal
glass plates. Allow the solution to fill the top width of the plates. While
maintaining a constant flow of solution, gently tap the glass plates to assist
the movement of solution to the bottom of the plates and to prevent the
formation of bubbles.
7.
Insert a 36-well sharkstooth comb or 34-well square-tooth comb between
the glass plates. Sharkstooth combs with 64 or 96 wells may also be used.
8.
Secure the comb with 3 evenly spaced clamps.
9.
Keep the remaining acrylamide solution as a polymerization control.
10. Allow polymerization to proceed for at least 2 hours. Check the
polymerization control to be sure that polymerization has occurred.
Note: The gel may be stored overnight if a paper towel saturated with
deionized water and plastic wrap are placed around the top and bottom to
prevent the gel from drying out (crystallization of the urea will destroy the
gel).
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 19
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 20
Instrument Preparation
1.
Open the ABI PRISM® 377 data collection software.
2.
Prepare a sample sheet as described in the GeneScan ® Analysis Software
User’s Manual. Enter the appropriate sample information in the “sample
info” column.
For lanes containing the PowerPlex® 16 Allelic Ladder Mix, insert the word
“ladder” in the “sample info” column for the blue dye color, yellow dye
color and green dye color. This information must be entered to successfully
analyze your data using the PowerTyper™ 16 Macro (Release 2.0).
3.
Create a new GeneScan® run, and use the following settings:
Plate Check Module:
PreRun Module:
Run Module:
Collect time:
Well-to-Read distance:
Plate Check A
PR GS 36A-2400
GS 36A-2400
3 hours
36cm
4.
Select the appropriate sample sheet and comb selection by using the pulldown menus.
5.
Select the appropriate gel matrix file (Section III.B).
Gel Pre-run
1.
Remove the clamps from the polymerized acrylamide gel. If necessary,
clean any excess acrylamide from the glass plates with paper towels
saturated with deionized water.
2.
Shave any excess polyacrylamide away from the comb, and remove the
comb. If using a sharkstooth comb, carefully insert the sharkstooth comb
teeth into the gel approximately 1–2mm.
3.
Position the gel/glass plate unit in the 377 cassette.
4.
Secure the cassette in the instrument, and perform a plate check as
recommended in the ABI PRISM® 377 DNA Sequencer User’s Manual. If the
horizontal line graph is not flat, remove the cassette, clean the plate surface
and repeat the plate check.
5.
Add TBE 1X buffer to the top and bottom buffer chambers of the instrument.
6.
Using a 60cc syringe filled with buffer, remove any air bubbles from the
well area of the gel, and place the lid on the upper buffer chamber. Using
a syringe fitted with a bent 18-gauge needle, remove any air bubbles from
the bottom of the gel.
7.
Attach the heating plate, connect the water tubing, attach all electrodes,
close the instrument door and select “PreRun”. Allow the gel to pre-run
for 15–20 minutes or until the gel temperature is at least 40°C. Open the
status window to monitor the temperature of the gel.
8.
Prepare the sample and allelic ladder samples during the gel pre-run.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 20
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 21
Sample Preparation and Loading
1.
Prepare a loading cocktail by combining and mixing the ILS 600 and Blue
Dextran Loading Solution as follows:
[(0.5µl ILS 600) × (# lanes)] + [(1.5µl Blue Dextran Loading Solution) × (# lanes)]
Note: The volume of internal lane standard used in the loading cocktail can
also be increased or decreased to adjust the intensity of the size standard
peaks.
2.
Vortex for 10–15 seconds.
3.
Combine 2.0µl of the prepared loading cocktail and 1.0µl of amplified
sample.
Note: Instrument detection limits vary; therefore, the amount of product
mixed with loading cocktail may need to be increased or decreased. If the
peak heights are higher than desired, the samples can be diluted in Gold
ST★R 1X Buffer before mixing with loading cocktail. This may result in
uneven allele peak heights across loci. For best results, use less template
DNA in the amplification reactions or reduce the number of cycles in the
amplification program by 2–4 cycles (i.e., 10/18 or 10/20 cycling).
4.
Combine 2.0µl of the prepared loading cocktail and 1.0µl of the PowerPlex®
16 Allelic Ladder Mix. Vortex the allelic ladder mix prior to pipetting.
5.
Briefly centrifuge the samples to bring the contents to the bottom of the
tubes.
6.
Just prior to loading the gel, denature the samples by heating at 95°C for
3 minutes, and immediately chill on crushed ice or in an ice-water bath.
Denature the samples just prior to loading the gel.
7.
After the 15- to 20-minute pre-run, pause the instrument by selecting
“Pause”. By pausing the pre-run, the water will continue to circulate,
keeping the gel warm during sample loading.
8.
Use a 60cc syringe filled with buffer and fitted with a bent 18-gauge needle
to flush the urea from the well area.
9.
Load 1.5µl of each denatured sample into the respective wells.
!
You may need to optimize the volume of sample loaded for individual
instruments. We recommend loading volumes of 1.0–2.0µl.
10. Place the lid on the upper buffer chamber, and close the instrument door.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 21
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 22
Gel Electrophoresis and Detection
1.
After loading, select “Cancel” to stop the pre-run. Make sure that the run
time is set at 3 hours, then select “Run” to begin electrophoresis.
2.
Monitor the electrophoresis by observing the gel image and status windows.
3.
Allow electrophoresis to proceed for 3 hours. The 600-base ILS fragment
will have migrated past the laser.
4.
Track and extract the gel lanes.
Reuse of Glass Plates
Separate the glass plates, and discard the gel. Clean the glass plates with hot
water and a detergent such as 1% Liqui-Nox® detergent. Rinse extremely well
with deionized water, and allow the plates to air-dry. Do not scrape the plates
with abrasive materials during this process.
Note: Soap and oil may build up on plates, resulting in gel extrusion or hazy
background. Soak the plates in 2N HCl for 15 minutes, then rinse thoroughly to
remove any buildup.
VI. Data Analysis
VI.A. PowerPlex® Panel and Bin Sets with GeneMapper ® ID, Version 3.2
To facilitate analysis of the data generated with the PowerPlex® 16 System, we
have created panel and bin files to allow automatic assignment of genotypes
using GeneMapper® ID software, version 3.2. We recommend that users of
GeneMapper ® ID software, version 3.2, complete the Applied Biosystems
GeneMapper ® ID Software Human Identification Analysis Tutorial to familiarize
themselves with the proper operation of the software. For GeneMapper® ID
software, version 3.1, users we recommend upgrading to version 3.2.
Getting Started
1.
Obtain the proper panel and bin files for use with GeneMapper® ID from
the Promega web site at: www.promega.com/geneticidtools/panels_bins/
2.
Enter your contact information, and select “GeneMapper ID version 3.2”.
Select “Submit”.
3.
Select the “PowerPlex® Panels & Bin Sets” link, and save the .zip file to
your computer.
4.
Open the files using the Windows® WinZip program, and save the
unzipped files to a known location on your computer.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 22
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:33 PM
Page 23
Importing Panel and Bin Files
These instructions loosely follow the Applied Biosystem GeneMapper® ID
software tutorial, pages 1–4.
1.
Open the GeneMapper® ID software, version 3.2.
2.
Select “Tools”, then “Panel Manager”.
3.
Highlight the Panel Manager icon in the upper left tile (navigation pane).
4.
Select “File”, then “Import Panels”.
5.
Navigate to the saved panel and bin files. Select
“Promega_Panels_ID3.2.X.txt”, where “X” refers to the most recent version
of the panel and bin files. Select “Import”.
6.
In the navigation pane, highlight the Promega_Panels_ID3.2.X folder that
you just imported.
7.
Select “File”, then “Import Bin Set”.
8.
Navigate to the saved panel and bin files. Select
“Promega_Bins_ID3.2.X.txt”, then “Import”.
9.
At the bottom of the Panel Manager window, select “Apply”, then “OK”.
The panel manager window will close automatically.
VI.B. Creating a Casework Analysis Method with GeneMapper ® ID Software
These instructions loosely follow the Applied Biosystem GeneMapper® ID
software tutorial, pages 1–11.
1.
Select “Tools”, then “GeneMapper Manager”.
2.
Select the Analysis Methods tab.
3.
Select “New”, and a new analysis method dialog box will open.
4.
Select “HID”, and select “OK”.
Note: If you do not see the HID option, you do not have the
GeneMapper® ID software. Contact Promega Technical Services by
e-mail: [email protected] for assistance.
5.
Enter a descriptive name for the analysis method, such as “PowerPlex16
advanced”.
6.
Select the Allele tab (Figure 2).
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 23
1/12/2007
12:33 PM
Page 24
5724TA
tmd012.0107.qxp
Figure 2. The Allele tab. Select the bin set “Promega_Bins_ID3.2.X.txt”, where “X” refers
to the most recent version of the bin set.
7.
Select the bin set corresponding to the PowerPlex System
“Promega_Bins_ID3.2.X”, where “X” refers to the most recent version of
the bin set.
8.
Ensure that the “Use marker-specific stutter ratio if available” box is
checked.
9.
Enter the values shown in Figure 2 for proper filtering of stutter peaks
when using the PowerPlex® 16 System. For an explanation of the proper
usage and effects of these settings, refer to the Applied Biosystems user
bulletin titled “Installation Procedures and New Features for GeneMapper ID
Software 3.2”.
Note: Some of these settings have been optimized and are different from
the recommended settings in the user bulletin.
10. Select the Peak Detector tab. We recommend the settings shown in Figure 3.
Note: Select full range or partial range for the analysis range. When using
a partial range, choose an appropriate analysis range based on the data.
Choose a start point after the primer peak and just before the first defined
internal lane standard peak to help ensure proper sizing of the internal
lane standard.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 24
Printed in USA.
Revised 1/07
1/12/2007
12:33 PM
Page 25
5723TA
tmd012.0107.qxp
Figure 3. The Peak Detector tab.
11. Select the Peak Quality tab. You may change the settings for peak quality.
Note: For Steps 11 and 12, see the GeneMapper® ID user’s manual for
more information.
12. Select the Quality Flags tab. You may also change these settings.
13. Select “OK” to save your settings.
Creating a Size Standard
1.
Select “Tools”, then “GeneMapper Manager”.
2.
Select the Size Standard tab.
3.
Select “New”.
4.
Select “Basic or Advanced” (Figure 4). The type of analysis method
selected must match the type of analysis method created earlier. Select “OK”.
5.
Enter a detailed name, such as “ILS 600 advanced”, in the “Size Standard
Editor” (Figure 5).
6.
Choose red as the color for the size standard dye.
7.
Enter the sizes of the internal lane standard fragments (see Section IX.E,
Figure 12).
8.
Select “OK”.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 25
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 26
5725TA
VI.B. Creating a Casework Analysis Method with GeneMapper ® ID Software
(continued)
5726TA
Figure 4. The Select Dye and Analysis Method window.
Figure 5. The Size Standard Editor.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 26
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 27
Processing Sample Data for Casework
1.
Import sample files into a new project as described in the Applied
Biosystems GeneMapper ® ID Software Human Identification Analysis Tutorial.
2.
In the “Sample Type” column, use the drop-down menu to select “Ladder”,
“Sample”, “Positive Control” or “Negative Control”. Every folder in the
project must contain at least one ladder that is designated as such for
proper genotyping.
3.
In the “Analysis Method” column, select the analysis method created
previously in the Creating a Casework Analysis Method section.
4.
In the “Panel” column, select “PowerPlex_16_ID3.2.X”, where “X” refers to
the most recent version of the panel files. This is the panel set that was
imported in Section VI.A.
5.
In the “Size Standard” column, select the size standard that was created in
the Creating a Size Standard section.
6.
If analyzing data from an ABI PRISM® 310 Genetic Analyzer or an
ABI PRISM® 377 DNA Sequencer, ensure that the appropriate matrix file is
selected in the “Matrix” column.
7.
Select “Analyze” (green arrow button) to start the data analysis.
VI.C. Creating a Databasing or Paternity Analysis Method with GeneMapper ® ID
Software
1.
Select “Tools”, then “GeneMapper Manager”.
2.
Select the Analysis Methods tab.
3.
Select “New”, and a new analysis method dialog box will open.
4.
Select “HID”, and select “OK”.
Note: If you do not see the HID option, you do not have the
GeneMapper® ID software. Contact Promega Technical Services by
e-mail: [email protected] for assistance.
5.
Enter a descriptive name for the analysis method, such as
“PowerPlex16_20%filter”.
6.
Select the Allele tab.
7.
Select the bin set corresponding to the PowerPlex® System
“Promega_Bins_ID3.2.X”, where “X” refers to the most recent version of
the bin set.
8.
Ensure that the “Use marker-specific stutter ratio if available” box is
checked.
9.
Enter the values shown in Figure 6 for proper filtering of peaks when using
the PowerPlex® 16 System. For an explanation of the proper usage and
effect of these settings, refer to the Applied Biosystems user bulletin titled
“Installation Procedures and New Features for GeneMapper ID Software 3.2”.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 27
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 28
5785TA
VI.C. Creating a Databasing or Paternity Analysis Method with GeneMapper ® ID
Software (continued)
Figure 6. The Allele tab with settings for using a 20% peak filter. Select the bin set
“Promega_Bins_ID3.2.X.txt”, where “X” refers to the most recent version of the bin set.
Creating a Size Standard
1.
Select “Tools”, then “GeneMapper Manager”.
2.
Select the Size Standard tab.
3.
Select “New”.
4.
Select “Basic or Advanced” (Figure 4). The type of analysis method
selected must match the type of analysis method created earlier. Select “OK”.
5.
Enter a detailed name, such as “ILS 600 advanced”, in the Size Standard
Editor (Figure 5).
6.
Choose red as the color for the size standard dye.
7.
Enter the sizes of the internal lane standard fragments (see Section IX.E,
Figure 12).
8.
Select “OK”.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 28
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 29
Processing Data for Databasing or Paternity Samples
1.
Import sample files into a new project as described in the Applied Biosystems
GeneMapper ® ID Software Human Identification Analysis Tutorial.
2.
In the “Sample Type” column, use the drop-down menu to select “Ladder”,
“Sample”, “Positive Control” or “Negative Control”. Every folder in the
project must contain at least one ladder that is designated as such for
proper genotyping.
3.
In the “Analysis Method” column, select the analysis method created in
the Creating a Databasing or Paternity Analysis Method section.
4.
In the “Panel” column, select “PowerPlex_16_ID3.2.X”, where “X” refers to
the most recent version of the panel files. This is the panel set that was
imported in Section VI.A.
5.
In the “Size Standard” column, select the size standard that was created in
the Creating a Size Standard section.
6.
If analyzing data from an ABI PRISM® 310 Genetic Analyzer or an
ABI PRISM® 377 DNA Sequencer, ensure that the appropriate matrix file is
selected in the “Matrix” column.
7.
Select “Analyze” (green arrow button) to start the data analysis.
VI.D. Sample Analysis Using the GeneScan® Software and PC Operating Systems
1.
Analyze the data using the GeneScan® analysis software.
2.
Review the raw data for one or more sample runs. Highlight the sample
file name, then under the “Sample” menu, select “raw data”. Move the
cursor so the crosshair is on the baseline to the right of the large primer
peak (before the first internal lane standard peak [red]). Use the X-value
number shown at the bottom left of the window for the start position in
the analysis parameters.
3.
The recommended analysis parameters are shown in Figure 7.
4.
The analysis parameters can be saved in the Params folder; in most
installations this is located at:
C:\AppliedBio\Shared\Analysis\Sizecaller\Params
5.
Apply the stored analysis parameters file to the samples.
6.
Assign a new size standard. Select a sample file, and highlight the arrow
next to size standard. Select “define new”. Assign the size standard peaks
as shown in Figure 12 in Section IX.E. Store the size standard in the Size
Standards folder at:
C:\AppliedBio\Shared\Analysis\Sizecaller\SizeStandards
7.
Apply the size standard file to the samples, then analyze the sample files.
See Section VI.F for additional information on the use of the PowerTyper™
16 Macro (Release 2.0) and Genotyper® software.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 29
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 30
Notes:
Peak heights outside the linear range of the instrument may generate
artifact peaks due to instrument saturation (i.e., overloading the sample).
Bleedthrough (pull-ups) from one color to another may be observed.
Saturated signal may also appear as two peaks (split peak).
2.
If the peak heights are not within the linear range of detection of the
instrument, the ratio of stutter peaks to real allele peaks increases, and
allele designations become difficult to interpret. The balance of the peak
heights may also appear less uniform.
3.
There may be variation between instruments regarding the relative
fluorescence levels detected using the same sample. Furthermore, different
instruments vary in the relative efficiency of color detection, affecting the
dye color-to-dye color balance.
5684TA
1.
Figure 7. The analysis parameters window. The start point of the analysis range, which
will vary, is defined in Section VI.D, Step 2.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 30
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 31
VI.E. Sample Analysis Using the GeneScan® Software and Macintosh® Operating
Systems
1.
Analyze the data using the GeneScan® analysis software.
2.
Review the raw data for one or more sample runs. Highlight the sample
file name, then under the “Sample” menu, select “raw data”. Move the
cursor so the crosshair is on the baseline to the right of the large primer
peak (before the first internal lane standard peak [red]). Use the X-value
number shown at the bottom left of the window for the start position in
the analysis parameters.
3.
The recommended analysis parameters are:
Analysis Range
Data Processing
Peak Detection
Size Call Range
Size Calling Method
Split Peak Correction
Start: Defined in Step 2
Stop: 10,000
Baseline: Checked
Multicomponent: Checked
Smooth Options: Light1
Peak Amplitude Thresholds2:
B:
Y:
G:
R:
Min. Peak Half Width: 2pts
Min: 60
Max: 600
Local Southern Method
None
1Smooth
options should be determined by individual laboratories. Occasionally the
TH01 alleles 9.3 and 10 will not be distinguished using heavy smoothing.
2The
peak amplitude thresholds are the minimum peak heights that the software
will call as a peak. Values for the peak amplitude thresholds are usually 50–200RFU
and should be determined by individual laboratories.
4.
The analysis parameters can be saved in the Params folder.
5.
Apply the stored analysis parameters file to the samples.
6.
Assign a new size standard. Select a sample file, highlight the arrow next
to size standard, then select “define new”. Assign the size standard peaks
as shown in Figure 12 in Section IX.E. Store the size standard in the Size
Standards folder.
7.
Apply the size standard file to the samples, then analyze the sample files.
See Section VI.F for additional information on the use of the
PowerTyper™ 16 Macro (Release 2.0) and Genotyper® software.
For additional information regarding the GeneScan® analysis software,
refer to the GeneScan® Analysis Software User’s Manual.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 31
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 32
Notes:
1.
Peak heights outside the linear range of the instrument may generate
artifact peaks due to instrument saturation (i.e., overloading the sample).
Bleedthrough (pull-ups) from one color to another may be observed.
Saturated signal may also appear as two peaks (split peak).
2.
If the peak heights are not within the linear range of detection of the
instrument, the ratio of stutter peaks to real allele peaks increases, and
allele designations become difficult to interpret. The balance of the peak
heights may also appear less uniform.
3.
There may be variation between instruments regarding the relative
fluorescence levels detected using the same sample. Furthermore, different
instruments vary in the relative efficiency of color detection, affecting the
dye color-to-dye color balance.
VI.F. Sample Analysis Using the Genotyper® Software and PowerTyper™ 16 Macro
To facilitate analysis of the data generated with the PowerPlex® 16 System, we
have created a file to allow automatic assignment of genotypes using the
Genotyper ® software. After samples are amplified, detected using the ABI PRISM®
310 or 3100 Genetic Analyzer (using data collection software, version 1.0.1 or 1.1)
or ABI PRISM® 377 DNA Sequencer, and analyzed using the GeneScan®
analysis software, the sample files can be imported into the Genotyper ®
program and analyzed using the PowerTyper™ 16 Macro (Release 2.0).
The PowerTyper™ 16 Macro (Release 2.0) is available upon request from
Promega. The PowerTyper™ 16 Macro (Release 2.0) is provided on the
PowerTyper™ Macros CD-ROM (Cat.# DG3470). The PowerTyper™ Macros
can also be downloaded from the Promega web site at:
www.promega.com/geneticidtools/
The PowerTyper™ 16 Macro (Release 2.0) is used in conjunction with Macintosh®
Genotyper ® software, version 2.5, and Windows NT® Genotyper® software,
version 3.6, or later. The Genotyper ® software must be installed on your
computer before the PowerTyper™ 16 Macro (Release 2.0) can be used.
Be certain the “sample info” (Macintosh® computers) or “color info”
(Windows NT® operating systems) column for each lane containing the allelic
ladder mix contains the word “ladder”. The macro uses the word “ladder” to
identify the sample file(s) containing allelic ladder. Sample info can be added or
modified after importing into the PowerTyper™ Macro. Highlight the sample,
then select “show dye/lanes window” under “Views”.
1.
Transfer the PowerTyper™ 16 Macro (Release 2.0) from the PowerTyper™
Macros CD-ROM (Cat.# DG3470) to a designated location on your
computer hard drive. Alternatively, download the PowerTyper™ 16 Macro
(Release 2.0) from the Promega web site.
2.
Open the Genotyper ® software, then the PowerTyper™ 16 Macro (Release
2.0). For questions about the Genotyper ® software, refer to the Genotyper ®
Analysis Software User’s Manual.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 32
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
3.
12:34 PM
Page 33
Under “File”, select “Import”, and import the GeneScan® project or sample
files to be analyzed. Import the blue, yellow, green and red dye colors.
Note: To select the dye colors to be imported, select “Set Preferences”
under the “Edit” menu.
4.
Double-click on the Check ILS macro. The macros are listed at the bottom
left corner of the active window. A plots window will be displayed to
show the internal lane standard (i.e., ILS 600) in the red dye color. Scroll
down to view, and confirm that the internal lane standard fragment sizes
are correct. If necessary, re-analyze samples using the GeneScan® software
and redefine the internal lane standard fragments.
Note: The software uses one ladder sample for determining allele sizes.
The macro uses the first ladder sample imported for allele designations.
5.
For casework, double-click on the POWER macro. The POWER macro
identifies the alleles in the ladder sample and calculates offsets for all of
the loci. This process may take several minutes. When completed, a plots
window will open to display the allelic ladders (i.e., Penta E, D18S51,
D21S11, TH01 and D3S1358).
Alternatively, for databasing or paternity, double-click on the POWER 20%
Filter macro. This macro has a higher level of filtering than the standard
POWER macro to reduce the need for manual editing of peak labels. The
POWER 20% Filter should not be used if mixtures may exist.
In general, the allelic ladders contain fragments of the same lengths as
many known alleles for the locus. The allelic ladder sizes and repeat units
are listed in Table 5 (Section IX.B). Analysis using GeneScan® analysis
software and Genotyper ® software allows allele determination by
comparing amplified sample fragments with allelic ladders and internal
lane standards. When using an internal lane standard, the calculated
lengths of the allelic ladder components may differ from those listed in
the table. This is due to differences in migration resulting from sequence
differences between the allelic ladder fragments and the internal size
standard and is not a matter of concern.
6.
Double-click on the Allelic Ladders macro. A plots window will open to
display the blue (fluorescein) dye allelic ladders (i.e., Penta E, D18S51,
D21S11, TH01 and D3S1358), the green (JOE) dye allelic ladders (i.e., Penta E,
CSF1PO, D16S539, D7S820, D13S317 and D5S818) and the yellow (TMR)
dye allelic ladders (i.e., FGA, TPOX, D8S1179, vWA and Amelogenin).
Confirm that the correct allele designations were assigned to the allelic
ladders (Figure 10 in Section VI.H).
Note: The software uses one ladder sample for determining the allele sizes.
The macro uses the first ladder sample imported for allele designations. If
the POWER macro is run a second time, the software will use the second
ladder; if the POWER macro is run a third time, the software will use the
third ladder, etc., until all ladders in the project are used. If an allelic ladder
fails to be analyzed or if many off-ladder alleles are found in the samples,
the samples should be re-analyzed using another ladder from the project.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 33
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 34
VI.F. Sample Analysis Using the Genotyper® Software and PowerTyper™ 16 Macro
(continued)
7.
Double-click on the Display Fluorescein Data macro to display the blue
dye for all the sample injections/lanes. Scroll down to observe and edit as
needed.
8.
Double-click on the Display TMR Data macro to display the yellow dye
for all sample injections/lanes. Scroll down to observe and edit as needed.
9.
Double-click on the Display JOE Data macro to display the green dye for
all sample injections/lanes. Scroll down to observe and edit as needed.
10. Create the appropriate table by selecting the “PowerTable”, “Make Allele
Table” or “Make CODIS Table” macro. The three available table formats
are shown below. The PowerTable option allows up to four alleles per
sample file. Additional information such as low peak signal or high peak
signal is also included. The Allele Table and CODIS Table options include
only two alleles per locus. If more than two alleles are present at a locus,
the smallest alleles identified are included. The Allele Table format
displays the categories (loci) in columns, while the CODIS table format
displays the categories in rows. These tables can be customized to fit
needs. To save data in tables, go to the Table drop-down menu, highlight
“Export to File...” and save the file with the desired name and location.
The saved file can be viewed and analyzed using Microsoft® Excel.
PowerTable Format
Sample Sample
Peak Peak Peak Peak Over- Low Satura- Edited Edited
Info
Comment Category 1
2
3
4
flow Signal tion
Label Row
Allele Table Format
Sample Category Category Category Category Category Category Category Category
Info
Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2
CODIS Table Format
Sample Info Category Peak 1
Peak 2
11. Save the analyzed data, go to the “File” menu and select “Save as”.
!
The PowerTyper™ Macro is a Genotyper ® file and can be overwritten if
“Save” is used instead of “Save as”.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 34
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 35
VI.G. Controls
1.
Observe the results for the negative control. The negative control should
be devoid of amplification products.
2.
Observe the results for the 9947A positive control DNA. Compare the
control DNA allelic repeat sizes with the locus-specific allelic ladder. The
expected 9947A DNA allele designations for each locus are listed in Table 6
(Section IX.B).
VI.H. Results
Representative results of the PowerPlex® 16 System are shown in Figure 8. The
PowerPlex® 16 Allelic Ladder Mix is shown in Figure 9.
A.
B.
C.
5683TA
D.
Figure 8. The PowerPlex® 16 System. A single template DNA (1.0ng) was amplified using the
PowerPlex® 16 10X Primer Pair Mix. The amplification products were mixed with the Internal Lane
Standard 600 and analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV, 5-second
injection. The results were analyzed using GeneMapper ® ID software, version 3.2. Panel A. An
electropherogram showing the peaks of the fluorescein-labeled loci: D3S1358, TH01, D21S11, D18S51
and Penta E. Panel B. An electropherogram showing the peaks of the JOE-labeled loci: D5S818,
D13S317, D7S820, D16S539, CSF1PO and Penta D. Panel C. An electropherogram showing the peaks
of the TMR-labeled loci: Amelogenin, vWA, D8S1179, TPOX and FGA. Panel D. An electropherogram
showing the 60bp to 500bp fragments of the Internal Lane Standard 600.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 35
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 36
A.
B.
5682TA
C.
Figure 9. The PowerPlex® 16 Allelic Ladder Mix. The PowerPlex® 16 Allelic Ladder Mix was analyzed
with an Applied Biosystems 3130 Genetic Analyzer using a 3kV, 5-second injection. The sample file
was analyzed with the GeneMapper ® ID software, version 3.2, and the PowerPlex® 16 panel and bin
files. Panel A. The fluorescein-labeled allelic ladder components and their allele designations. Panel B.
The JOE-labeled allelic ladder components and their allele designations. Panel C. The TMR-labeled
allelic ladder components and their allele designations.
Artifacts and Stutter
Stutter bands are a common amplification artifact associated with STR analysis.
Stutter products are often observed one repeat unit below the true allele peak
and, occasionally, two repeat units smaller or one repeat unit larger than the
true allele peak. Frequently, alleles with a greater number of repeat units will
exhibit a higher percent stutter. The pattern and intensity of stutter may differ
slightly between primer sets for the same loci. Stutter was determined and
published as part of the validation of PowerPlex® 16 System (9).
In addition to stutter peaks, other artifact peaks can be observed at some of the
PowerPlex® 16 System loci. Low-level products can be seen in the n–2 and n+2
positions (two bases below and above the true allele peak, respectively) with
some loci such as D21S11. Samples may show low-level artifacts in the noncalling
regions between the D7S820 and D13S317 allele ranges and between the
D3S1358 and TH01 allele ranges. Occasionally an off-ladder artifact can be seen
in the 270–271bp position in the JOE dye channel. One or more extra peaks that
are not directly related to amplification may be observed at positions 8–26 bases
smaller than TPOX alleles and 6–21 bases smaller than vWA alleles. These extra
peaks occur when the amplified peaks are particularly intense (high signal level
or template amount), formamide, polymer or capillary was of poor quality or
denaturation was ineffective. Please see Section VII for more information about
how to minimize these artifacts.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 36
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 37
VII. Troubleshooting
For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact
information available at: www.promega.com. E-mail: [email protected]
VII.A. Amplification and Fragment Detection
Symptoms
Faint or absent allele peaks
Causes and Comments
Impure template DNA. Because of the small amount of
template used, this is rarely a problem. Depending on the DNA
extraction procedure used and the sample source, inhibitors
may be present in the DNA sample.
Insufficient template. Use the recommended amount of
template DNA.
Insufficient template. Low-copy-number (LCN) analysis using
capillary electrophoresis may benefit from reducing competing
charged particles during injection. This can be accomplished
with post-PCR cleanup or desalting, lower-conductivity
formamide or reduced amounts of ILS 600. In-house validation
should be performed for any of these methods.
Insufficient enzyme activity. Use the recommended amount of
AmpliTaq Gold® DNA polymerase. Check the expiration date
on the tube label.
Incorrect amplification program. Confirm the amplification
program.
High salt concentration or altered pH. If the DNA template is
stored in TE buffer that is not pH 8.0 or contains a higher EDTA
concentration, the DNA volume should not exceed 20% of the
total reaction volume. Carryover of K+, Na+, Mg2+ or EDTA
from the DNA sample can negatively affect PCR. A change in
pH may also affect PCR. Store DNA in TE–4 buffer (10mM Tris
HCl [pH 8.0], 0.1mM EDTA) or nuclease-free water.
Thermal cycler, plate or tube problems. Review the thermal
cycling protocols in Section IV.B. We have not tested other
reaction tubes, plates or thermal cyclers. Calibrate the thermal
cycler heating block, if necessary.
Primer concentration was too low. Use the recommended
primer concentration. Mix the 10X PowerPlex® 16 Primer Pair
for 15 seconds using a vortex mixer before use.
Poor capillary electrophoresis injection (ILS 600 peaks also
affected). Re-inject the sample. Check the syringe for leakage.
Check the laser power.
Poor-quality formamide was used. Use only Hi-Di™ formamide
when running samples.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 37
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 38
VII.A. Amplification and Fragment Detection (continued)
Symptoms
Extra peaks visible in one or all
of the color channels
Causes and Comments
Contamination with another template DNA or previously
amplified DNA. Cross-contamination can be a problem. Use
aerosol-resistant pipette tips, and change gloves regularly.
Samples were not completely denatured. Heat denature the
samples for the recommended time, and cool on crushed ice
or in an ice-water bath immediately prior to loading the gel or
capillary.
Artifacts of STR amplification. PCR amplification of STR
systems sometimes generates artifacts that appear as faint
peaks one repeat unit smaller than the allele. Stutter band
peak heights can be high if the samples are overloaded.
Artifacts of STR amplification. PCR amplification of STR
systems can result in artifacts that appear as peaks one base
smaller than the allele due to incomplete addition of the 3´ A
residue. Be sure to perform the 30-minute extension step at
60°C after thermal cycling (Section IV.B).
High background. Load less amplification product or decrease
the injection time. See Section V.
Capillary electrophoresis (CE)-related artifacts (“spikes”).
Minor voltage changes or urea crystals passing by the laser can
cause “spikes” or unexpected peaks. Spikes sometimes appear
in one color but often are easily identified by their presence in
more than one color. Re-inject the samples to confirm.
CE-related artifacts (contaminants). Contaminants in the water
used with the instrument or to dilute the 10X genetic analyzer
buffer may generate peaks in the blue and green dye colors.
Use autoclaved water, change vials and wash buffer reservoir.
Excessive amount of DNA. Amplification of >2ng template can
result in a higher number of stutter bands. Use less template
DNA, or reduce the number of cycles in the amplification
program by 2–4 cycles (10/20 or 10/18 cycling).
Pull-up or bleedthrough. Pull-up can occur when peak heights
are too high or if a poor or incorrect matrix has been applied
to the samples.
• For the ABI PRISM® 310 Genetic Analyzer and 377 DNA
Sequencer, generate a new matrix, and apply it to the
samples. For the ABI PRISM® 3100 and 3100-Avant Genetic
Analyzers and Applied Biosystems 3130 and 3130xl
Genetic Analyzers, perform a new spectral calibration and
re-run the samples.
• Sensitivities of instruments may vary. Optimize the
injection or gel loading conditions. See Section V.
Long-term storage of amplified sample in formamide can
result in degradation. Repeat preparation of samples using
fresh formamide.
The CE polymer was beyond its expiration date, or polymer
was stored at room temperature for more than one week.
Maintain instrumentation on a daily or weekly basis, as
recommended by the manufacturer.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 38
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 39
VII.A. Amplification and Fragment Detection (continued)
Symptoms
Allelic ladder not running the
the same as the sample
Peak height imbalance
Causes and Comments
Allelic ladder and primer pair mix were not compatible. Ensure
that the allelic ladder is from the same kit as the primer pair
mix.
Buffer incompatibility. Samples were diluted in the wrong
buffer. Use Gold ST★R 1X Buffer to dilute samples.
Poor-quality formamide. Use only Hi-Di™ formamide when
analyzing samples.
Be sure the allelic ladder and samples are from the same
instrument run.
Migration of samples changed slightly over the course of a
CE run with many samples. This may be due to changes in
temperature or the CE column over time. Use a different
injection of the allelic ladder to determine sizes.
Poor injection of allelic ladder. Include more than one ladder
per instrument run.
Excessive amount of DNA. Amplification of >1ng of template
can result in an imbalance with yields with smaller loci
showing more product than the larger loci. Use less template,
or reduce the number of cycles in the amplification program
by 2–4 cycles (10/20 or 10/18 cycling) to improve locus-tolocus balance.
Note: Dilution of overamplified samples can result in dropout
of larger loci.
Use of FTA® paper. Results may be similar to those obtained
with excess amounts of DNA template. Reduce the number of
cycles in the amplification program by 2–4 cycles (10/20 or
10/18 cycling) to improve locus-to-locus balance.
Degraded DNA sample. DNA template is degraded, and the
larger loci show diminished yield. Repurify the template DNA.
Insufficient template DNA. Use the recommended amount of
template DNA. Stochastic effects can occur when amplifying
low amounts of template.
Miscellaneous balance problems. Thaw the 10X Primer Pair
Mix and Gold ST★R 10X Buffer completely, and vortex for
15 seconds before using. Do not centrifuge the 10X Primer
Pair Mix after mixing. Calibrate thermal cyclers and pipettes
routinely. Using a 59°C annealing temperature instead of 60°C
has been shown to improve balance in some instances.
Impure template DNA. Inhibitors that may be present in
forensic samples can lead to allele dropout or imbalance.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 39
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 40
VII.B. GeneMapper® ID Analysis Software
Symptoms
Alleles not called
Off-ladder alleles
Size standard not called
correctly (Figure 11)
Peaks in size standard missing
Causes and Comments
To analyze samples with GeneMapper ® ID software, the
analysis parameters and size standard must both have “Basic
or Advanced” as the analysis type. If they are different, an
error is obtained (Figure 10).
An insufficient number of ILS 600 fragments was defined. Be
sure to define at least one ILS 600 fragment smaller than the
smallest sample peak and at least one ILS 600 fragment larger
than the largest sample peak.
Run was too short, and larger peaks in ILS were not captured.
Not all of the ILS 600 peaks defined in the size standard were
detected during the run.
• Create a new size standard using the internal lane standard
fragments present in the sample.
• Re-run the samples using a longer run time.
An allelic ladder from a different run than the samples was
used. Re-analyze the samples with an allelic ladder from the
same run.
The GeneMapper® ID software requires that the allelic ladder
be imported from the same folder as the sample. Be sure that
the allelic ladder is in the same folder as the sample. Create a
new project and re-analyze, as described in Section VI.B or VI.C.
Panel file selected for analysis was incorrect for the STR system
used. Assign correct panel file that corresponds to the STR
system used for amplification.
The allelic ladder was not identified as an allelic ladder in the
sample type column.
The wrong analysis type was chosen for the analysis method.
Be sure to use the HID analysis type.
The internal lane standard was not properly identified in the
sample. Manually redefine the sizes of the size standard
fragments in the sample.
Starting data point was incorrect for the partial range chosen
in Section VI.B. Adjust the starting data point in the analysis
method. Alternatively, use a full range for the analysis.
Extra peaks in advanced mode size standard. Open the size
match editor. Highlight the extra peak, select “Edit” and select
“delete size label”. Select “auto adjust sizes”.
Run was too short, and larger peaks in ILS were not captured.
Not all of the ILS 600 peaks defined in the size standard were
detected during the run.
• Create a new size standard using the internal lane standard
fragments present in the sample.
• Re-run the samples using a longer run time.
If peaks are below threshold, decrease the peak threshold in
the analysis method for the red channel to include peaks.
If peaks are low-quality, redefine the size standard for the
sample to skip these peaks.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 40
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 41
VII.B. GeneMapper® ID Analysis Software (continued)
Symptoms
Error message:
“Either panel, size standard,
or analysis method is invalid”
No alleles called, but no error
message appears
Error message:
“Both the Bin Set used in the
Analysis Method and the Panel
must belong to the same
Chemistry Kit”.
Significantly raised baseline
Red bar appears during analysis
of samples, and the following
error message appears when data
are displayed: “Some selected
sample(s) do not contain analysis
data. Those sample(s) will not be
shown”.
Error message after attempting
to import panel and bin files:
“Unable to save panel data:
java.SQLEException:
ORA-00001: unique constraint
(IFA.CKP_NNN) violated”.
Causes and Comments
The size standard and analysis method were not in the same
mode (“Classic” vs. “Basic or Advanced”). Be sure both files
are set to the same mode, either Classic or Basic or Advanced
mode.
Panel was not selected for sample. In “Panel” column, select
the appropriate panel set for the STR system that was used.
No size standard was selected. In the size standards column,
be sure to select the appropriate size standard.
Size standard was not correctly defined or size peaks were
missing. Redefine size standard to include only peaks present
in your sample. Terminating analysis early or using short run
times will cause larger ladder peaks to be missing. This will
cause your sizing quality to be flagged as “red”, and no allele
sizes will be called.
The bin set assigned to the analysis method may have been
deleted. In the GeneMapper ® Manager, select the Analysis
Methods tab, and open the analysis method of interest. Select
the Alleles tab, and select an appropriate bin set.
The wrong bin set was chosen in the analysis method Allele
tab. Be sure to choose the appropriate bin set, as shown in
Figure 2.
• Poor spectral calibration for the ABI PRISM® 3100 and 3100Avant Genetic Analyzers and Applied Biosystems 3130 and
3130xl Genetic Analyzers. Perform a new spectral
calibration and re-run the samples.
• Poor matrix for the ABI PRISM® 310 Genetic Analyzer.
Re-run and optimize the matrix.
Use of Classic mode analysis method. Use of Classic mode
analysis on samples can result in baselines with more noise
than those analyzed using the Basic or Advanced mode
analysis method. Advanced mode analysis methods and size
standards are recommended.
If none of the samples had matrices applied when run on the
ABI PRISM® 310 Genetic Analyzer, no data will be displayed.
Apply a matrix file during analysis in the GeneMapper ® ID
software and re-analyze.
There was a conflict between different sets of panel and bin
files. Delete all panel and bin sets, and re-import files in a
different order.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 41
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 42
VII.B. GeneMapper® ID Analysis Software (continued)
Symptoms
Peaks in the allelic ladder are
labeled off-ladder
Causes and Comments
GeneMapper ® ID software was not used or microsatellite
analysis settings were used instead of HID analysis settings.
GeneMapper ® software does not use the same algorithms as
GeneMapper ® ID software and cannot correct for sizing
differences using the allelic ladder. Promega recommends
using GeneMapper ® ID software to analyze PowerPlex®
reactions. If using GeneMapper ® ID software, version 3.1 or 3.2,
be sure that the analysis method selected is an HID method.
This can be verified by opening the analysis method using the
GeneMapper ® Manager, then selecting the General tab. The
analysis type cannot be changed. If the method is not HID, it
should be deleted and a new analysis method created.
VII.C. PowerTyper™ 16 Macro
Symptoms
File does not open
on your computer
Error message:
“Could not complete the
“Run Macro” command because
no dye/lanes are selected”
Error message:
“Could not complete the
“Run Macro” command
because the labeled peak
could not be found”
Causes and Comments
Genotyper ® software was not installed. Be certain that the
Genotyper ® software, version 2.5 (Macintosh®) or version 3.6
or higher (Windows NT®), is installed.
Incorrect version of Genotyper ® software. The PowerTyper™
16 Macro will not work with Genotyper ® software versions
prior to version 2.5.
The CD-ROM may have been damaged during shipment.
Contact Technical Services by e-mail: [email protected]
The file was corrupted during download or transfer. Download
the file again, or obtain the file on CD-ROM.
Allelic ladder sample files were not identified. Be certain the
“sample info” or “color info” column for each lane containing
the PowerPlex® 16 Allelic Ladder Mix contains the word
“ladder”. The macro uses the word “ladder” to identify the
sample files containing the allelic ladder.
All four dye colors were not imported. For Genotyper ®
software, versions 2.5 and 3.5 or higher, set the preferences
(under “Edit”) to import the blue, green, yellow and red colors.
The peak heights for one or more of the alleles in the allelic
ladder sample file were below 150RFU. The allelic ladder
categories are defined as having a minimum peak height of
150RFU. If the peak heights of the ladder alleles are below
150RFU, the software will not be able to locate the allele peak.
Re-run the allelic ladder using more sample or longer injection
time to assure peak heights above 150RFU.
CE spikes in the allelic ladder sample were identified as alleles
by the macro. Use a different injection of the allelic ladder.
TH01 9.3 and 10 alleles were not separated when using heavy
smoothing in the GeneScan® analysis parameters. Use light
smoothing in the GeneScan® analysis parameters.
Allelic ladder data was not compatible with the PowerTyper™
file used. Confirm that the PowerTyper™ Macro file matches
the allelic ladder being used.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 42
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 43
VII.C. PowerTyper™ 16 Macro (continued)
Symptoms
Error message:
“Could not complete the
“Run Macro” command
because the labeled peak
could not be found”
(continued)
The plots window or allele
table does not display all data
The Check ILS macro
displays an empty plot
window
Off-ladder peaks
Causes and Comments
The base-pair size of the alleles in the allelic ladder are outside
of the defined category range. Be sure the internal lane
standard fragments are correctly sized. Redefine the internal
lane standard fragments, and re-analyze the sample using
GeneScan® software. Compare the size of the smallest allele in
the allelic ladder with the base-pair size and range listed in the
categories for the same alleles. If necessary, increase the
category start range (in the category window) to greater than
± 6bp, and save the macro under a new name.
Allelic ladder peaks were too high, causing stutter peaks to be
called as allele peaks. Use a shorter injection time, decrease
the amount of allelic ladder used or re-analyze the allelic
ladder sample using increased peak amplitude thresholds in
the GeneScan® analysis parameters.
The macros were not run in the proper order. Use the POWER
or POWER 20% Filter macro option.
All four dye colors were not imported. For Genotyper ®
software, versions 2.5 and 3.5 or higher, set the preferences
(under “Edit”) to import the blue, green, yellow and red colors.
All four dye colors were not imported. For Genotyper ®
software, versions 2.5 and 3.5 or higher, set the preferences
(under “Edit”) to import the blue, green, yellow and red colors.
Migration of samples changed slightly over the course of a
CE run with many samples. This may be due to changes in
temperature or the CE column over time. Use a different
injection of the allelic ladder to determine sizes in the
PowerTyper™ 16 Macro (Release 2.0). Do not use the first
injection on a new column for the ladder sample.
The base-pair size of the alleles was incorrect because incorrect
fragment sizes were assigned to the internal lane standard.
Confirm that the internal lane standard fragment sizes are
assigned correctly. Re-analyze the sample using GeneScan®
software, and redefine the internal lane standard fragments.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 43
1/12/2007
12:34 PM
Page 44
5685TA
tmd012.0107.qxp
5686TA
Figure 10. The error message that appears in the GeneMapper® ID software when the
analysis parameters and the size standard have different analysis types.
Figure 11. An example showing improper assignment of size standard fragments in the
GeneMapper® ID software.
VIII. References
1.
Edwards, A. et al. (1991) DNA typing with trimeric and tetrameric tandem repeats: Polymorphic loci,
detection systems, and population genetics. In: The Second International Symposium on Human
Identification 1991, Promega Corporation, 31–52.
2.
Edwards, A. et al. (1991) DNA typing and genetic mapping with trimeric and tetrameric tandem
repeats. Am. J. Hum. Genet. 49, 746–56.
3.
Edwards, A. et al. (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four
human population groups. Genomics 12, 241–53.
4.
Warne, D. et al. (1991) Tetranucleotide repeat polymorphism at the human β-actin related pseudogene 2
(actbp2) detected using the polymerase chain reaction. Nucleic Acids Res. 19, 6980.
5.
Ausubel, F.M. et al. (1996) Unit 15: The polymerase chain reaction. In: Current Protocols in Molecular
Biology, Vol. 2, John Wiley and Sons, NY.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 44
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 45
6.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Chapter 14: In vitro amplification of DNA by the
polymerase chain reaction. In: Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York.
7.
PCR Technology: Principles and Applications for DNA Amplification (1989) Erlich, H.A., ed., Stockton
Press, New York, NY.
8.
PCR Protocols: A Guide to Methods and Applications (1990) Innis, M.A. et al., eds., Academic Press, San
Diego, CA.
9.
Krenke, B. et al. (2002) Validation of a 16-locus fluorescent multiplex system. J. Forensic Sci. 47, 773–85.
10.
Budowle, B. et al. (2001) STR primer concordance study. Forensic Sci. Int. 124, 47–54.
11.
Presley, L.A. et al. (1992) The implementation of the polymerase chain reaction (PCR) HLA DQ alpha
typing by the FBI laboratory. In: The Third International Symposium on Human Identification 1992,
Promega Corporation, 245–69.
12.
Hartmann, J.M. et al. (1991) Guidelines for a quality assurance program for DNA analysis. Crime
Laboratory Digest 18, 44–75.
13.
Budowle, B. et al. (1991) Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution
PAGE. Am. J. Hum. Genet. 48, 137–44.
14.
Nakamura, Y. et al. (1987) Variable number of tandem repeat (VNTR) markers for human gene
mapping. Science 235, 1616–22.
15.
Budowle, B. and Monson, K.L. (1989) In: Proceedings of an International Symposium on the Forensic
Aspects of DNA Analysis, Government Printing Office, Washington, DC.
16.
Levinson, G. and Gutman, G.A. (1987) Slipped-strand mispairing: A major mechanism for DNA
sequence evolution. Mol. Biol. Evol. 4, 203–21.
17.
Schlotterer, C. and Tautz, D. (1992) Slippage synthesis of simple sequence DNA. Nucleic. Acids Res. 20,
211–5.
18.
Smith, J.R. et al. (1995) Approach to genotyping errors caused by nontemplated nucleotide addition by
Taq DNA polymerase. Genome Res. 5, 312–7.
19.
Magnuson, V.L. et al. (1996) Substrate nucleotide-determined non-templated addition of adenine by
Taq DNA polymerase: Implications for PCR-based genotyping. BioTechniques 21, 700–9.
20.
Walsh, P.S., Fildes, N.J. and Reynolds, R. (1996) Sequence analysis and characterization of stutter
products at the tetranucleotide repeat locus vWA. Nucleic. Acids Res. 24, 2807–12.
21.
Moller, A., Meyer, E. and Brinkmann, B. (1994) Different types of structural variation in STRs:
HumFES/FPS, HumVWA and HumD21S11. Int. J. Leg. Med. 106, 319–23.
22.
Brinkmann, B., Moller A. and Wiegand, P. (1995) Structure of new mutations in 2 STR systems.
Int. J. Leg. Med. 107, 201–3.
23.
Griffiths, R. et al. (1998) New reference allelic ladders to improve allelic designation in a multiplex STR
system. Int. J. Legal Med. 111, 267–72.
24.
Bär, W. et al. (1997) DNA recommendations: Further report of the DNA Commission of the ISFH
regarding the use of short tandem repeat systems. Int. J. Legal Med. 110, 175–6.
25.
Gill, P. et al. (1997) Considerations from the European DNA Profiling Group (EDNAP) concerning STR
nomenclature. Forensic Sci. Int. 87, 185–92.
26.
Levadokou, E.N. et al. (2001) Allele frequencies for fourteen STR loci of the PowerPlex® 1.1 and 2.1
multiplex systems and Penta D locus in Caucasians, African-Americans, Hispanics, and other
populations of the United States of America and Brazil. J. Forensic Sci. 46, 736–61.
27.
Lins, A.M. et al. (1998) Development and population study of an eight-locus short tandem repeat (STR)
multiplex system. J. Forensic Sci. 43, 1168–80.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 45
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 46
VIII. References (continued)
28.
Puers, C. et al. (1993) Identification of repeat sequence heterogeneity at the polymorphic STR locus
HUMTH01[AATG]n and reassignment of alleles in population analysis using a locus-specific allelic
ladder. Am. J. Hum. Genet. 53, 953–8.
29.
Hammond, H. et al. (1994) Evaluation of 13 short tandem repeat loci for use in personal identification
applications. Am. J. Hum. Genet. 55, 175–89.
30.
Bever, R.A. and Creacy, S. (1995) Validation and utilization of commercially available STR multiplexes
for parentage analysis. In: Proceedings from the Fifth International Symposium on Human Identification
1994. Promega Corporation, 61–8.
31.
Sprecher, C.J. et al. (1996) General approach to analysis of polymorphic short tandem repeat loci.
BioTechniques 20, 266–76.
32.
Lins, A.M. et al. (1996) Multiplex sets for the amplification of polymorphic short tandem repeat loci—
silver stain and fluorescent detection. BioTechniques 20, 882–9.
33.
Jones, D.A. (1972) Blood samples: Probability of discrimination. J. Forensic Sci. Soc. 12, 355–9.
34.
Brenner, C. and Morris, J.W. (1990) In: Proceedings from the International Symposium on Human
Identification 1989, Promega Corporation, 21–53.
35.
Mandrekar, P.V., Krenke, B.E. and Tereba, A. (2001) DNA IQ™: The intelligent way to purify DNA.
Profiles in DNA 4(3), 16.
36.
Mandrekar, M.N. et al. (2001) Development of a human DNA quantitation system. Profiles in DNA
4(3), 9–12.
37.
Greenspoon, S. and Ban, J. (2002) Robotic extraction of sexual assault samples using the Biomek® 2000
and the DNA IQ™ System. Profiles in DNA 5(1), 3–5.
Additional STR references can be found at: www.promega.com/geneticidentity/
IX. Appendix
IX.A. Advantages of STR Typing
STR typing is more tolerant of degraded DNA templates than other typing
methods because the amplification products are less than 500bp long, much
smaller than the material detected using AMP-FLP (13) or VNTR (14) analysis.
STR typing is also amenable to a variety of rapid DNA purification techniques,
which are compatible with PCR but do not provide enough DNA of appropriate
quality for Southern blot-based analyses.
Amplification products generated with Promega STR products are generally of
discrete and separable lengths. This allows the construction of allelic ladders
containing fragments of the same lengths as several or all known alleles for
each locus. Visual or software-based comparison between the allelic ladder and
amplified samples of the same locus allows rapid and precise assignment of
alleles. Results obtained using the PowerPlex® 16 System can be recorded in a
digitized format, allowing direct comparison with stored databases. Population
analyses do not require the use of arbitrarily defined fixed bins for population
data (15).
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 46
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 47
IX.B. Advantages of Using the Loci in the PowerPlex® 16 System
The loci included in the PowerPlex® 16 System (Tables 4 and 5) have been
selected because they satisfy the needs of several major standardization bodies
throughout the world. For example, the United States Federal Bureau of
Investigation (FBI) has selected 13 STR core loci for typing prior to searching or
including (submitting) samples in CODIS (Combined DNA Index System), the
U.S. national database of convicted offender profiles. The PowerPlex® 16 System
amplifies all CODIS core loci in a single reaction.
The PowerPlex® 16 System also contains two low-stutter, highly polymorphic
pentanucleotide repeat loci, Penta E and Penta D. These additional loci add
significantly to the discrimination power of the system, making the PowerPlex®
16 System a single-amplification system with a power of exclusion sufficient to
resolve paternity disputes definitively. In addition, the extremely low level of
stutter seen with Penta E and Penta D makes them ideal loci for evaluation of
DNA mixtures often encountered in forensic casework. Finally, the Amelogenin
locus is included in the PowerPlex® 16 System to allow gender identification of
each sample. Table 6 lists the PowerPlex® 16 System alleles revealed in
commonly available standard DNA templates.
We have carefully selected STR loci and primers to avoid or minimize artifacts,
including those associated with Taq DNA polymerase, such as repeat slippage
and terminal nucleotide addition. Repeat slippage (16,17), sometimes called
“n–4 bands”, “stutter” or “shadow bands”, is due to the loss of a repeat unit
during DNA amplification, somatic variation within the DNA in sample
material, or both. The amount of this artifact observed depends primarily on the
locus and the DNA sequence being amplified.
Terminal nucleotide addition (18,19) occurs when Taq DNA polymerase adds a
nucleotide, generally adenine, to the 3´ ends of amplified DNA fragments in a
template-independent manner. The efficiency with which this occurs varies with
different primer sequences. Thus, an artifact band one base shorter than expected
(i.e., missing the terminal addition) is sometimes seen. We have modified primer
sequences and added a final extension step of 60°C for 30 minutes (20) to the
amplification protocols to provide conditions for essentially complete terminal
nucleotide addition when recommended amounts of template DNA are used.
The presence of microvariant alleles (alleles differing from one another by
lengths other than the repeat length) complicates interpretation and assignment
of alleles. There appears to be a correlation between a high degree of
polymorphism, a tendency for microvariants and increased mutation rate
(21,22). Thus, FGA and D21S11 display numerous, relatively common
microvariants. For reasons yet unknown, the highly polymorphic Penta E locus
does not display frequent microvariants (Table 5).
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 47
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 48
Table 4. The PowerPlex® 16 System Locus-Specific Information.
STR Locus
Penta E
Label
FL
Chromosomal Location
15q
GenBank® Locus and
Locus Definition
NA
Repeat Sequence1
→ 3´
5´→
AAAGA
HUMUT574
AGAA (23)
D18S51
FL
18q21.3
D21S11
FL
21q11–21q21
HUMD21LOC
TCTA Complex (23)
TH01
FL
11p15.5
AATG (23)
D3S1358
FL
3p
HUMTH01, human tyrosine
hydroxylase gene
NA
FGA
TMR
4q28
TPOX
TMR
2p23–2pter
D8S1179
TMR
8q
vWA
TMR
12p12–pter
Amelogenin2
TMR
Xp22.1–22.3 and Y
Penta D
JOE
CSF1PO
JOE
D16S539
JOE
D7S820
D13S317
D5S818
TCTA Complex
HUMFIBRA, human
TTTC
fibrinogen alpha chain gene
Complex (23)
HUMTPOX, human thyroid
AATG
peroxidase gene
NA
TCTA Complex (23)
HUMVWFA31, human von
Willebrand factor gene
HUMAMEL, human Y
chromosomal gene for
Amelogenin-like protein
TCTA
Complex (23)
NA
21q
NA
AAAGA
5q33.3–34
HUMCSF1PO, human c-fms
proto-oncogene for CSF-1
receptor gene
AGAT
16q24–qter
NA
GATA
JOE
7q11.21–22
NA
GATA
JOE
13q22–q31
NA
TATC
JOE
5q23.3–32
NA
AGAT
1The
August 1997 report (24,25) of the DNA Commission of the International Society for Forensic
Haemogenetics (ISFH) states, “1) for STR loci within coding genes, the coding strand shall be used
and the repeat sequence motif defined using the first possible 5´ nucleotide of a repeat motif; and
2) for STR loci not associated with a coding gene, the first database entry or original literature
description shall be used”.
2Amelogenin
is not an STR but displays a 106-base, X-specific band and a 112-base, Y-specific band.
9947A DNA (female) displays only the 106-base, X-specific band.
TMR = carboxy-tetramethylrhodamine
FL = fluorescein
JOE = 6-carboxy-4´,5´-dichloro-2´,7´-dimethoxyfluorescein
NA = not applicable
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 48
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 49
Table 5. The PowerPlex® 16 System Allelic Ladder Information.
STR Locus
Penta E
Label
FL
Size Range of Allelic
Ladder Components1,2
(bases)
379–474
Repeat Numbers of Allelic
Ladder Components
5–24
D18S51
FL
290–366
8–10, 10.2, 11–13, 13.2, 14–27
D21S11
FL
203–259
24, 24.2, 25, 25.2, 26–28, 28.2,
29, 29.2, 30, 30.2, 31, 31.2, 32,
32.2, 33, 33.2, 34, 34.2, 35,
35.2, 36–38
TH01
FL
156–195
4–9, 9.3, 10–11, 13.3
D3S1358
FL
115–147
12–20
FGA
TMR
322–444
16–18, 18.2, 19, 19.2, 20, 20.2,
21, 21.2, 22, 22.2, 23, 23.2, 24,
24.2, 25, 25.2, 26–30, 31.2, 43.2,
44.2, 45.2, 46.2
TPOX
TMR
262–290
6–13
D8S1179
TMR
203–247
7–18
vWA
TMR
123–171
10–22
Amelogenin5
TMR
106, 112
X, Y
Penta D
JOE
376–449
2.2, 3.2, 5, 7–17
CSF1PO
JOE
321–357
6–15
D16S539
JOE
264–304
5, 8–15
D7S820
JOE
215–247
6–14
D13S317
JOE
176–208
7–15
D5S818
JOE
119–155
7–16
1The
Repeat Numbers of
Alleles Not Present
in Allelic Ladder 3,4
20.3
length of each allele in the allelic ladder has been confirmed by sequence analyses.
2When
using an internal lane standard, such as the Internal Lane Standard 600, the calculated sizes of
allelic ladder components may differ from those listed. This occurs because different sequences in
allelic ladder and ILS components may cause differences in migration. The dye label also affects
migration of alleles.
3The
alleles listed are those with a frequency of >1/1000.
4For
a current list of microvariants, see the Variant Allele Report published at the U.S. National
Institute of Standards and Technology (NIST) web site at: www.cstl.nist.gov/div831/strbase/
5Amelogenin
is not an STR but displays a 106-base, X-specific band and a 112-base, Y-specific band.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 49
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 50
Table 6. The PowerPlex® 16 System Allele Determinations in Commonly Available
Standard DNA Templates.
Standard DNA Templates1
STR Locus
Penta E
K5622
14, 5
9947A
13, 12
99483
11, 11
D18S51
16, 15
19, 15
18, 15
D21S11
31, 30, 29
30, 30
30, 29
TH01
9.3, 9.3
9.3, 8
9.3, 6
D3S1358
16, 16
15, 14
17, 15
FGA
24, 21
24, 23
26, 24
TPOX
9, 8
8, 8
9, 8
D8S1179
12, 12
13, 13
13, 12
vWA
16, 16
18, 17
17, 17
Amelogenin
X, X
X, X
X, Y
Penta D
13, 9
12, 12
12, 8
CSF1PO
10, 9
12, 10
12, 11, 10
D16S539
12, 11
12, 11
11, 11
D7S820
11, 9
11, 10
11, 11
D13S317
8, 8
11, 11
11, 11
D5S818
12, 11
11, 11
13, 11
1Information
on strains 9947A, 9948 and K562 is available online at: locus.umdnj.edu/nigms/
Strain K562 is available from the American Type Culture Collection: www.atcc.org (Manassas, VA).
2Strain
K562 displays three alleles at the D21S11 locus.
3Strain
9948 displays three alleles at the CSF1PO locus. The peak height for allele 12 is much lower
than those for alleles 10 and 11.
IX.C. Power of Discrimination
The fifteen STR loci amplified with the PowerPlex® 16 System provide powerful
discrimination. Population statistics for these loci and their various multiplex
combinations are displayed in Tables 7–9. These data were developed as part of
a collaboration (26) with The Bode Technology Group (Springfield, VA), North
Carolina Bureau of Investigation (Raleigh, NC), Palm Beach County Sheriff’s
Office (West Palm Beach, FL), Virginia Division of Forensic Science (Richmond,
VA) and Charlotte/Mecklenburg Police Department Laboratory (NC).
Generation of these data included analysis of over 200 individuals from
African-American, Caucasian-American and Hispanic-American populations.
Data for Asian-Americans includes analysis of over 150 individuals. For
additional population data for STR loci, see references 27–32.
Table 7 shows the matching probability (33) for the PowerPlex® 1.2 and 16
Systems in various populations. The matching probability of the PowerPlex® 16
System ranges from 1 in 1.83 × 1017 for Caucasian-Americans to 1 in 1.41 × 1018
for African-Americans.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 50
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 51
A measure of discrimination often used in paternity analyses is the paternity
index (PI), a means for presenting the genetic odds in favor of paternity given
the genotypes for the mother, child and alleged father (34). The typical paternity
indices for the PowerPlex® 1.2 and 16 Systems are shown in Table 8. The
PowerPlex® 16 System provides typical paternity indices exceeding 500,000 in
each population group. An alternative calculation used in paternity analyses is
the power of exclusion (34). This value, calculated for the PowerPlex® 16 System,
exceeds 0.999998 in all populations tested (Table 9).
Table 7. Matching Probabilities of the PowerPlex® 1.2 and 16 Systems in Various
Populations.
Matching Probability
STR System
African-American Caucasian-American Hispanic-American Asian-American
®
PowerPlex 1.2
System (8 STR loci)
1 in 2.77 × 108
1 in 1.15 × 108
1 in 1.45 × 108
1 in 1.32 × 108
®
PowerPlex 16
System (15 STR loci) 1 in 1.41 × 1018
1 in 1.83 × 1017
1 in 2.93 × 1017
1 in 3.74 × 1017
Table 8. Typical Paternity Indices of the PowerPlex® 1.2 and 16 Systems in Various
Populations.
Typical Paternity Index
STR System
PowerPlex® 1.2
System
PowerPlex® 16
System
African-American Caucasian-American Hispanic-American Asian-American
497
262
318
471
2,510,000
1,520,000
522,000
4,110,000
Table 9. Power of Exclusion of the PowerPlex® 1.2 and 16 Systems in Various
Populations.
Power of Exclusion
STR System
PowerPlex® 1.2
System
PowerPlex® 16
System
African-American Caucasian-American Hispanic-American Asian-American
0.9982042
0.9968863
0.9973367
0.9981793
0.9999996
0.9999994
0.9999983
0.9999998
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 51
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 52
IX.D. DNA Extraction and Quantitation Methods
The DNA IQ™ System (Cat.# DC6700) is a DNA isolation and quantitation
system designed specifically for forensic and paternity samples (35). This novel
system uses paramagnetic particles to prepare clean samples for STR analysis
easily and efficiently and can be used to extract DNA from stains or liquid
samples, such as blood or solutions. The DNA IQ™ Resin eliminates PCR
inhibitors and contaminants frequently encountered in casework samples. With
larger samples, the DNA IQ™ System delivers a consistent amount of total
DNA. The system has been used to isolate and quantify DNA from routine
sample types including buccal swabs, stains on FTA® paper and liquid blood.
Additionally, DNA has been isolated from casework samples such as tissue,
differentially separated sexual assault samples and stains on support materials.
The DNA IQ™ System has been tested with the PowerPlex® Systems to ensure
a streamlined process. See Section IX.I for ordering information.
For applications requiring human-specific DNA quantification, the AluQuant®
Human DNA Quantitation System (Cat.# DC1010) has been developed (36). See
Section IX.I for ordering information.
The DNA IQ™ System and AluQuant® Human DNA Quantitation System have
been fully automated on the Beckman Coulter Biomek® 2000 Laboratory
Automation Workstation (37). For information about automation of laboratory
processes on Beckman Coulter or other workstations, contact your local
Promega Branch Office or Distributor (contact information available at:
www.promega.com/worldwide/) or e-mail: [email protected]
IX.E. The Internal Lane Standard 600
The Internal Lane Standard (ILS) 600 contains 22 DNA fragments of 60, 80, 100,
120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
550 and 600 bases in length (Figure 12). Each fragment is labeled with carboxyX-rhodamine (CXR) and may be detected separately (as a fourth color) in the
presence of PowerPlex® 16-amplified material. The ILS 600 is designed for use
in each gel lane or capillary electrophoresis (CE) injection to increase precision
in analyses when using the PowerPlex® 16 System. Protocols for preparation
and use of this internal lane standard are provided in Section V.
1,200
100
200
400
300
600
500
1,000
800
600
60 80
120 140 160 180
225 250 275
325 350 375
425 450 475
550
400
5751TA
200
0
Figure 12. Internal Lane Standard 600. An electropherogram showing the fragments of
the Internal Lane Standard 600.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 52
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 53
IX.F. Preparing the PowerPlex® 16 System Master Mix
A worksheet to calculate the required amount of each component of the PCR
master mix is provided in Table 10. Multiply the volume (µl) per reaction by the
total number of reactions to obtain the final master mix volume (µl).
Table 10. Master Mix for the PowerPlex® 16 System.
PCR Master Mix Component
Gold ST★R 10X Buffer
PowerPlex® 16 10X
Primer Pair Mix
AmpliTaq Gold®
DNA polymerase1
nuclease-free
water 2
Per tube
template DNA volume2
(0.25–1ng)
total reaction volume
Volume Per
Reaction
×
2.5µl
×
=
2.5µl
×
=
0.8µl (4u)
×
=
×
=
×
=
up to 19.2µl
×
=
25µl
×
=
µl
Number of
Reactions
=
Final Volume
(µl)
1Assumes
the AmpliTaq Gold® DNA polymerase is at 5u/µl. If the enzyme concentration
is different, the volume of enzyme must be adjusted accordingly.
2The
master mix volume and template DNA volume should total 25µl. Consider the
volume of template DNA, and add nuclease-free water to the master mix to bring the
final volume of the final reaction to 25µl.
IX.G. Agarose Gel Electrophoresis of Amplification Products (Optional)
This procedure is optional if PCR is routinely performed in your laboratory.
Agarose gel electrophoresis can be used to rapidly confirm the success of the
amplification reaction prior to performing polyacrylamide gel or capillary
electrophoresis.
Materials to Be Supplied by the User
(Solution compositions are provided in Section IX.H.)
• TAE 1X buffer
• agarose
• 5X loading solution
• ethidium bromide solution, 0.5µg/ml
1.
Prepare a 2% agarose gel (approximately 150cm2) by adding 2.0g of agarose
to 100ml of TAE 1X buffer. Mark the liquid level on the container, then
boil or heat in a microwave oven to dissolve the agarose. Add preheated
(60°C) deionized water to make up for any volume lost due to evaporation.
2.
Cool the agarose to 55°C before pouring into the gel tray. Be sure that the
gel tray is level. Pour the agarose into the tray, insert the gel comb and
allow to set for 20–30 minutes.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 53
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 54
3.
Prepare the samples by mixing 10µl of each amplified sample with 2.5µl of
5X loading solution.
4.
Prepare 1 liter of TAE 1X buffer for the electrophoresis running buffer.
5.
Place the gel and tray in the electrophoresis gel box. Pour enough running
buffer into the tank to cover the gel to a depth of at least 0.65cm. Gently
remove the comb.
6.
Load each sample mixed with 5X loading solution (prepared in Step 3).
7.
Set the voltage at 5 volts/cm (measured as the distance between the two
electrodes). Allow the gel to run for 2 hours.
8.
After electrophoresis, stain the gel in TAE 1X buffer containing 0.5µg/ml
ethidium bromide. Gently rock for 20 minutes at room temperature.
Remove the ethidium bromide solution, and replace with deionized water.
Allow the gel to destain for 20 minutes.
9.
Photograph the gel using a UV transilluminator (302nm).
Note: When analyzing the data, do not be alarmed by extra bands in
addition to the alleles. DNA heteroduplexes can be expected when
performing nondenaturing agarose gel electrophoresis. The sole purpose
of the agarose gel is to confirm the success of the PCR reaction.
IX.H. Composition of Buffers and Solutions
10% ammonium persulfate
Add 0.05g of ammonium persulfate
to 500µl of deionized water.
Blue Dextran Loading Solution
88.25%
15mg/ml
4.1mM
formamide
blue dextran
EDTA (pH 8.0)
ethidium bromide solution
(10mg/ml)
1.0g
ethidium bromide
Dissolve ethidium bromide in 100ml
of deionized water. Wrap in
aluminum foil or transfer the solution
to a dark bottle, and store at room
temperature.
!
★R 10X Buffer
Gold ST★
500mM
100mM
15mM
1%
2mM
1.6mg/ml
KCl
Tris-HCl
(pH 8.3 at 25°C)
MgCl2
Triton® X-100
each dNTP
BSA
TAE 50X buffer (pH 7.2)
242g
57.1ml
100ml
Tris base
glacial acetic acid
0.5M EDTA stock
Add the Tris base and EDTA stock to
500ml of deionized water. Add the
glacial acetic acid. Bring the volume
to 1 liter with deionized water.
Caution: Ethidium bromide is a
powerful mutagen. Wear gloves
when working with the dye, and
wear a mask when weighing it.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 54
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 55
IX.H. Composition of Buffers and Solutions (continued)
TBE 10X buffer
107.8g
7.44g
~55.0g
Tris base
EDTA
(Na2EDTA • 2H2O)
boric acid
Dissolve Tris base and EDTA in
800ml of deionized water. Slowly
add the boric acid, and monitor the
pH until the desired pH of 8.3 is
obtained. Bring the final volume to
1 liter with deionized water.
TE-4 buffer (10mM Tris-HCl, 0.1mM
EDTA [pH 8.0])
2.21g
0.037g
Tris base
EDTA
(Na2EDTA • 2H2O)
Dissolve Tris base and EDTA in
900ml of deionized water. Adjust to
pH 8.0 with HCl. Bring the final
volume to 1 liter with deionized
water.
IX.I. Related Products
Fluorescent STR Multiplex Systems
Product
PowerPlex® 16 Monoplex System, Penta E
(Fluorescein)
PowerPlex® 16 Monoplex System, Penta D (JOE)
PowerPlex® ES Monoplex System, SE33 (JOE)
PowerPlex® 1.2 System
PowerPlex® 16 BIO System
PowerPlex® ES System
PowerPlex® Y System
Size
Cat.#
100 reactions
100 reactions
100 reactions
100 reactions
100 reactions
400 reactions
100 reactions
400 reactions
50 reactions
200 reactions
DC6591
DC6651
DC6751
DC6101
DC6541
DC6540
DC6731
DC6730
DC6761
DC6760
Size
50µl (each dye)
25µl (each dye)
1 CD-ROM
150µl
1.2ml
12ml
50ml (2 × 25ml)
Cat.#
DG4640
DG4650
DG3470
DG2611
DM2411
DY1151
P1193
Not for Medical Diagnostic Use.
Accessory Components
Product
PowerPlex® Matrix Standards, 310*
PowerPlex® Matrix Standards, 3100/3130*
PowerTyper™ Macros*
Internal Lane Standard 600**
Gold ST★R 10X Buffer**
Mineral Oil
Nuclease-Free Water**
*Not for Medical Diagnostic Use.
**For Laboratory Use.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 55
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 56
Sample Preparation Systems
Product
DNA IQ™ System**
Size
100 reactions
400 reactions
50 samples
200 samples
Cat.#
DC6701
DC6700
DC6801
DC6800
80 determinations
400 determinations
10 pack
DC1010
DC1011
V1391
Size
25g
1L
1kg
3ml
Cat.#
V3131
V4251
V3171
DV4351
Size (tips/pack)
960
960
960
960
960
960
960
800
Cat.#
DY1051
DY1061
DY1071
DY1081
DY1101
DY1111
DY1121
DY1131
Differex™ System*
AluQuant® Human DNA Quantitation
System*
Slicprep™ 96 Device**
*Not for Medical Diagnostic Use.
**For Laboratory Use.
Polyacrylamide Gel Electrophoresis Reagents
Product
Ammonium Persulfate
TBE Buffer, 10X
Urea
Blue Dextran Loading Solution*
*For Laboratory Use.
ART® Aerosol-Resistant Tips
Product
ART® 10 Ultramicro Pipet Tip
ART® 20E Ultramicro Pipet Tip
ART® 20P Pipet Tip
ART® GEL Gel Loading Pipet Tip
ART® 100 Pipet Tip
ART® 100E Pipet Tip
ART® 200 Pipet Tip
ART® 1000E Pipet Tip
Volume
0.5–10µl
0.5–10µl
20µl
100µl
100µl
100µl
200µl
1,000µl
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TMD012
Page 56
Printed in USA.
Revised 1/07
tmd012.0107.qxp
1/12/2007
12:34 PM
Page 57
(a)STR loci are the subject of U.S. Pat. No. RE 37,984, German Pat. No. DE 38 34 636 C2 and other
patents issued to the Max-Planck-Gesellschaft zur Förderung der Wissenschaften, e.V.,
Germany. The development and use of STR loci are covered by U.S. Pat. No. 5,364,759,
Australian Pat. No. 670231 and other pending patents assigned to Baylor College of Medicine,
Houston, Texas.
Patents for the foundational PCR process, European Pat. Nos. 201,184 and 200,362, expired on
March 28, 2006. In the U.S., the patents covering the foundational PCR process expired on
March 29, 2005.
(b)U.S.
Pat. Nos. 6,238,863 and 6,767,703 have been issued to Promega Corporation for materials
and methods for identifying and analyzing intermediate tandem repeat DNA markers. Other
patents are pending.
(c)U.S. Pat. Nos. 5,843,660, 6,479,235, 6,221,598 and 7,008,771, Australian Pat. No. 724531,
Canadian Pat. No. 2,118,048, Korean Pat. No. 290332, Singapore Pat. No. 57050 and Japanese
Pat. No. 3602142 have been issued to Promega Corporation for multiplex amplification of STR
loci. Other patents are pending.
(d)The purchase of this product does not convey a license to use AmpliTaq Gold® DNA
polymerase. You should purchase AmpliTaq Gold® DNA polymerase licensed for the forensic
and human identity field directly from your authorized enzyme supplier.
© 2000–2007 Promega Corporation. All Rights Reserved.
AluQuant and PowerPlex are registered trademarks of Promega Corporation. Differex, DNA IQ,
PowerTyper and Slicprep are trademarks of Promega Corporation.
ABI PRISM, GeneMapper, GeneScan, Genotyper and MicroAmp are registered trademarks of
Applera Corporation. AmpliTaq Gold and GeneAmp are registered trademarks of Roche
Molecular Systems, Inc. ART is a registered trademark of Molecular Bio-Products, Inc. Biomek
is a registered trademark of Beckman Coulter, Inc. FTA is a registered trademark of Flinders
Technologies, Pty, Ltd., and is licensed to Whatman. GenBank is a registered trademark of the
U.S. Dept. of Health and Human Services. Hi-Di and POP-4 are trademarks of Applera
Corporation. Liqui-Nox is a registered trademark of Alconox, Inc. Long Ranger and Long Ranger
Singel are registered trademarks of Cambrex Corporation. Macintosh is a registered trademark
of Apple Computer, Inc. Microsoft, Windows and Windows NT are registered trademarks of
Microsoft Corporation. Nalgene is a registered trademark of Nalge Nunc International. Triton
is a registered trademark of Union Carbide Chemicals and Plastics Technology Corporation.
Products may be covered by pending or issued patents or may have certain limitations. Please
visit our Web site for more information.
All prices and specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the
Promega online catalog for the most up-to-date information on Promega products.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/07
Part# TMD012
Page 57
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement