PowerPlex® 2.1 System

PowerPlex® 2.1 System
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Technical Manual
PowerPlex® 2.1 System
INSTRUCTIONS FOR USE OF PRODUCTS DC6470 AND DC6471
PRINTED IN USA
Revised 7/08
Part# TMD011
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PowerPlex® 2.1 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]
1.
Description..................................................................................................................................2
2.
Product Components and Storage Conditions ....................................................................3
3.
Before You Begin .......................................................................................................................4
4.
Protocols for DNA Amplification Using the PowerPlex® 2.1 System ............................5
A. Amplification Setup.........................................................................................................5
B. Amplification Thermal Cycling .....................................................................................7
5.
Detection of Amplified Fragments Using the
Hitachi FMBIO® and FMBIO® II Fluorescence Imaging Systems ..................................9
A. Polyacrylamide Gel Preparation....................................................................................9
B. Gel Pre-Run.....................................................................................................................11
C. Sample Preparation and Loading................................................................................12
D. Gel Electrophoresis ........................................................................................................13
E. Detection..........................................................................................................................13
F.
Reuse of Glass Plates .....................................................................................................14
6.
Data Analysis ...........................................................................................................................15
A
Controls ...........................................................................................................................15
B. Allelic Ladders................................................................................................................15
C. Results..............................................................................................................................16
7.
Troubleshooting.......................................................................................................................20
8.
References .................................................................................................................................22
9.
Appendix ...................................................................................................................................25
A. Advantages of STR Typing...........................................................................................25
B. Advantages of Using the Loci in the PowerPlex® 2.1 System.................................25
C. Fluorescent STR Products .............................................................................................28
D. Power of Discrimination ...............................................................................................29
E. Methods for Polyacrylamide Gel Reuse .....................................................................31
F.
DNA Extraction and Quantitation Methods..............................................................33
G. The Internal Lane Standard 600...................................................................................33
H. Agarose Gel Electrophoresis of Amplification Products (Optional)......................34
I.
Composition of Buffers and Solutions........................................................................35
J.
Related Products ............................................................................................................36
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 7/08
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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® 2.1 System(a–d) allows co-amplification and three-color detection of
nine STR loci. The PowerPlex® 2.1 System contains the loci Penta E, D18S51, D21S11,
TH01, D3S1358, FGA, TPOX, D8S1179 and vWA. One of the two primers for Penta E,
D18S51, D21S11, TH01 and D3S1358 is labeled with fluorescein (FL) and one primer
specific for FGA, TPOX, D8S1179 and vWA is labeled with carboxytetramethylrhodamine (TMR). All nine loci are amplified simultaneously in a single
tube and analyzed in a single gel lane.
The PowerPlex® 16 Monoplex System, Penta E (Fluorescein) (Cat.# DC6591) is
available to amplify the Penta E locus. This 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 also can
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 quality.
The PowerPlex® 2.1 System is customized for use with the Hitachi FMBIO® and
FMBIO® II Fluorescence Imaging Systems.
The PowerPlex® 2.1 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® 2.1
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. Protocols for operation of the fluorescencedetection instruments should be obtained from the instrument manufacturer.
Information on other Promega fluorescent STR systems is available upon request
from Promega or online at: www.promega.com. For information about detecting
amplified STR fragments using silver staining (9) refer to the GenePrint® STR
Systems Technical Manual #TMD004.
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# TMD011
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Product Components and Storage Conditions
Product
PowerPlex® 2.1 System
Size
100 reactions
Cat.#
DC6471
Not For Medical Diagnostic Use. Cat.# DC6471 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® 2.1 10X Primer Pair Mix
3μg
K562 DNA High Molecular Weight (10ng/μl)
Postamplification Components Box (Beige Label)
1 × 150μl
PowerPlex® 2.1 Allelic Ladder Mix
1× 150μl
Internal Lane Standard (ILS) 600
1 × 1ml
Bromophenol Blue Loading Solution
250μl
Gel Tracking Dye
1
Protocol
Product
PowerPlex® 2.1 System
Size
400 reactions
Cat.#
DC6470
Not For Medical Diagnostic Use. Cat.# DC6470 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® 2.1 10X Primer Pair Mix
3μg
K562 DNA High Molecular Weight (10ng/μl)
Postamplification Components Box (Beige Label)
4 × 150μl
PowerPlex® 2.1 Allelic Ladder Mix
4× 150μl
Internal Lane Standard (ILS) 600
2 × 1ml
Bromophenol Blue Loading Solution
250μl
Gel Tracking Dye
1
Protocol
The PowerPlex® 2.1 Allelic Ladder Mix is provided in a separate, sealed bag for
shipping. This component should be moved to the postamplification box after opening.
Storage Conditions: Store all components at –20°C in a nonfrost-free freezer. The
PowerPlex® 2.1 10X Primer Pair Mix, PowerPlex® 2.1 Allelic Ladder Mix and Internal
Lane Standard 600 are light-sensitive and must be stored in the dark. The
postamplification components are packaged separately to prevent crosscontamination. We strongly recommend that pre-amplification and postamplification
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
Printed in USA.
Revised 7/08
Part# TMD011
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Product Components and Storage Conditions (continued)
Available Separately
Product
Internal Lane Standard 600
Size
150μl
Cat.#
DG1701
For Laboratory Use.
Additional fluorescent STR multiplex product information and ordering information
for accessory components and related products is provided in Section 9.J and is
available on the Internet at: www.promega.com or upon request from Promega.
3.
Before You Begin
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
(10,11).
The quality of the purified DNA, as well as small changes in buffers, ionic strength,
primer concentrations, choice of thermal cycler and thermal cycling conditions, can
affect PCR success. 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, PowerPlex® 2.1 10X
Primer Pair Mix and K562 DNA High Molecular Weight) should be stored separately
from those used following amplification (PowerPlex® 2.1 Allelic Ladder Mix, Internal
Lane Standard 600, Bromophenol Blue Loading Solution and Gel Tracking Dye).
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 9.J).
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.
Table 1. Hazardous Reagents.
Reagent
Hazard
acrylamide
ammonium persulfate
bisacrylamide
formamide (contained in the Bromophenol
Blue Loading Solution and Gel Tracking Dye)
TEMED
urea
suspected carcinogen, toxic
oxidizer, corrosive
toxic, irritant
irritant, teratogen
corrosive, flammable
irritant
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
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Protocols for DNA Amplification Using the PowerPlex® 2.1 System
Materials to Be Supplied by the User
• model 480 or GeneAmp® PCR System 9600, 9700 or 2400 thermal cycler
(Applied Biosystems)
• microcentrifuge
• 0.5ml GeneAmp® or 0.2ml thin-walled MicroAmp® reaction 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 (Section 9.J)
• 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 1–2ng of template DNA in a 25μl reaction volume using the
protocols detailed below. Preferential amplification of smaller loci can occur. Expect
to see more intense bands for smaller loci and relatively less intense bands for 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.
Primer concentrations are optimized for use with the GeneAmp® PCR systems 9600
thermal cycler and AmpliTaq Gold® DNA polymerase as described in the
amplification protocol provided below. Thermal cycling conditions are also provided
for the GeneAmp® PCR system 9700 and 2400 and Perkin-Elmer model 480 thermal
cyclers. Use of AmpliTaq Gold® DNA polymerase is always recommended.
DNA template of 5ng or greater results in an imbalance in more intense bands from
locus to locus. The smaller loci show greater amplification yield than larger loci.
Reduce the number of cycles in the amplification program by 2 or 4 cycles (i.e.,
10/18 or 10/16 cycling) to improve locus-to-locus balance.
4.A. Amplification Setup
The use of gloves and aerosol-resistant pipette tips is highly recommended to
prevent cross-contamination. Keep all pre-amplification and postamplification
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 7.
1.
Thaw the Gold ST★R 10X Buffer and PowerPlex® 2.1 10X Primer Pair Mix.
Notes:
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.
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 7/08
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4.A. Amplification Setup (continued)
2.
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.
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® reaction tubes. For the Perkin-Elmer model
480 thermal cycler, we recommend 0.5ml GeneAmp® thin-walled reaction
tubes.
4.
In the order listed, add the final volume of each reagent listed in Table 2
into a sterile, 1.5ml amber-colored tube. Mix gently.
Table 2. PCR Master Mix for the PowerPlex® 2.1 System.
PCR Master Mix Component1
nuclease-free water
Gold ST★R 10X Buffer
PowerPlex®
Volume Per
Number of
Final
×
=
Reactions
Volume
Reaction
17.05μl
2.5μl
2.1 10X Primer Pair Mix
2.5μl
AmpliTaq Gold® DNA polymerase2
0.45μl (2.25u)
total volume
22.5μl
1Add
nuclease-free water to the PCR master mix first, then add Gold ST★R 10X
Buffer, PowerPlex® 2.1 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.
Note: If the volume of AmpliTaq Gold® DNA polymerase added to the
master mix is less than 0.5μl, you may wish to dilute the enzyme with 1X
Gold ST★R Buffer first and add a larger volume. The amount of NucleaseFree Water in the reaction should be adjusted accordingly so that the final
volume of master mix per reaction is 22.5μl. Do not store diluted
AmpliTaq Gold® DNA polymerase.
5.
Pipet 22.5μl of PCR master mix into each reaction tube, and place at room
temperature.
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
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Pipet 2.5μl of template DNA (1–2ng) for each sample into the respective
tube containing 22.5μl of PCR master mix.
Note: If the template DNA is stored in TE buffer, the volume of DNA
added should not exceed 20% of the final reaction volume. 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
extraction procedure used.
7.
For the positive amplification control, dilute K562 DNA to 0.4–0.8ng/μl.
Pipet 2.5μl (1–2ng) of diluted K562 DNA into a reaction tube containing
22.5μl of PCR master mix.
8.
For the negative amplification control, pipet 2.5μl of nuclease-free water
(instead of template DNA) into a reaction tube containing 22.5μl of 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.
10. Centrifuge samples briefly to contents to the bottom of the tube.
4.B. Amplification Thermal Cycling
This manual contains protocols for use of the PowerPlex® 2.1 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]
1.
Place tubes 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
Perkin-Elmer model 480 thermal cycler are provided below.
Note: Ramp settings displayed in the cycling profile indicate the ramp to
the temperature that follows.
3.
After completion of the thermal cycling protocol, store samples at –20°C in
a light-protected box.
Note: Storage of amplified samples at 4°C or higher may produce
degradation products.
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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 20 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 20 cycles, then:
60°C for 30 minutes
60°C for 30 minutes
4°C soak
4°C soak
GeneAmp®
Protocol for the
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 20 cycles, then:
90°C for 1 minute
60°C for 1 minute
70°C for 1.5 minutes
for 20 cycles, then:
60°C for 30 minutes
60°C for 30 minutes
4°C soak
4°C soak
1When
GeneAmp®
using the
PCR System 9700 thermal cycler, the ramp rates indicated in the
cycling program must be set, and the program must be run in 9600 ramp mode.
The ramp rates are set in the Ramp Rate Modification screen. While viewing the cycling program,
navigate to the Ramp Rate Modification screen by selecting "More", then "Modify". On the Ramp
Rate Modification screen the default rates for each step are 100%. The rate under each hold step is
the rate at which the temperature will change to that hold temperature. Figure 1 shows the ramp
rates for the GeneAmp® PCR System 9700 thermal cycler.
The ramp mode is set after “start” has been selected for the thermal cycling run. A Select Method
Options screen appears. Select 9600 ramp mode, and enter the reaction volume.
94.0°C
100%
70.0°C
23%
60.0°C
29%
3 tmp 22 cycles
90.0°C
100%
70.0°C
23%
60.0°C
29%
7486MA
3 tmp 10 cycles
Figure 1. The ramp rates for the GeneAmp® PCR System 9700 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
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Detection of Amplified Fragments Using the Hitachi FMBIO® and FMBIO® II
Fluorescence Imaging Systems
Materials to Be Supplied by the User
(Solution compositions are provided in Section 9.I.)
• polyacrylamide gel electrophoresis apparatus
• power supply (4,000 volts)
• dry heating block, water bath or thermal cyclers
• squaretooth comb, 35cm, 60 wells (cut in half for 30 wells/gel), 0.4mm thick
(Owl Scientific Cat.# S2S-60A) or vinyl doublefine sharkstooth comb, 14cm,
49 point, 0.4mm thick
• Nalgene® tissue culture filter (0.2 micron)
• aerosol-resistant pipette tips (Section 9.J)
• low-fluorescence glass plates: 43cm × 19cm × 0.4mm (The Gel Company
Cat.# GG047-B0505S)
• spacers (0.4mm)
• SA-43 extension (Lab Repco Cat.# 31096423) for use with 43cm glass plates
• clamps (e.g., large office binder clamps)
• diamond pencil for marking glass plates
• 50% Long Ranger® gel solution or Long Ranger Singel® pack for ABI sequencers
377-36cm (Cambrex)
• TBE 10X buffer
• 10% Ammonium Persulfate (Cat.# V3131)
• Urea (Cat.# V3171)
• TEMED
• bind silane (methacryloxypropyltrimethoxysilane), for use with squaretooth
combs
• Liqui-Nox® detergent
5.A. Polyacrylamide Gel Preparation
!
Caution: Acrylamide 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.
Note: To reduce the time of preparing gels or the expense of precast gels, we have
developed a rapid method to reuse gels between 2 and 8 times over a period of
several days (12). Methods for polyacrylamide gel reuse are provided in Section 9.E.
1.
Etch each glass plate on one side in one corner with a diamond pencil to
distinguish the gel sides of the glass plates. Thoroughly clean the glass
plates twice with 95% ethanol and Kimwipes® tissues.
Note: If using a squaretooth comb, the shorter glass plate requires bind
silane treatment (see below). The plates do not require a special silane
treatment when using a sharkstooth comb.
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5.A. Polyacrylamide Gel Preparation (continued)
Bind Silane Treatment of Glass Plate
Prepare fresh binding solution in a chemical fume hood. Add 1μl of bind
silane to a 1.5ml microcentrifuge tube containing 0.5ml of 0.5% acetic acid
in 95% ethanol. Wipe the etched side of the shorter glass plate in the comb
region using a Kimwipes® tissue saturated with freshly prepared binding
solution. Wait 5 minutes for the binding solution to dry. Wipe the shorter
glass plate 3–4 times with 95% ethanol and Kimwipes® tissues in the comb
area to remove excess binding solution.
2.
Assemble the glass plates by placing 0.4mm side spacers between the front
and rear glass plates, using clamps to hold them in place (3–4 clamps on
each side). A bottom spacer is neither required nor recommended. Place
the assembly horizontally on a test tube rack or similar support.
3.
Prepare a 5% Long Ranger® acrylamide solution by combining the
ingredients listed in Table 3 for Long Ranger® acrylamide gels.
Table 3. Preparation of 5% Long Ranger® Polyacrylamide Gels.
Component
5% Gel
Final Concentration
urea
deionized water
10X TBE Buffer
50% Long Ranger ® gel solution
total volume
18.0g
26.0ml
5.0ml
5.0ml
50ml
6M
—
1X
5%
Note: Long Ranger Singel® Packs may also be used.
Note: While standard 4% or 5% polyacrylamide gels can be used, the
performance of the Hitachi FMBIO® II Fluorescence Imaging System
band-finding software is better when 5% Long Ranger® acrylamide is used.
4.
Filter the acrylamide solution through a 0.2 micron filter (e.g., Nalgene®
tissue culture filter). Degas the Long Ranger® acrylamide for 5 minutes.
5.
Pour the filtered acrylamide solution into a squeeze bottle.
6.
Add the appropriate amounts of TEMED and 10% ammonium persulfate
listed in Table 4 to the acrylamide solution, and mix gently.
Table 4. Amounts of TEMED and 10% Ammonium Persulfate for
5% Long Ranger® Polyacrylamide Gels.
7.
Component
5% Long Ranger® Gel (50ml)
TEMED
10% ammonium persulfate
18.0g
26.0ml
Pour the gel by starting at the well end of the plates and carefully pouring
the acrylamide between the horizontal glass plates. Allow the solution to
fill the top width of the plates. Slightly tilt the plates to assist movement of
the solution to the bottom of the plates while maintaining a constant flow
of solution. When the solution begins to flow out from the bottom,
position the plates horizontally.
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Revised 7/08
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8.
Insert the squaretooth comb between the glass plates until the teeth are
almost completely inserted into the gel, or insert one 14cm doublefine
(49 point) sharkstooth comb, straight side into the gel, between the glass
plates (6mm of the comb should be between the two glass plates).
9.
Secure the comb with three evenly spaced clamps.
10. Pour the remaining acrylamide solution into a disposable conical tube as a
polymerization control. Rinse the squeeze bottle, including the spout, with
water.
11. Allow polymerization to proceed for at least 1 hour. Check the
polymerization control to be sure that polymerization has occurred.
Note: The gel may be stored overnight if a paper towel saturated with 1X
TBE or water and plastic wrap are placed around the top and bottom of
the gel to prevent the gel from drying out (crystallization of the urea will
destroy the gel).
5.B. Gel Pre-Run
1.
Remove clamps from the polymerized acrylamide gel, and clean the glass
plates with paper towels saturated with deionized water.
2.
Shave any excess polyacrylamide away from the comb, and remove the
comb.
3.
Add 1X TBE buffer to the bottom chamber of the electrophoresis apparatus.
4.
Gently lower the gel/glass plates unit into the buffer with the longer plate
facing out and the well-side on top.
5.
Secure the glass plates to the gel electrophoresis apparatus.
6.
Add 1X TBE buffer to the top buffer chamber of the electrophoresis
apparatus.
7.
Using a 50–100cc syringe filled with buffer, remove the air bubbles on the
top of the gel. Be certain the well area is devoid of air bubbles and small
pieces of polyacrylamide. Using a syringe with a bent 18-gauge needle,
remove any air bubbles from the bottom of the gel.
8.
Pre-run the gel to achieve a gel surface temperature of approximately
50°C. Consult the manufacturer's instruction manual for the recommended
electrophoresis conditions.
Note: As a reference, we generally use 60 watts for 45–60 minutes for a
43cm gel. The gel running conditions may need to be adjusted to reach a
temperature of 50°C.
9.
Prepare samples and allelic ladder samples during the gel pre-run.
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5.C. Sample Preparation and Loading
The Internal Lane Standard 600 is included in the PowerPlex® 2.1 System for
those who wish to use an internal size marker for three-color detection and
analysis of amplified samples. With this approach, only 2–3 lanes of the
PowerPlex® 2.1 Allelic Ladder are required per gel. Alternatively, the two-color
detection method may be employed in which the Internal Lane Standard 600 is
not used, and the PowerPlex® 2.1 Allelic Ladder Mix is loaded as often as every
third gel lane.
1.
Prepare amplified samples and ladders as described below, depending on
the detection method employed (either three-color detection or two-color
detection).
For three-color detection (using the Internal Lane Standard 600)
a.
Prepare a loading cocktail by combining and mixing Internal Lane
Standard 600 and Bromophenol Blue Loading Solution as follows:
[(1μl ILS 600) × (# lanes)] + [(3μl Bromophenol Blue Loading Solution)
× (# lanes)]
b.
Combine 4μl of prepared loading cocktail and 2μl of amplified
sample.
Note: If the fluorescent signal on the gel is too intense, dilute samples
in Gold ST★R 1X Buffer before mixing with loading cocktail or use
less DNA template in the amplification reactions.
c.
Combine 4μl of prepared loading cocktail and 2μl of PowerPlex® 2.1
Allelic Ladder Mix.
For two-color detection (not using the Internal Lane Standard 600)
a.
Combine 2.5μl of Bromophenol Blue Loading Solution and 2.5μl of
amplified sample.
Note: If the fluorescent signal is too intense, dilute samples in Gold
ST★R 1X Buffer before mixing with loading solution or use less DNA
template in the amplification reactions.
b.
Combine 2.5μl of Bromophenol Blue Loading Solution and 2.5μl of
PowerPlex® 2.1 Allelic Ladder Mix.
2.
(Optional) Place 6μl of Gel Tracking Dye in one tube. The Gel Tracking
Dye contains both bromophenol blue and xylene cyanol. This dye is
loaded in the outermost lane of the gel at least three lanes from the nearest
sample and is used as a visual indicator of migration.
3.
Briefly centrifuge samples to bring contents to the bottom of the tubes.
4.
Denature samples by heating at 95°C for 2 minutes, and immediately chill
on crushed ice or in an ice-water bath. Denature samples just prior to
loading the gel.
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5.
After the pre-run use a 50–100cc syringe filled with buffer to flush urea
from the well area. If using a squaretooth comb, do not reinsert the comb,
as the samples will be loaded directly into the wells. If using a sharkstooth
comb, insert the teeth into the gel approximately 1–2mm, and leave the
comb inserted in the gel during both gel loading and electrophoresis.
6.
Load 3μl of each denatured sample into the respective wells. The loading
process should take no longer than 20 minutes to prevent the gel from
cooling.
5.D. Gel Electrophoresis
1.
After loading, run the gel using the same conditions as in Section 5.B.
Observe the lane containing Gel Tracking Dye to monitor sample
migration. In a 5% Long Ranger® acrylamide gel, xylene cyanol dye
migrates at approximately 190 bases, and bromophenol blue dye migrates
at less than 80 bases.
2.
Based on size ranges for each locus (Table 7) and migration characteristics
of the dyes contained in the Gel Tracking Dye, stop electrophoresis before
the smallest locus (i.e., D3S1358) has reached the bottom of the gel.
Note: For an SA-43 gel, run the gel until the leading edge of xylene cyanol
is approximately 17.5cm from the bottom of the gel. Run time will vary
with the power supply used. For a 5% Long Ranger® gel, run time is
approximately 1 hour, 30 minutes.
5.E. Detection
1.
After electrophoresis, remove the gel/glass plate unit from the apparatus.
Remove the comb and side spacers, but do not separate the glass plates.
2.
The plates must be thoroughly cleaned before scanning. Clean both sides
of the gel/glass plate unit with deionized water and lint-free paper.
Ethanol should not be used to clean the plate unit; ethanol fluoresces and
may be detected as background by the FMBIO® instrument.
3.
Scan the gel according to the parameters listed in Table 5. Use the 505nm
filter to detect fluorescein-labeled fragments, the 585nm filter to detect
TMR-labeled fragments and the 650nm filter to detect the Internal Lane
Standard 600. Different laboratories may wish to modify these parameters
according to their personal preferences.
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Table 5. Instrument Parameters for the Hitachi FMBIO® and FMBIO® II Fluorescence
Scanners and PowerPlex® 2.1 System.
Hitachi FMBIO®
Fluorescence Scanner
Hitachi FMBIO® II
Fluorescence Scanner
Material Type
acrylamide gel
acrylamide gel
Resolution:
Horizontal
Vertical
150dpi
150dpi
150dpi
150dpi
Rate
0.1024s/line
NA
Repeat
1 time
256 times
Gray Level Correction Type
range
range
Cutoff Threshold:
Low Background
High Signal
50%
1%
50%
1%
80%
100% (505nm channel)
80% (585nm channel)
100% (650nm channel)
Reading Sensitivity
Focusing
Point1
NA
0.00mm
NA = not applicable
1Focusing point of 0.00mm is based on use of 5mm glass plates. If using precast gels or
thinner glass plates, the focusing point may need to be adjusted.
5.F. Reuse of Glass Plates
Separate the glass plates, and discard the gel. Clean glass plates with deionized
water and 1% Liqui-Nox® detergent. The use of Liqui-Nox® detergent is
extremely important, as other kinds of soap can build up on the glass plates.
This will result in low signal and high background on the gels. If the glass plates
have a build-up of soap residue on them, we recommend soaking in 10%
sodium hydroxide for 1 hour and rinsing well in deionized water.
If bind silane is used to fix the gel to the smaller glass plate, soak the plate in
10% sodium hydroxide for 1 hour (or until the gel comes off the plate) and
clean as described.
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Data Analysis
View and analyze the gel image to determine allele designations as recommended in
the FMBIO® user’s manual. For information regarding the use of FMBIO® Analysis
Software, contact the Hitachi Technical Support Group (800-624-6176, extension
7508). Perform the multicolor separation for all gels containing material amplified
using the PowerPlex® 2.1 System. Display the gel image using green for the
fluorescein-labeled loci (Penta E, D18S51, D21S11, TH01 and D3S1358), red for the
TMR-labeled loci (FGA, TPOX, D8S1179 and vWA) and cyan for the Internal Lane
Standard 600.
6.A. Controls
Observe the lanes containing the negative controls. They should be devoid of
amplification products.
Observe the lanes containing the K562 DNA positive controls. Compare K562
DNA allelic repeat sizes with the locus-specific allelic ladder. The expected
K562 DNA allele sizes for each locus are listed in Table 8 (Section 9.B). Figure 5
shows an example of results obtained after amplification of K562 DNA using
the PowerPlex® 2.1 System. K562 DNA contains imbalanced alleles at several
loci due to the unusual chromosome content of this cell line. This is not a
function of PowerPlex® 2.1 System performance.
6.B. Allelic Ladders
In general, allelic ladders contain fragments of the same lengths as most or all
known alleles for the locus. Allelic ladder sizes and repeat units are listed in
Table 7 (Section 9.B). Visual comparison between allelic ladder and amplified
samples of the same locus allows precise assignment of alleles. Analysis using
specific instrumentation also allows allele determination by comparing
amplified sample fragments with either allelic ladders, internal size standards
or both (see software documentation from instrument manufacturer). When
using an internal lane standard, the calculated lengths of allelic ladder
components will differ from those listed in Table 7. This is due to differences in
migration resulting from sequence differences between allelic ladder fragments
and internal lane standard fragments.
Note: It may prove helpful to confirm that your gel analysis software identifies
the correct number of alleles present in the allelic ladder lanes prior to analysis
of sample lanes. The PowerPlex® 2.1 System Allelic Ladder has 86 alleles in the
fluorescein channel (20 Penta E alleles, 22 D18S51 alleles, 25 D21S11 alleles,
10 TH01 alleles and 9 D3S1358 alleles), and 52 alleles in the TMR channel
(19 FGA alleles, 8 TPOX alleles, 12 D8S1179 alleles and 13 vWA alleles).
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6.C. Results
Figures 2–5 show typical results achieved using the PowerPlex® 2.1 System in
the three-color detection format as described in Section 5.E.
A.
505nm Scan—Fluorescein
L 1 2 3 4 5 6 L 7 8 9 10 11 12 L
B.
585nm Scan—TMR
L 1 2 3 4 5 6 L 7 8 9 10 11 12 L
–24
–46.2
– 43.2
Penta E
–31.2
–5
–27
FGA
–17
D18S51
–13
–8
TPOX
–38
–6
–18
D8S1179
D21S11
–7
–24
–13.3
–11
–22
TH01
–4
–20
D3S1358
vWA
2652TA
–10
–12
Figure 2. The PowerPlex® 2.1 System. Twelve DNA samples (lanes 1–12) were amplified with the
PowerPlex® 2.1 Primer Pair Mix using 1ng of DNA template. Lanes labeled L contain allelic ladders
for each of the nine loci contained in the PowerPlex® 2.1 System. Panel A. A scan using a 505nm
filter, showing the fluorescein-labeled loci, Penta E, D18S51, D21S11, TH01 and D3S1358. Panel B. A
scan using a 585nm filter, showing the TMR-labeled loci, FGA, TPOX, D8S1179 and vWA. In panels
A and B, each allelic ladder is labeled to the right with the number of copies of the repeated
sequence contained within its corresponding largest and smallest alleles. Two sets, 17 to 31.2 and
43.2 to 46.2, are shown for the locus FGA. The rare allele 13.3 is also shown for the locus TH01. All
materials were separated using a 5% denaturing Long Ranger® polyacrylamide gel and detected
using the Hitachi FMBIO® II Fluorescence Imaging System.
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Internal Lane Standard 600
600
550
500
475
450
425
400
375
350
325
300
275
250
225
200
180
160
140
100
2653TA
120
Figure 3. The Internal Lane Standard 600. The Internal Lane Standard 600 contains fragments
ranging from 60 to 600 bases in length. It was mixed with the samples shown in Figure 2 before
loading the gel. Following separation, the Internal Lane Standard 600 was detected using a 650nm
filter with the Hitachi FMBIO® II Fluorescence Imaging System. Fragments smaller than 100 bases are
not shown on this gel. Fragment sizes are shown to the right of the gel. The 100-, 200-, 300-, 400-, 500and 600-base fragments display double the intensity of the others.
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A.
Page 18
505nm Scan—Fluorescein
B.
585nm Scan—TMR
L 1 2 3 4 5 6 7 8 L
L 1 2 3 4 5 6 7 8 L
Penta E
FGA
D18S51
TPOX
D8S1179
D21S11
TH01
vWA
2654TA
D3S1358
Figure 4. Amplification of various amounts of template with the PowerPlex® 2.1 System. A single
DNA template (10, 5, 2, 1, 0.5, 0.2 or 0.1ng) was amplified. Results are shown in lanes 1–7, respectively.
Lane 8 shows amplification results with no DNA template. Lanes labeled L contain allelic ladders for
each of the nine loci. Panel A. A scan using a 505nm filter shows the fluorescein-labeled loci, Penta E,
D18S51, D21S11, TH01 and D3S1358. Panel B. A scan using a 585nm filter shows the TMR-labeled
loci, FGA, TPOX, D8S1179 and vWA. All materials were separated using a 5% denaturing Long
Ranger® polyacrylamide gel and detected using the Hitachi FMBIO® II Fluorescence Imaging System.
A lane trace of lane 4 (1ng template) is shown to the right of each panel.
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505nm Scan—Fluorescein
B.
585nm Scan—TMR
L K562
L K562
Penta E
FGA
D18S51
TPOX
D8S1179
D21S11
TH01
vWA
2655TA
D3S1358
Figure 5. K562 DNA amplified using the PowerPlex® 2.1 System. K562 DNA (1ng) was amplified.
Panel A. A scan using a 505nm filter shows the fluorescein-labeled loci, Penta E, D18S51, D21S11,
TH01 and D3S1358. Panel B. A scan using a 585nm filter shows the TMR-labeled loci, FGA, TPOX,
D8S1179 and vWA. All materials were separated using a 5% denaturing Long Ranger®
polyacrylamide gel and detected using the Hitachi FMBIO® II Fluorescence Imaging System. A lane
trace is shown to the right of each panel. The lane traces show imbalance in the heterozygous alleles in
several loci. This occurs primarily because the two copies of each chromosome are not present in equal
amounts within this cell line. The K562 DNA also shows a three-band pattern for the locus D21S11.
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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]
Symptoms
Faint or absent allele bands
Bands are fuzzy throughout
the lanes
n–1 bands are present
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. Diluting the template in
TE-4 buffer or water prior to amplification may improve
results.
Insufficient template. Use the recommended amount of
template DNA.
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 or tube problems. Review the thermal cycling
protocols in Section 4.B. We have not tested other reaction
tubes 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® 2.1 Primer Pair
for 15 seconds using a vortex mixer before use.
Samples were not completely denatured. Heat-denature
samples at 95°C for 2 minutes, and immediately chill on
crushed ice or in an ice-water bath prior to loading.
Poor-quality polyacrylamide gel. Prepare acrylamide and
buffer solutions using high-quality reagents. We strongly
recommend 5% Long Ranger® acrylamide (Cambrex).
Electrophoresis temperature was too high. Run gel at lower
temperature (40–50°C).
Following amplification, lengthen the final extension step from
30 minutes at 60°C to 45 minutes. n–1 bands may be generated
when more than 1ng template DNA is used. This is most
commonly observed with the vWA and D3S1358 amplification
products. Reduce the amount of template DNA used or
reduce the number of cycles by 2 or 4 cycles (10/18 or
10/16 cycles).
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Symptoms
Extra bands visible in
one or all lanes
Imbalance of band
intensities across loci
Page 21
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
samples for the recommended time, and cool on crushed ice
or in an ice-water bath immediately prior to loading the gel.
Artifacts of STR amplification. PCR amplification of STR
systems sometimes generates artifacts that appear as faint
bands one repeat unit smaller than the allele. Refer to
Section 9.B for locus-specific information regarding this
event. Stutter band intensities can be high if samples are
overloaded. Use 1–2ng DNA template.
Artifacts of STR amplification. PCR amplification of STR
systems can result in artifacts that appear as bands 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 4.B).
High background. Load less amplification product.
Bleedthrough due to poor color separation. If samples lanes are
dark, repeat color separation. Rerun gel with less sample DNA.
Gel not run in reverse long enough when reusing gels. Gels
should be run in reverse 15–30 minutes longer than the
previous run. Longer time may be required if using a different
gel rig or power supply.
Allelic ladder contamination. Keep pre- and postamplification
components separate.
Excessive amount of DNA. The system is balanced using 1ng
of DNA template. Amplification of >5ng of template can result
in an imbalance with smaller loci showing more product than
larger loci. Use less template, or reduce the number of cycles
in the amplification program.
Too much template from cards or membrane punches of
bloodstains. Cards or membranes that bind DNA tightly can
contain more template DNA than recommended. This will
lead to overrepresented smaller alleles and underrepresented
larger alleles. Use the recommended amount of template by
using a smaller punch of the membrane. Alternately, fewer
cyles of amplification can compensate for this type of
unevenness of product yield (i.e., 10/18 or 10/16 cycling).
The band intensity of the Penta E locus is generally about 65%
the intensity of the other loci. Use of twice the recommended
amount of AmpliTaq Gold® DNA polymerase can compensate
for the lower yield of the Penta E locus.
Poor-quality polyacrylamide gel. Prepare acryamide and
buffer solutions using high-quality reagents. We recommend
5% Long Ranger® acrylamide (Cambrex).
Too many cycles in the amplification protocol. Use the
recommended amplification program; confirm the number of
cycles.
Too much enzyme was present. Use the recommended
amount of AmpliTaq Gold® DNA polymerase.
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7.
Page 22
Troubleshooting (continued)
Symptoms
Imbalance of band
intensities across loci
(continued)
Poor separation of alleles in
ladder lanes or difficulty
resolving microvariant alleles
White background with low
signal intensity
Dark, grainy background with
low signal intensity
8.
Causes and Comments
Degraded DNA sample. DNA template is degraded, and the
larger loci show diminished yield. Confirm the DNA integrity
by running an aliquot on an agarose gel. Repurify the template
DNA if necessary.
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.
Gel was not run long enough. Run gel as long as possible
without running the 100-base ILS fragment off the bottom of
the gel. If necessary, run the gel for additional time after the
first scan, then scan the gel a second time to achieve better
separation of larger alleles.
Scanning resolution was too low. Default scanning resolution
is 150dpi. If necessary, this resolution can be increased to
300dpi, which should help sharpen bands.
Part of the spacers were scanned. Rescan the gel, being careful
not to scan any portion of the spacers.
Focusing point may need to be adjusted. Confirm that 5mm
plates are being used. Adjust focusing point if necessary.
Plates were improperly washed. Improper washing of plates
can cause a soap residue to build up on the plates, which can
cause background fluorescence. Plate may be soaked in
1N NaOH to clean off residue. Do not use ethanol to clean
plates prior to scanning.
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
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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.
Bassam, B.J., Caetano-Anolles, G. and Gresshoff, P.M. (1991) Fast and sensitive silver staining of DNA
in polyacrylamide gels. Anal. Biochem. 196, 80–3.
10.
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.
11.
Hartmann, J.M. et al. (1991) Guidelines for a quality assurance program for DNA analysis. Crime
Laboratory Digest 18, 44–75.
12.
Tereba, A., Micka, K.A. and Schumm, J.W. (1998) Reuse of denaturing polyacrylamide gels for short
tandem repeat analysis. BioTechniques 25, 892–7.
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
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8.
Page 24
References (continued)
28.
Micka, K. et al. (1996) Validation of multiplex polymorphic STR amplification sets developed for
personal identification applications. J. Forensic Sci. 41, 582–90.
29.
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.
30.
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.
31.
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.
32.
Sprecher, C.J. et al. (1996) General approach to analysis of polymorphic short tandem repeat loci.
BioTechniques 20, 266–76.
33.
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.
34.
Jones, D.A. (1972) Blood samples: Probability of discrimination. J. Forensic Sci. Soc. 12, 355–9.
35.
Brenner, C. and Morris, J.W. (1990) In: Proceedings from the International Symposium on Human
Identification 1989, Promega Corporation, 21–53.
36.
Mandrekar, P.V., Krenke, B.E. and Tereba, A. (2001) DNA IQ™: The intelligent way to purify DNA.
Profiles in DNA 4(3), 16.
37.
Krenke, B.E. et al. (2005) Development of a novel, fluorescent, two-primer approach to quantitative
PCR. Profiles in DNA 8(1), 3–5.
38.
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.
39.
McLaren, B., Bjerke, M. and Tereba, A. (2006) Automating the DNA IQ™ System on the Biomek® 3000
Laboratory Automation Workstation. Profiles in DNA 9(1), 11–13.
40.
Cowan, C. (2006) The DNA IQ™ System on the Tecan Freedom EVO® 100. Profiles in DNA 9(1), 8–10.
41.
Bjerke, M. et al. (2006) Forensic application of the Maxwell™ 16 Instrument. Profiles in DNA 9(1), 3–5.
42.
Mandrekar, P. et al. (2007) Introduction to Maxwell® 16 low elution volume configuration for forensic
casework. Profiles in DNA 10(2), 10–12.
The fields of forensic and paternity analysis are changing rapidly. For this reason, it
is difficult to keep our manuals up-to-date regarding new technologies and new
publications. However, a substantial reference list of publications describing STRs
and much related information can be found at an Internet web site created by the
National Institutes of Science and Technology (NIST) Biotechnology Division
(www.cstl.nist.gov/biotech/strbase/). This web site is occasionally updated and has
numerous links to many other useful sites.
Additional STR references also can be found at: www.promega.com/geneticidentity/
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
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Appendix
9.A. Advantages of STR Typing
STR typing is more tolerant of degraded DNA templates than other typing
methods because amplification products are less than 500bp long, much smaller
than material detected using AMP-FLP (13) or VNTR (14) analysis. STR typing
is also amenable to a variety of rapid DNA purification techniques that 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 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® 2.1 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).
9.B. Advantages of Using the Loci in the PowerPlex® 2.1 System
The loci included in the PowerPlex® 2.1 System (Tables 6 and 7) have been
selected because they satisfy the needs of several major standardization bodies
throughout the world. INTERPOL, the European police network, has established
a set of four STR loci (FGA, D21S11, TH01 and vWA) as a pan-European
standard. The European Network of Forensic Science Institutes (ENFSI) has
completed a multilaboratory study of STR loci and recommend that European
laboratories employ the following seven STR loci plus Amelogenin: FGA, TH01,
vWA, D3S1358, D8S1179, D18S51 and D21S11. The loci amplified in the
PowerPlex® 2.1 System include both of these STR standard sets.
The United States Federal Bureau of Investigation (FBI) has selected 13 STR loci
to be typed prior to inclusion of sample profiles in or searching of the U.S.
national database of convicted offender profiles, CODIS (COmbined DNA
Index System). Eight of the PowerPlex® 2.1 System loci (D18S51, D21S11, TH01,
D3S1358, FGA, TPOX, D8S1179 and vWA) are included in this core set of
13 STR loci. When used in combination with the PowerPlex® 1.1 System, all
CODIS core loci can be analyzed in two amplification reactions. Three of the
same loci (TH01, TPOX and vWA) are amplified in both systems to minimize
possibilities of undetected sample mix-up when performing the two
amplifications.
The PowerPlex® 2.1 System also contains a low-stutter, highly polymorphic
pentanucleotide repeat locus, Penta E. This additional locus adds significantly
to the discrimination power of the system, making the PowerPlex® 2.1 System a
single-amplification system with a power of exclusion sufficient to resolve
nearly all paternity disputes definitively. In addition, the extremely low level of
stutter seen with Penta E makes it an ideal locus to evaluate DNA mixtures
often encountered in forensic casework.
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9.B. Advantages of Using the Loci in the PowerPlex® 2.1 System (continued)
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, as well as genetic artifacts called microvariant
alleles. 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, or both. The amount of this artifact observed
depends primarily on the locus and DNA sequence being amplified. Individual
laboratories should develop independent guidelines regarding acceptable cutoff
values for repeat slippage, as well as standards for background level and allele
intensity. The average percent of repeat slippage (stutter) for each locus present
in the PowerPlex® 2.1 System has been determined (Table 6). Note the
comparatively low stutter of Penta E.
Table 6. The PowerPlex® 2.1 System Locus-Specific Information.
STR
Locus
Label
Chromosomal
Location
GenBank® Locus and
Locus Definition
Repeat Sequence1
Average
→ 3´
Percent Stutter
5´→
Penta E
FL
15q
NA
AAAGA
1–2%
D18S51
FL
18q21.3
HUMUT574
AGAA (23)
>6%
D21S11
FL
21q11–21q21
HUMD21LOC
TCTA Complex (23)
>6%
TH01
FL
11p15.5
HUMTH01, human tyrosine
hydroxylase gene
NA
AATG (23)
1–2%
D3S1358
FL
3p
FGA
TMR
4q28
TPOX
TMR
2p24–2pter
D8S1179
TMR
8q
vWA
TMR
12p12–pter
TCTA Complex
>6%
HUMFIBRA, human
TTTC
fibrinogen alpha chain gene
Complex (23)
HUMTPOX, human thyroid
AATG
peroxidase gene
NA
TCTA Complex (23)
4–6%
HUMVWFA31, human von
Willebrand factor gene
>6%
TCTA
Complex (23)
2–4%
4–6%
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
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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). For example, the FGA and D21S11 loci are highly polymorphic and
display numerous relatively common microvariants. For reasons yet unknown,
the highly polymorphic Penta E locus does not display frequent microvariants.
Table 8 lists the PowerPlex® 2.1 System alleles revealed in commonly available
standard DNA templates.
Table 7. The PowerPlex® 2.1 System Allelic Ladder Information.
STR Locus
Label
Size Range of Allelic
Ladder Components1,2
(bases)
Penta E
D18S51
D21S11
FL
FL
FL
379–474
290–366
203–259
TH01
D3S1358
FGA
FL
FL
TMR
156–195
115–147
322–444
TPOX
D8S1179
vWA
TMR
TMR
TMR
262–290
203–247
123–171
1The
Repeat Numbers of Allelic
Ladder Components
Repeat Numbers of
Alleles Not Present
in Allelic Ladder 3,4
5–24
8–10, 10.2, 11–13, 13.2, 14–27
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
4–9, 9.3, 10–11, 13.3
12–20
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
6–13
7–18
10–22
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
D21S11 alleles correspond to alleles: 53–57, 59, 61–77, 79, 81 as defined by the Forensic Science
Service using a different nomenclature.
4The
alleles listed are those with a frequency of >1/1000.
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Table 8. The PowerPlex® 2.1 System Allele Determinations in Commonly Available Standard
DNA Templates.
Standard DNA Templates1
STR Locus
K562
9947A
9948
Penta E
5, 14
12, 13
11, 11
D18S51
15, 16
15, 19
15, 18
D21S11
29, 30, 31
30, 30
29, 30
TH01
9.3, 9.3
8, 9.3
6, 9.3
D3S1358
16, 16
14, 15
15, 17
FGA
21, 24
23, 24
24, 26
TPOX
8, 9
8, 8
8, 9
D8S1179
12, 12
13, 13
12, 13
vWA
16, 16
17, 18
17, 17
1Strains
9947A and 9948 are available from the NIGMS Human Genetic Mutant Cell Repository
(Cornell Institute, Camden, NJ). Strain K562 is available from the American Type Culture Collection
(Manassas, VA). Information 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).
9.C. Fluorescent STR Products
Table 9 lists the fluorescent STR multiplex systems available from Promega.
Three quadriplexes (the GammaSTR®, CTTv and FFFL multiplexes) have been
developed with fluorescein labels. The combination of the PowerPlex® 1.1
System (26,27) and PowerPlex® 2.1 System provides analysis of 14 STR loci. Use
of the PowerPlex® 1.1 System, the PowerPlex® 2.1 System and the FFFL System
(26,27) provides analysis of 18 STR loci in three amplification reactions. Each
STR locus contained in the GammaSTR®, CTTv and FFFL multiplexes is also
available as a fluorescein-labeled monoplex system.
Each of the fluorescent STR systems contains all the materials required to
perform amplification reactions except for Taq DNA polymerase and sample
DNA. The corresponding allelic ladders are included with all systems. The
Fluorescent Ladder (CXR), 60–400 Bases, is used as an internal lane standard
(ILS) and is included with the PowerPlex® 1.1 System. The Internal Lane
Standard 600 (60–600 bases) is included with the PowerPlex® 2.1 System. The
internal lane standards may be purchased separately.
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Table 9. Loci and Labels for the Fluorescent STR Muliplex Systems.
CTTv
Multiplex
STR Locus
CFS1PO
TPOX
TH01
vWA
F13A01
FESFPS
F13B
LPL
D16S539
D7S820
D13S317
D5S818
Amelogenin
Penta E
D18S51
D21S11
D3S1358
FGA
D8S1179
FFFL
Multiplex
GammaSTR®
Multiplex
FL
FL
FL
FL
PowerPlex® 1.1 PowerPlex® 2.1
System
System
TMR
TMR
TMR
TMR
TMR
FL
TMR
FL
FL
FL
FL
FL
FL
FL
FL
FL
FL
FL
FL
TMR1
FL
FL
FL
FL
TMR
TMR
FL = 5´-terminal fluorescein label
TMR = 5´-terminal carboxy-tetramethylrhodamine label
1Amelogenin is not included in the PowerPlex® 1.1 System but may be amplified using an additional
system.
9.D. Power of Discrimination
The nine STR loci amplified with the PowerPlex® 2.1 System provide powerful
discrimination. Population statistics for these loci and their various multiplex
combinations are displayed in Tables 10–12. These data were generated as part
of a collaboration (26) with The Bode Technology Group (Springfield, VA). Data
generation included analysis of over 200 individuals from each of the three
major racial and ethnic groups in the United States. For additional population
data for STR loci, see references 27 and 29–33.
Table 10 shows the matching probability (34) for the PowerPlex® 2.1 System in
various populations. The matching probability of the PowerPlex® 2.1 System
ranges from 1 in 84,600,000,000 for Caucasian-Americans to 1 in 300,000,000,000
for African-Americans. The matching probability of the PowerPlex® 2.1 System
in combination with the PowerPlex® 1.1 System is 1 in 10,700,000,000,000,000 for
Caucasian-Americans and 1 in 41,900,000,000,000,000 for African-Americans.
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9.D. Power of Discrimination (continued)
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 (35). The typical paternity
indices for the PowerPlex® 2.1 System and PowerPlex® 1.1 System in
combination with the FFFL multiplex are shown in Table 11. The PowerPlex®
2.1 System alone provides typical paternity indices exceeding 3,250 in each
population group, enough to satisfy routine requirements for paternity
determination. When the PowerPlex® 1.1 System and PowerPlex® 2.1 System
are combined, the values exceed 163,000 in all groups.
Table 10. Matching Probabilities of the PowerPlex® Systems in Various Populations.
STR System
PowerPlex® 1.1
System (8 STR loci)
PowerPlex® 2.1 System
(9 STR loci)
AfricanAmerican
Matching Probability
CaucasianHispanicAmerican
American
AsianAmerican
1 in 2.74 × 108
1 in 1.14 × 108
1 in 1.45 × 108
1 in 1.32 × 108
1 in 3.0 × 1011
1 in 8.46 × 1010
1 in 1.02 × 1011
1 in 1.52 × 1011
PowerPlex® 1.1 System
plus PowerPlex® 2.1
System (14 STR loci)
1 in 4.19 × 1016
1 in 1.07 × 1016
1 in 1.59 × 1016
1 in 2.17 × 1016
PowerPlex® 1.1 System
System plus
PowerPlex® 2.1 System
plus FFFL muliplex (18
STR loci)
1 in 7.03 × 1020
1 in 2.84 × 1019
1 in 5.21 × 1019
1 in 1.14 × 1018
Table 11. Typical Paternity Indices of the PowerPlex® Systems in Various Populations.
STR System
AfricanAmerican
Typical Paternity Index
CaucasianHispanicAmerican
American
AsianAmerican
PowerPlex® 1.1 System
(8 STR loci)
PowerPlex® 2.1 System
(9 STR loci)
498
260
319
471
13,130
13,199
3,250
41,800
PowerPlex® 1.1 System
plus PowerPlex® 2.1
System (14 STR loci)
6.11 × 105
4.08 × 105
1.63 × 105
2.02 × 106
1.03 × 107
6.24 × 106
1.34 × 106
4.55 × 106
PowerPlex®
1.1 System
System plus PowerPlex®
2.1 System plus FFFL
Multiplex (18 STR loci)
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An alternative calculation used in paternity analyses is the power of exclusion
(35). This value, calculated for the PowerPlex® 2.1 System, exceeds 0.9997 in all
populations tested (Table 12). In combination with the PowerPlex® 1.1 System,
the values exceed 0.9999951, demonstrating the usefulness of these two systems
for paternity analyses as well as for forensic determinations.
Note: The data in Tables 10, 11 and 12 for the PowerPlex® 1.1 System and FFFL
multiplex were published in Lins et al. (26). Data for the PowerPlex® 2.1 System
were generated as part of a collaborative effort between The Bode Technology
Group and Promega Corporation.
Table 12. Power of Exclusion of the PowerPlex® Systems in Various Populations.
Power of Exclusion
CaucasianHispanicAmerican
American
STR System
AfricanAmerican
PowerPlex®
0.9982125
0.9968853
0.9973337
0.9981793
0.9999219
0.9999242
0.9997134
0.9999759
0.9999988
0.9999982
0.9999951
0.9999995
0.9999999
0.9999999
0.9999995
0.9999999
1.2 System
PowerPlex® 16 System
1.1 System
plus PowerPlex® 2.1
System (14 STR loci)
Asian-American
PowerPlex®
PowerPlex®
1.1 System
System plus
PowerPlex® 2.1 System
plus FFFL Muliplex
(18 STR loci)
9.E. Methods for Polyacrylamide Gel Reuse
A simple technique has been developed to allow reuse of gels while effectively
eliminating the previous samples from the gel (12). With this technique, a gel has
been successfully reused up to eight times over a period of several days. With the
exception of the first and last lanes, the second and third runs are as good as, if
not better than, the first run. The bands continue to remain sharp on subsequent
runs but edge effects (frowning of the outside lanes) become progressively worse
and ultimately limit gel reuse if more than 3/4 of the gel is to be analyzed.
Gel sizes: 17cm × 43cm × 0.4cm; 17cm × 32cm × 0.4cm.
Combs: 15.2cm, 34-well flat-bottom; 16.3cm, 30-well flat-bottom.
Electrophoresis apparatus: Life Technologies Model SA; EC600 power supply
(E-C Apparatus Corporation).
Electrophoretic conditions: Maintain a plate temperature of 45 to 50°C
(60V/cm at a constant power of 55W for 43cm gels).
Analysis: Hitachi FMBIO® II Fluorescence Imaging System.
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9.E. Methods for Polyacrylamide Gel Reuse (continued)
Plate Preparation
1.
To ensure that the wells and gel remain firmly fixed to the plate, treat both
plates with methacryloxypropyltrimethoxysilane (9μl in 3ml 0.5% acetic
acid in 95% ethanol).
2.
Remove excess solution with 4 washes of ethanol. Only the top portion of
the short plate needs to be treated if three or fewer runs were performed
and the acrylamide solution was degassed.
Acrylamide Gel Solution Preparation
See Section 5.A–D for acrylamide gel preparation, gel pre-run, sample
preparation and gel electrophoresis.
Scanning
Scan the gel on a Hitachi FMBIO® II Fluorescence Imaging System, leaving the
plates, spacers and comb intact.
Removal of First-Run Samples from Gel and Second Pre-Run
Remove samples from the first run from the gel by reverse electrophoresis
(electrodes reversed). Use new buffer for this reverse electrophoresis, and
increase the reverse run time to 15–30 minutes longer than the first run at the
same wattage. This electrophoresis serves to preheat the gel for the second run.
It is not necessary to change the running buffer again between reverse
electrophoresis of the first run and loading of the second run; simply rinse wells
with running buffer and proceed with sample loading. If another run is not
planned for the same day, the gel may be stored as described below after
scanning. Gels with samples may be stored up to 3 days with no problem.
Longer periods have not yet been attempted. Different electrophoresis
apparatus can produce slightly different running conditions so it is advisable to
either use the same apparatus for both the forward and reverse electrophoresis
to ensure that the previous samples are removed in the pre-run or ensure that
different apparatus give comparable results.
Gel Storage
Following the final run of the day, store gels by placing plastic wrap around the
gel. Paper towels wetted with water should be placed inside the plastic wrap at
the top and bottom of the gel to help keep the gels from drying out. Reuse of
gels that are older than one week is not recommended.
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9.F. 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 (36). 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 9.J for ordering information.
For applications requiring human-specific DNA quantification, the Plexor® HY
System (Cat.# DC1000) has been developed (37). See Section 9.J for ordering
information.
The DNA IQ™ System has been fully automated on the Beckman Coulter
Biomek® 2000 Laboratory Automation Workstation (38), Biomek® 3000
Laboratory Automation Workstation (39) and Tecan Freedom EVO® Liquid
Handler (40). In addition, the DNA IQ™ Reference Sample Kit for Maxwell® 16
(Cat.# AS1040) and DNA IQ™ Casework Sample Kit for Maxwell® 16 are
available (41,42). For information about automation of laboratory processes on
automated workstations, contact your local Promega Branch Office or
Distributor (contact information available at: www.promega.com/worldwide/)
or e-mail: [email protected]
9.G. 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. Each fragment is labeled with carboxy-X-rhodamine
(CXR) and may be detected separately (as a third color) in the presence of
PowerPlex® 2.1-amplified material using the Hitachi FMBIO® and FMBIO® II
Fluorescence Imaging Systems. The ILS 600 is designed for use in each gel lane
to increase precision in analyses when using the PowerPlex® 2.1 System. This
practice reduces the number of allelic ladder lanes needed per gel and, therefore,
increases the number of lanes available for amplified sample materials. A
protocol for preparation and use of this internal lane standard is provided in
Section 5.C.
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 7/08
Part# TMD011
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9.H. Agarose Gel Electrophoresis of Amplification Products (Optional)
This procedure is optional if PCR is performed routinely in your laboratory.
Agarose gel electrophoresis can be used to rapidly confirm amplification
success prior to performing polyacrylamide gel or capillary electrophoresis.
Materials to Be Supplied by the User
(Solution compositions are provided in Section 9.I.)
• 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.
3.
Prepare 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 PCR success.
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# TMD011
Page 34
Printed in USA.
Revised 7/08
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9.I. Composition of Buffers and Solutions
40% acylamide:bis (19:1)
Dissolve 380g of acrylamide and
20g of bisacrylamide in 500ml of
deionized water. Bring volume to
1 liter with deionized water.
10% ammonium persulfate
Add 0.5g of ammonium persulfate to
5ml of deionized water. Use 200μl of
10% ammonium persulfate for each
30ml of acrylamide gel solution.
Store the remaining volume in 200μl
aliquots at –20°C.
Bromophenol Blue Loading
Solution
10mM
95%
0.05%
NaOH
formamide
bromophenol blue
0.5M EDTA (pH 8.0) stock
186.1g
Na2EDTA • 2H2O
Add EDTA to 800ml of deionized
water with vigorous stirring. Adjust
the pH to 8.0 with NaOH (about 20g
of NaOH pellets). Dispense into
aliquots and sterilize by autoclaving.
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.
Caution: Ethidium bromide is a
powerful mutagen. Wear gloves
when working with the dye, and
wear a mask when weighing it.
Gel Tracking Dye
10mM
95%
0.05%
0.05%
NaOH
formamide
bromophenol blue
xylene cyanol FF
★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
5X loading solution
5%
0.1%
0.1%
100mM
10mM
Ficoll® 400
bromophenol blue
xylene cyanol
EDTA
(Na2EDTA • 2H2O)
Tris-HCl (pH 7.5)
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.
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.
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 7/08
Part# TMD011
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9.J. Related Products
Fluorescent STR Multiplex Systems
Product
PowerPlex® 16 Monoplex System, Penta E (Fluorescein)
PowerPlex® 16 Monoplex System, Penta D (JOE)
PowerPlex® 1.1 System
GenePrint ® GammaSTR® Multiplex (Fluorescein)
GenePrint ® Fluorescent STR Multiplex—
CSF1PO, TPOX, TH01, vWA (Fluorescein)
(CTTv Multiplex)
GenePrint ® Fluorescent STR Multiplex—
F13A01, FESFPS, F13B, LPL (Fluorescein)
(FFFL Multiplex)
PowerPlex® 16 System
PowerPlex® ES System
PowerPlex® Y System
Size
100 reactions
100 reactions
100 reactions
400 reactions
100 reactions
400 reactions
Cat.#
DC6591
DC6651
DC6091
DC6090
DC6071
DC6070
100 reactions
400 reactions
DC6301
DC6300
100 reactions
400 reactions
100 reactions
400 reactions
100 reactions
400 reactions
50 reactions
200 reactions
DC6311
DC6310
DC6531
DC6530
DC6731
DC6730
DC6761
DC6760
Size
Cat.#
100 reactions
DC5171
100 reactions
DC6171
Size
150μl
65μl
150μl
150μl
150μl
Cat.#
DG1701
DG6221
DG3291
DG2121
DG2131
Not for Medical Diagnostic Use.
GenePrint ® Sex Identification Systems
Product
GenePrint ® Fluorescent Sex Identification
System—Amelogenin (Fluorescein)
GenePrint ® Fluorescent Sex Identification
System—Amelogenin (TMR)
Not for Medical Diagnostic Use.
Allelic Ladders
Product
Internal Lane Standard 600
Fluorescent Ladder (CXR), 60–400 Bases
GammaSTR® Allelic Ladder Mix (Fluorescein)
CTTv Allelic Ladder Mix (Fluorescein)
FFFL Allelic Ladder Mix (Fluorescein)
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
Part# TMD011
Page 36
Printed in USA.
Revised 7/08
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Page 37
Accessory Components
Product
Bromophenol Blue Loading Solution*
Gel Tracking Dye*
Gold ST★R 10X Buffer*
Mineral Oil
Nuclease-Free Water*
Size
3ml
(3 × 1ml)
1ml
(4 × 250μl)
1.2ml
12ml
50ml (2 × 25ml)
Cat.#
DV4371
DM2411
DY1151
P1193
Size
100 reactions
400 reactions
50 samples
200 samples
1 each
48 preps
48 preps
800 reactions
200 reactions
10 pack
Cat.#
DC6701
DC6700
DC6801
DC6800
AS2000
AS1040
AS1210
DC1000
DC1001
V1391
DV4361
*For Laboratory Use.
Sample Preparation Systems
Product
DNA IQ™ System**
Differex™ System*
Maxwell® 16 Instrument**
DNA IQ™ Reference Sample Kit for Maxwell® 16***
DNA IQ™ Casework Sample Kit for Maxwell® 16***
Plexor® HY System*
Slicprep™ 96 Device**
*Not for Medical Diagnostic Use.
**For Laboratory Use.
***For Research Use Only. Not for use in diagnostic procedures.
Polyacrylamide Gel Electrophoresis Reagents
Product
Ammonium Persulfate
TBE Buffer, 10X
Urea
Size
25g
1L
1kg
Cat.#
V3131
V4251
V3171
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
Size (tips/pack)
960
960
960
960
960
960
960
800
Cat.#
DY1051
DY1061
DY1071
DY1081
DY1101
DY1111
DY1121
DY1131
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 7/08
Part# TMD011
Page 37
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(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 and Korean Pat. No. 691195 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 and 6,221,598, Australian Pat. No. 724531, Canadian Pat. No.
2,118,048 and Korean Pat. No. 290332 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.
© 1999–2008 Promega Corporation. All Rights Reserved.
GammaSTR, GenePrint, Maxwell, Plexor and PowerPlex are registered trademarks of Promega
Corporation. Differex, DNA IQ and Slicprep are trademarks of Promega 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. Ficoll is a registered trademark of GE Healthcare Bio-sciences. FMBIO is
a registered trademark of Hitachi Software Engineering Company, Ltd. Freedom EVO is a
registered trademark of Tecan AG Corporation. 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. Kimwipes is a registered trademark of KimberlyClark. 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. MicroAmp is a registered trademark of Applera 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
Part# TMD011
Page 38
Printed in USA.
Revised 7/08
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