GenePrint STR Systems (Silver Stain Detection) TMD004

GenePrint STR Systems (Silver Stain Detection) TMD004
TECHNICAL MANUAL
GenePrint® STR Systems
(Silver Stain Detection)
InstrucƟons for use of Products
DC1191, DC4001, DC4011, DC4021, DC4031, DC4041, DC4051,
DC4061, DC4061, DC4071, DC4080, DC4081, DC6000, DC6001,
DC6031, DC6451, DG2141 and DG2101
Revised 10/15
TMD004
GenePrint ® STR Systems
(Silver Stain Detection)
All technical literature is available on the Internet at: www.promega.com/protocols
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 ....................................................................2
3.
Before You Begin .......................................................................................................................4
4.
Amplification .............................................................................................................................5
A. Choice of Thermal Cycling Protocol .............................................................................5
B. Amplification Setup.........................................................................................................9
C. Amplification Thermal Cycling ...................................................................................11
5.
Polyacrylamide Gel Preparation...........................................................................................12
A. Notes ................................................................................................................................12
B. Procedure.........................................................................................................................13
6.
Polyacrylamide Gel Electrophoresis....................................................................................15
A. Gel Pre-Run.....................................................................................................................15
B. Sample Preparation........................................................................................................15
C. Sample Loading..............................................................................................................16
D. Gel Electrophoresis ........................................................................................................16
7.
Silver Staining..........................................................................................................................17
A. Procedure.........................................................................................................................17
B. Reuse of Glass Plates .....................................................................................................18
8.
Exposure of Film......................................................................................................................19
9.
Data Analysis ...........................................................................................................................20
A. pGEM® DNA Markers...................................................................................................21
B. Controls ...........................................................................................................................21
C. STR Ladders ....................................................................................................................21
10.
Representative STR Data .......................................................................................................22
11.
Troubleshooting.......................................................................................................................26
12.
References .................................................................................................................................28
13.
Appendix ...................................................................................................................................30
A. Advantages of STR Typing...........................................................................................30
B. Advantages of Using the Loci in the GenePrint ® STR Systems...............................30
C. Power of Discrimination ...............................................................................................34
D. DNA Extraction and Quantitation Methods..............................................................35
E. Agarose Gel Electrophoresis of Amplification Products (Optional)......................36
F.
Composition of Buffers and Solutions........................................................................37
G. Population Data..............................................................................................................38
H. Organizational Sheets....................................................................................................39
I.
Related Products ............................................................................................................42
14.
Summary of Changes..............................................................................................................43
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 10/15
Part# TMD004
Page 1
1.
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 often
may be detected using PCR (5–8). Alleles of these loci are differentiated by the
number of copies of the repeat sequence contained within the amplified region and
are distinguished from one another using silver stain or fluorescent detection
following electrophoretic separation.
The GenePrint ® STR Systems provide all of the materials required to perform 100 or
400 amplification reactions except for Taq DNA polymerase and sample DNA.
Accessory components are available to simplify many of the procedures related to
STR analysis (Section 13.I).
This manual describes methods we have evaluated and recommend for sample
preparation, amplification of sample, separation of amplified products and silver
detection of separated material. All of the GenePrint ® STR Systems can be amplified
using either the Perkin-Elmer model 480 or 9600 thermal cyclers, but slight
differences in yield or balance between loci might be observed if the system is not
first optimized on that particular thermal cycler.
Information about allele frequencies for African-Americans, Caucasian-Americans and
Hispanic-Americans for all currently available STR Systems is available in Section
13.G. Additional population data for STR loci can be found in Edwards et al. (3), Puers
et al. (9), Hammond et al. (10), Bever et al. (11), Sprecher et al. (12) and Lins et al. (13).
2.
Product Components and Storage Conditions
GenePrint ® STR Multiplex Systems (Silver Stain Detection)
Each system contains the appropriate locus-specific primer pair and allelic
ladder in addition to STR 10X Buffer, K562 DNA, STR 2X Loading Solution and
pGEM® DNA Markers. Multiplex STR Systems include a single tube containing
all required 10X primer pairs as a mixture for simultaneous amplification of
more than one locus and another tube containing a mixture of the allelic ladders
for the same set of loci.
Product
GenePrint ® SilverSTR® III System(a)
Size
Cat.#
100 reactions
DC6451
400 reactions
DC6450
Not For Medical Diagnostic Use. Cat.# DC6450 contains sufficient reagents for
400 reactions of 25µl each. Includes:
•
•
•
•
•
•
•
1ml
600µl
1.2ml
3µg
2ml
3µg
1
SilverSTR® III 10X Primer Pair Mix
SilverSTR® III Allelic Ladder Mix
STR 10X Buffer
K562 DNA High Molecular Weight (10ng/µl)
STR 2X Loading Solution
pGEM® DNA Markers (20ng/µl)
Protocol
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Part# TMD004
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Printed in USA.
Revised 10/15
2.
Product Components and Storage Conditions (continued)
Product
GenePrint ® STR Multiplex System—
CSF1PO, TPOX, TH01 Multiplex(a)
GenePrint ® STR Multiplex System—
F13A01, FESFPS, vWA Multiplex
Size
Cat.#
100 reactions
400 reactions
DC6001
DC6000
100 reactions
400 reactions
DC6031
DC6030
Not For Medical Diagnostic Use.
GenePrint ® STR Monoplex Systems (Silver Stain Detection)
Each system contains the specific primer pair and ladder plus other components
sufficient to perform the specified number of reactions.
Product
GenePrint ® Sex Identification System
Amelogenin (Silver Detection)
GenePrint ® STR System—CSF1PO
GenePrint ® STR System—F13A01
GenePrint ® STR System—F13B
GenePrint ® STR System—FESFPS
GenePrint ® STR System—HPRTB
GenePrint ® STR System—LPL
GenePrint ® STR System—TH01
GenePrint ® STR System—TPOX
GenePrint ® STR System—vWA
Not For Medical Diagnostic Use.
Size
Cat.#
100 reactions
100 reactions
100 reactions
100 reactions
100 reactions
100 reactions
100 reactions
100 reactions
100 reactions
100 reactions
DC4081
DC4011
DC4041
DC4001
DC4021
DC4061
DC4071
DC1191
DC4051
DC4031
Size
150µl
150µl
Cat.#
DG2101
DG2141
Allelic Ladders(a)
Product
CTT Allelic Ladder Mix
FFv Allelic Ladder Mix
Storage Conditions: Store all components at –20°C. The pre- and postamplification components (Allelic Ladder Mix, STR 2X Loading Solution
and pGEM® DNA Markers) are sealed in separate packages to prevent crosscontamination. We strongly recommend that pre-amplification and postamplification reagents be stored separately and handled with different pipettes,
tube racks, etc. Store amplified material at –20°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 10/15
Part# TMD004
Page 3
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
(14,15). 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 amplification. We suggest strict adherence to
recommended procedures for amplification, denaturing gel electrophoresis, silver
stain analysis and recording of data on film.
PCR-based STR analysis is subject to contamination by very small amounts of
nontemplate human DNA. Extreme care should be taken to avoid cross-contamination
when preparing sample DNA, handling primer pairs, setting up amplification
reactions and analyzing amplification products. Reagents and materials used prior to
amplification (STR 10X Buffer, K562 Control DNA and 10X Primer Pairs) are provided
in a separate box and should be stored separately from those used following
amplification (Allelic Ladders, STR 2X Loading Solution and pGEM® DNA Markers).
Always include a negative control reaction (i.e., no template) to detect reagent
contamination. We highly recommend the use of gloves and aerosol-resistant pipet
tips.
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
acetic acid (fix/stop solution)
corrosive, hygroscopic
acrylamide
ammonium persulfate
suspected carcinogen, toxic
oxidizer, corrosive
bisacrylamide
toxic, irritant
formaldehyde (staining solution
and developer solution)
formamide (STR 2X Loading Solution)
highly toxic, suspected
carcinogen
irritant, teratogen
methacryloxypropyltrimethoxysilane (bind silane)
toxic, moisture sensitive
silver nitrate (staining solution)
highly toxic, oxidizer
sodium thiosulfate (developer solution)
irritant, hygroscopic
TEMED
corrosive, flammable
urea
irritant
xylene cyanol FF (STR 2X Loading Solution)
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
Part# TMD004
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Printed in USA.
Revised 10/15
4.
Amplification
The GenePrint ® STR Systems have been developed for amplification without artifacts
using standard Taq DNA polymerase. Special enzymes such as AmpliTaq Gold®
DNA polymerase are not required for peak performance. However, if using
AmpliTaq Gold® DNA polymerase, use the GoldST*R 10X Buffer (Cat.# DM2411,
available separately) instead of the STR 10X Buffer. The STR 10X Buffer (pH 9.0) is
not compatible with AmpliTaq Gold® DNA polymerase because the modified Taq
DNA polymerase is optimized at pH 8.3. Also, when using AmpliTaq Gold® DNA
polymerase, incorporate an additional incubation at 95°C for 11 minutes prior to
initiation of the thermal cycling program.
Materials to Be Supplied by the User
(Solution compositions are provided in Section 13.F.)
• thermal cycler, model 480 or GeneAmp® system 9600 (Perkin-Elmer)
• microcentrifuge
• Taq DNA polymerase
• Nuclease-Free Water (Cat.# P1193 or equivalent)
• Mineral Oil (Cat.# DY1151 or equivalent)
• 0.5ml or 0.2ml microcentrifuge tubes (compatible with thermal cycler)
• 1.5ml microcentrifuge tubes
• BSA Fraction V (optional)
• aerosol-resistant pipet tips
• crushed ice
4.A. Choice of Thermal Cycling Protocol
The CTT and FFv multiplexes, their corresponding monoplexes, the GenePrint ®
Sex Identification System—Amelogenin and the GenePrint ® STR System—HPRTB
are optimized for use with Perkin-Elmer GeneAmp® reaction tubes and the
Perkin-Elmer model 480 thermal cycler. The SilverSTR® III System is optimized
for use with MicroAmp® tubes and the GeneAmp® PCR system 9600 thermal
cycler. However, each system may be used with either thermal cycler.
Please refer to Tables 2 and 3 for recommended and alternative protocols for
each system and thermal cycler. The cycling conditions for each protocol are
given in Table 4. When using a thermal cycler on which a system was not
optimized, there may be a small loss in product yield or sensitivity, and the
balance between loci may change slightly in the multiplex systems. Meticulous
care must be taken to ensure successful amplification. A guide to amplification
troubleshooting is provided in Section 11.
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Printed in USA.
Revised 10/15
Part# TMD004
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Table 2. Protocol Options for the Model 480 Thermal Cycler.
Recommended
Protocols1
Alternative
Protocols2
CTT Multiplex
2
1
CTT Multiplex with Amelogenin
2
1
FFv Multiplex
7
1
7
NA
Amelogenin
2
1
CSF1PO, F13A01, TH01 or TPOX
2
NA
F13B, LPL or vWA
7
1
FESFPS or HPRTB
1
NA
GenePrint ® STR System
Multiplexes
SilverSTR®
III
Multiplex3
Individual Systems
NA = not applicable.
1Recommended protocols offer similar performance characteristics.
2Alternative protocols also work but may trade off performance characteristics, such as
greater speed or convenience, for less sensitivity.
3Performance variation of thermal cyclers may cause extraneous bands to be generated
above the allele range. See Section 9.
Table 3. Protocol Options for the GeneAmp® PCR System 9600 Thermal Cycler.
Recommended
Protocols1
Alternative
Protocols2
CTT Multiplex
5,6
12
CTT Multiplex with Amelogenin
5,6
12
FFv Multiplex
8,9
3,4
SilverSTR® III Multiplex3
10
NA
Individual Systems
Amelogenin, CSF1PO, F13A01,
TH01 or TPOX
5,6
NA
F13B, FESFPS, HPRTB, LPL or vWA
3,4
NA
GenePrint ® STR System
Multiplexes
NA = not applicable.
1Recommended protocols offer similar performance characteristics.
2Alternative protocols also work but may trade off performance characteristics, such as
greater speed or convenience, for less sensitivity.
3Performance variation of thermal cyclers may cause extraneous bands to be generated
above the allele range. See Section 9.
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# TMD004
Page 6
Printed in USA.
Revised 10/15
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
Printed in USA.
Revised 10/15
Part# TMD004
Page 7
Protocol
Number
1
(Refer to
Note 1)
2
(Refer to
Note 1)
3
(Refer to
Note 2)
4
(Refer to
Note 3)
5
(Refer to
Note 2)
6
(Refer to
Note 3)
7
(Refer to
Note 1)
8
(Refer to
Note 2)
Initial
Incubation2
96°C for
2 minutes
96°C for
2 minutes
96°C for
2 minutes
96°C for
2 minutes
96°C for
2 minutes
96°C for
2 minutes
96°C for
2 minutes
96°C for
2 minutes
Thermal
Cycler1
480
480
9600
9600
9600
9600
480
9600
Table 4. Amplification Protocols.
Cycling for
First 10 Cycles
94°C, 1 minute
60°C, 1 minute
70°C, 1.5 minutes
None
94°C, 1 minute
64°C, 1 minute
70°C, 1.5 minutes
None
94°C, 1 minute
60°C, 1 minute
70°C, 1.5 minutes
50 seconds to 94°C, 1 minute
34 seconds to 60°C, 1 minute
25 seconds to 70°C, 1.5 minutes
None
94°C, 1 minute
64°C, 1 minute
70°C, 1.5 minutes
50 seconds to 94°C, 1 minute
30 seconds to 64°C, 1 minute
15 seconds to 70°C, 1.5 minutes
None
94°C, 1 minute
60°C, 1 minute
70°C, 1.5 minutes
None
94°C, 1minute
60°C, 1 minute
70°C, 1.5 minutes
Programmed
Ramp Times
None
Cycling for
Last 20 Cycles
90°C, 1 minute
60°C, 1 minute
70°C, 1.5 minutes
None
90°C, 1 minute
64°C, 1 minute
70°C, 1.5 minutes
None
90°C, 1 minute
60°C, 1 minute
70°C, 1.5 minutes
45 seconds to 90°C, 1 minute
30 seconds to 60°C, 1 minute
25 seconds to 70°C, 1.5 minutes
None
90°C, 1 minute
64°C, 1 minute
70°C, 1.5 minutes
45 seconds to 90°C, 1 minute
26 seconds to 64°C, 1 minute
15 seconds to 70°C, 1.5 minutes
None
90°C, 1 minute
60°C, 1 minute
70°C, 1.5 minutes
None
90°C, 1 minute
60°C, 1 minute
70°C, 1.5 minutes
Programmed
Ramp Times
None
60°C for
30 minutes
60°C for
30 minutes
None
None
None
None
None
Extension
Step
None
4°C
4°C
4°C
4°C
4°C
4°C
4°C
Hold
Step
4°C
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Revised 10/15
Initial
Incubation2
96°C for
2 minutes
96°C for
1 minute
96°C for
2 minutes
96°C for
2 minutes
Thermal
Cycler1
9600
9600
9600
9600
Programmed
Cycling for
Ramp Times
First 10 Cycles
50 seconds to 94°C, 30 seconds
34 seconds to 60°C, 1 minute
25 seconds to 70°C, 1.5 minutes
Default ramp to 94°C, 30 seconds
68 seconds to 60°C, 30 seconds
50 seconds to 70°C, 45 seconds
50 seconds to 94°C, 1 minute
34 seconds to 60°C, 1 minute
25 seconds to 70°C, 1.5 minutes
50 seconds to 94°C, 1 minute
30 seconds to 64°C, 1 minute
15 seconds to 70°C, 1.5 minutes
Programmed
Cycling for
Ramp Times
Last 20 Cycles
45 seconds to 90°C, 1 minute
30 seconds to 60°C, 1 minute
25 seconds to 70°C, 1.5 minutes
Default ramp to 90°C, 30 seconds
60 seconds to 60°C, 30 seconds
50 seconds to 70°C, 45 seconds
45 seconds to 90°C, 1 minute
30 seconds to 60°C, 1 minute
25 seconds to 70°C, 1.5 minutes
45 seconds to 90°C, 1 minute
26 seconds to 64°C, 1 minute
15 seconds to 70°C, 1.5 minutes
None
60°C for
30 minutes
60°C for
30 minutes
Extension
Step
60°C for
30 minutes
4°C
4°C
4°C
Hold
Step
4°C
refers to the Perkin-Elmer model 480 thermal cycler; 9600 refers to the Perkin-Elmer GeneAmp® PCR system 9600 thermal cycler.
2Initial incubation performed using AmpliTaq® DNA polymerase. When using AmpliTaq Gold® DNA polymerase, include an additional incubation at 95°C for 11 minutes
prior to initiation of the thermal cycling program. Also when using AmpliTaq® DNA polymerase, be sure to use the GoldST*R 10X Buffer.
1480
Protocol
Number
9
(Refer to
Note 3)
10
(Refer to
Note 4)
11
(Refer to
Note 4)
12
(Refer to
Note 4)
Table 4. Amplification Protocols (continued).
Notes for Table 4:
1. Use GeneAmp® reaction tubes, and overlay all reactions with mineral oil.
2. Use GeneAmp® reaction tubes in combination with the GeneAmp® thinwalled tray. This reduces the maximum number of simultaneous reactions
to 48 due to the spacing of holes in the tray. Add mineral oil to all reactions.
3. Use MicroAmp® reaction tubes in the MicroAmp® 9600 tray. This allows a
maximum of 96 simultaneous reactions. Add mineral oil to all reactions.
not cover the reactions with the system 9600 thermal cycler lid. Cover
! Do
the reaction tubes loosely with aluminum foil.
Optional: Add BSA Fraction V (final concentration 60µg/ml) to all reactions.
This may result in a slight increase in yield. It will also produce higher silver
background in the gel lanes. We recommend Sigma BSA (Cat.# A2153).
Performance may vary depending on the source of this component.
4. Use MicroAmp® reaction tubes in the MicroAmp® 9600 tray. This allows a
maximum of 96 simultaneous reactions. No mineral oil is needed.
! Cover reactions with the system 9600 thermal cycler lid.
Optional: Add BSA Fraction V (final concentration 60µg/ml) to all reactions.
This may result in a slight increase in yield. It will also produce higher silver
background in the gel lanes. We recommend Sigma BSA (Cat.# A2153).
Performance may vary depending on the source of this component.
4.B. Amplification Setup
The use of gloves and aerosol-resistant pipet tips is highly recommended to
prevent cross-contamination. Helpful organizational sheets are provided in
Section 13.H.
1.
Thaw the STR 10X Buffer and 10X Primer Pair(s), and place on ice.
Notes:
1. Mix reagents by vortexing for 15 seconds before each use.
2. If using AmpliTaq Gold® DNA polymerase, use the GoldST*R 10X
Buffer (Cat.# DM2411) instead of the STR 10X Buffer.
2.
Place one clean, autoclaved 0.5ml reaction tube for each reaction into a
rack, and label appropriately.
Note: If using the GeneAmp® PCR system 9600 thermal cycler, refer to the
notes for Table 4 for tube selection.
3.
Determine the number of reactions to be set up. This should include a
positive and negative control reaction. 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.
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Part# TMD004
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4.B. Amplification Setup (continued)
4.
Calculate the required amount of each component of the PCR master mix
(Table 5). Multiply the volume (µl) per sample by the total number of
reactions (from Step 3) to obtain the final volume (µl).
Note: The CTT Multiplex and Amelogenin locus can be amplified
simultaneously.
5.
In the order listed in Table 5, add the final volume of each reagent to a
sterile tube. Mix gently (do not vortex), and place on ice.
Note: The volume given assumes a Taq DNA polymerase concentration of
5u/µl. For different enzyme concentrations, the volume of enzyme added
must be adjusted accordingly. If the final volume of Taq DNA polymerase
added to the master mix is less than 0.5µl, you may wish to dilute the
enzyme with STR 1X Buffer, and add a larger volume. The amount of
sterile water should be adjusted accordingly so that the final volume per
reaction is 25µl. Do not store diluted Taq DNA polymerase.
Table 5. PCR Amplification Reaction Setup.
Multiplex Reactions Containing Three Loci
PCR Master Mix Component
sterile water
STR 10X Buffer
Multiplex 10X Primer Pair Mix
Taq DNA polymerase (at 5u/µl)
total volume
Volume Per
Sample (µl)
17.35
2.50
2.50
0.15 (0.75u)
22.50
×
Number of
Final
=
Reactions
Volume (µl)
Combined CTTv Multiplex and Amelogenin Reactions
PCR Master Mix Component
sterile water
STR 10X Buffer
CTT Multiplex 10X Primer Pair Mix
Amelogenin 10X Primer Pair
Taq DNA polymerase (at 5u/µl)
total volume
Volume Per
Sample (µl)
14.85
2.50
2.50
2.50
0.15 (0.75u)
22.50
×
Number of
Final
=
Reactions
Volume (µl)
Monoplex or Amelogenin-Only Reactions
PCR Master Mix Component
sterile water
STR 10X Buffer
locus-specific 10X primer pair
Taq DNA polymerase (at 5u/µl)
total volume
Volume Per
Sample (µl)
17.45
2.50
2.50
0.05 (0.25u)
22.50
×
Number of
Reactions
=
Final
Volume (µl)
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Revised 10/15
6.
!
7.
Add 22.5µl of PCR master mix to each tube, and place on ice.
Failure to keep the reagents and samples on ice can produce imbalanced
amplification of multiplexed loci. If using AmpliTaq Gold® DNA
polymerase, it is not necessary to keep the reactions on ice.
Pipet 2.5µl of each sample into the respective tube containing 22.5µl of
PCR master mix.
Notes:
1.
For the multiplex CTT and SilverSTR® III Systems, use 1–5ng of
template DNA.
For the multiplex FFv System, use 5–10ng of template DNA.
2.
If the template DNA is stored in TE buffer (10mM Tris-HCl, 1mM
EDTA [pH 7.5]), the volume of the DNA sample added should not
exceed 20% of the final reaction volume. PCR amplification efficiency
and quality can be altered greatly by changes in pH (due to added
Tris-HCl) or available magnesium concentration (due to chelation by
EDTA). DNA samples stored or diluted in sterile, deionized water are
not subject to this caution.
8.
For the positive amplification control, pipet 2.5µl (5ng) of K562 DNA (diluted
to 2ng/µl) into a 0.5ml reaction tube containing 22.5µl of PCR master mix.
9.
For a negative amplification control, pipet 2.5µl of sterile water (instead of
template DNA) into a 0.5ml reaction tube containing 22.5µl of PCR master
mix.
10. If recommended by the cycling protocol, add 1 drop of mineral oil to each
tube. Close the tubes.
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.
11. Centrifuge the samples briefly to bring the contents to the bottom of the
tube.
4.C. Amplification Thermal Cycling
1.
Place the tubes in a thermal cycler.
2.
Select and run a recommended protocol from Table 2 or 3 (Section 4.A).
3.
After completion of the thermal cycling protocol, store the samples at
–20°C.
Note: Storage of amplified samples at 4°C or above may produce
degradation products.
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Printed in USA.
Revised 10/15
Part# TMD004
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5.
Polyacrylamide Gel Preparation
Materials to Be Supplied by the User
(Solution compositions are provided in Section 13.F.)
• 40% acrylamide:bis (19:1) and TEMED
• 10X TBE Buffer (Cat.# V4251)
• 10% Ammonium Persulfate (Cat.# V3131)
• Urea (Cat.# V3171)
• bind silane (methacryloxypropyltrimethoxysilane)
• Gel Slick® solution (Cambrex Cat.# 50640)
• 0.5% acetic acid in 95% ethanol
• Nalgene® tissue culture filter (0.2 micron)
• polyacrylamide gel electrophoresis apparatus for gels ≥30cm (e.g., SA32 or S2)
• glass plates and side spacers for polyacrylamide gel ≥30cm
• 14cm vinyl doublefine sharkstooth comb(s), 49 point, 0.4mm thick; or square-tooth
comb, 35cm, 60 wells (cut in half for 30 wells/gel), 0.4mm thick (Owl Scientific
Cat.# S2S-60A)
• power supply
• Liqui-Nox® detergent (Use of Liqui-Nox® detergent is extremely important, as
other kinds of detergent can build up on the glass plates.)
• clamps (e.g., large office binder clips)
• diamond pencil for marking glass plates
5.A. Notes
1.
Use 6% acrylamide for the GenePrint ® SilverSTR® III System. In a 4% gel,
DNA strand separation in the locus D16S539 are such that the top strand
of one allele overlaps with the bottom strand of the next larger allele. See
Figure 2.
2.
Unpolymerized acrylamide is a neurotoxin and suspected carcinogen; avoid
inhalation and contact with skin. Read the warning label, and take the
necessary precautions when handling this substance. Always wear gloves
and safety glasses when working with acrylamide powder or solutions.
3.
Bind silane is toxic and should be used in a chemical fume hood.
4.
The longer glass plate will be treated with Gel Slick® solution to prevent the
gel from sticking, and the shorter glass plate will be treated with bind silane
to bind the gel. The two plates must be kept apart at all times to prevent
cross-contamination.
5.
All cleaning utensils (sponges) for the longer glass plates should be kept
separate from those for the shorter glass plates to prevent crosscontamination of the binding solution.
6.
The shorter glass plate preparation must be repeated for each gel. The
longer glass plate preparation must be repeated after every four gels.
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7.
To remove the glass plate treatments (Gel Slick® solution or bind silane)
immerse the plate(s) in 10% NaOH solution for 1 hour. Thoroughly rinse
the plate(s) with deionized water, and clean with a detergent. The same
10% NaOH solution may be used for multiple gels.
8.
New glass plates should be soaked in 10% NaOH for 1 hour and then
rinsed thoroughly with deionized water before use. New plates also should
be etched with a diamond pencil in the corner of one side to distinguish the
sides of the plates in contact with the gel.
5.B. Procedure
The following protocol is for the preparation of a denaturing polyacrylamide
gel with the dimensions of 31.0cm wide × 38.5cm high × 0.4mm thick (e.g., S2
sequencing gel electrophoresis apparatus, Whatman Cat.# 21105-010). Use onehalf of the volumes described here for a gel with the dimensions of 17cm wide
× 32cm high × 0.4mm thick (e.g., SA32 sequencing gel apparatus, Whatman
Cat.# 31096-019).
1.
Etch each glass plate on one side in one corner with a diamond pencil to
distinguish the treated sides of the glass plates. Thoroughly clean the shorter
and longer glass plates twice with 95% ethanol and Kimwipes® tissues.
Note: The gel side is the etched side of the glass plate.
2.
Using gloves, apply 3ml of Gel Slick® solution onto the etched side of the
longer glass plate. With a dry paper towel, spread the Gel Slick® solution
using a circular motion over the entire surface.
3.
Wait 5 minutes for the Gel Slick® solution to dry. Remove the excess Gel
Slick® solution with a paper towel saturated with deionized water. Finally,
dry the glass plate with Kimwipes® tissue.
4.
In a chemical fume hood, prepare fresh binding solution by adding 3µl of
bind silane to 1ml of 0.5% acetic acid in 95% ethanol in a 1.5ml tube. Wipe
the etched side of the shorter glass plate using a Kimwipes® tissue saturated
with the freshly prepared binding solution. Be certain to wipe the entire
plate surface with the saturated tissue.
5.
Wait 5 minutes for the binding solution to dry. Wipe the shorter glass
plate 3–4 times with 95% ethanol and Kimwipes® tissues to remove the
excess binding solution.
!
Failure to wipe excess binding solution from the shorter glass plate will
cause the gel to stick to both plates, and the gel will be destroyed upon
separation of the glass plates after electrophoresis.
6.
Take special care not to allow the treated surfaces to touch each other.
Assemble the glass plates by placing 0.4mm side spacers and a 0.4mm
bottom spacer (optional) between the plates and using clamps to hold
them in place. Lean the assembled plates against a test tube rack or other
similar support.
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5.B. Procedure (continued)
7.
Prepare a 4% or 6% acrylamide solution (total of 75ml) by combining the
ingredients listed below.
Component
urea
deionized water
10X TBE buffer
40% acrylamide:bis (19:1)
total volume
4% Gel
31.50g
40.00ml
3.75ml
7.50ml
75ml
6% Gel
31.50g
36.25ml
3.75ml
11.25ml
75ml
Final
Concentration
7M
–
0.5X
4% or 6%
Note: If preparing multiple gels on a daily basis, a larger 4% or 6% stock
solution may be prepared, filtered as in Step 8 below, and stored at 4°C in
the dark for up to one month. To prepare a single gel, remove 75ml of this
stock solution, and continue with Step 9.
8.
Filter the acrylamide solution through a 0.2 micron filter (e.g., Nalgene®
tissue culture filter).
9.
Pour the filtered acrylamide solution into a squeeze bottle.
10. Add 50µl of TEMED and 500µl of 10% ammonium persulfate to the
acrylamide solution, and mix gently.
11. Carefully pour the acrylamide solution between the glass plates. To
prevent bubble formation, start pouring at one side of the assembled
plates and maintain a constant flow of solution.
12. Position the gel horizontally, resting it on two test tube racks or other
similar supports. Remove any bubbles that may have formed.
13. Insert one or two 14cm doublefine (49 point) sharkstooth combs, straight side
into the gel, between the glass plates (6mm of the comb should be between
the two glass plates). If using a square-tooth comb, insert the comb between
the glass plates until the teeth are almost completely inserted into the gel.
14. Secure the comb(s) with 2–3 clamps each.
15. Pour the remaining acrylamide solution into a disposable conical tube as a
polymerization control. Rinse the squeeze bottle, including the spout, with
water.
16. 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
deionized water and plastic wrap are placed around the well end of the
gel to prevent the gel from drying out. If no bottom spacer is used, the
bottom of the gel should be wrapped.
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6.
Polyacrylamide Gel Electrophoresis
6.A. Gel Pre-Run
1.
Remove the 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. Remove the comb
and bottom spacer.
3.
Add 0.5X TBE to the bottom chamber of the electrophoresis apparatus.
4.
Gently lower the gel and glass plates into the buffer with the longer plate
facing out and the well-side on top.
5.
Secure the glass plates to the sequencing gel apparatus.
6.
Add 0.5X TBE 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. Use a syringe with a bent 19-gauge needle to
remove the air bubbles between the glass plates on 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 60–65 watts for a 40cm
polyacrylamide gel, 40–45 watts for a 32cm gel. The gel running conditions
may have to be adjusted in order to reach a temperature of 50°C.
6.B. Sample Preparation
1.
Prepare the PCR samples by mixing 2.5µl of each sample with 2.5µl of STR
2X Loading Solution.
Note: The sample alleles may appear more intense than ladder alleles on
the gel, but this should not interfere with allele determination. For more
even band intensities, mix 1µl of each sample with 4µl of a premix
containing 2.5µl of STR 2X Loading Solution and 1.5µl of STR 1X Buffer.
2.
Add 2.5µl (50ng) of pGEM® DNA Markers to 2.5µl of STR 2X Loading
Solution for each marker lane.
Note: We recommend loading pGEM® DNA Markers into the first and last
lanes of the gel.
3.
Add 2.5µl of the Allelic Ladder Mix to 2.5µl of STR 2X Loading Solution for
each allelic ladder lane. The number of allelic ladder lanes used depends on
personal preference.
Note: For combined CSF1PO, TPOX, TH01 multiplex and Amelogenin
reactions, mix the corresponding allelic ladder mixes 1:1, then add 2.5µl of
allelic ladder mix to 2.5µl of STR 2X Loading Solution.
4.
Briefly centrifuge the samples in a microcentrifuge to bring the contents to
the bottom of the tube.
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6.C. Sample Loading
1.
Denature the samples by heating at 95°C for 2 minutes, then immediately
chill on crushed ice or in an ice-water bath.
Note: Denature the samples just prior to loading the instrument.
2.
After the pre-run (Section 6.A), use a 50–100cc syringe filled with buffer to
flush the urea from the well area. If using a sharkstooth comb, carefully
insert the comb teeth into the gel approximately 1–2mm. Leave the comb
inserted in the gel during both gel loading and electrophoresis.
3.
Load 3µl of each sample into the respective wells. The loading process
should take no longer than 20 minutes to prevent the gel from cooling.
Note: An organizational sheet for loading a gel is provided in Section 13.H.
6.D. Gel Electrophoresis
1.
Once loading is complete, run the gel using the same conditions as in
Section 6.A.
If you are loading the gel multiple times, allow the gel to run 20–30 minutes
before loading the next set of samples (Figure 4). This will prevent the
samples from overlapping during electrophoresis. (Do not do this with
multiplex systems.)
Note: In a 6% gel, bromophenol blue migrates at approximately 25 bases
and xylene cyanol migrates at approximately 105 bases. In a 4% gel,
bromophenol blue migrates at approximately 40 bases and xylene cyanol
migrates at approximately 170 bases.
2.
Knowing the size ranges for each locus (Tables 7 and 8, Section 13.B) and
migration characteristics of the dyes (Step 1, above), stop electrophoresis
any time after the locus of interest has passed the midpoint of the gel. If
running more than one locus or a multiplex, be careful not to run the
smallest locus off the bottom of the gel.
3.
Proceed to Section 7 for silver stain detection.
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7.
Silver Staining
This protocol describes the use of the SILVER SEQUENCE™ Staining Reagents (Cat.#
Q4132). One system contains sufficient reagents to stain 10 sequencing size gels and
includes:
•
•
•
•
•
500µl
20g
60ml
10ml
600g
Bind Silane
Silver Nitrate (10 × 2g)
Formaldehyde, 37% (20 × 3ml)
Sodium Thiosulfate, 10mg/ml (10 × 1ml)
Sodium Carbonate (10 × 60g)
Materials to Be Supplied by the User
(Solution compositions are provided in Section 13.F.)
• fix/stop solution
• staining solution
• developer solution (chilled to 4–10°C)
• Nalgene® wash tubs (54.1 × 43.5 × 13cm or appropriate size for your system)
• orbital shaker or rocker platform
Use 2 liters of each solution per gel for each step (for a 54.1 × 43.5 × 13cm tray).
7.A. Procedure
1.
After electrophoresis, empty the buffer chambers and carefully loosen the
gel clamps. Remove the glass plates from the apparatus.
2.
Place the gel and glass plates on a flat surface. Remove the comb and side
spacers. Use a plastic wedge to carefully separate the two glass plates. The
gel should be strongly affixed to the shorter glass plate.
3.
Place the gel (attached to the shorter plate) in a shallow plastic tray (e.g.,
Nalgene® wash tub).
4.
To silver stain, follow Steps a–h. Gently agitate during each step.
!
Steps involving solutions containing formaldehyde should be performed
in a chemical hood.
Step
a.
b.
c.
d.
e.
f.
Solution
fix/stop solution (See Note 1)
deionized water
repeat Step b, twice
staining solution
deionized water (See Note 2)
developer solution
g.
h.
fix/stop solution (See Note 3)
deionized water
Time
20 minutes
2 minutes
2 × 2 minutes
30 minutes
10 seconds
up to 5 minutes (until alleles
and ladders are visible)
5 minutes
2 minutes
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7.A. Procedure (continued)
Notes:
5.
1.
Save the fix/stop solution from Step 4a, to use in Step 4g.
2.
The duration of Step 4e is important. The total time from immersion
in deionized water to immersion in developer solution should be less
than 20 seconds. If the deionized water rinse step does exceed
20 seconds, repeat Step 4d.
3.
Add fix/stop solution directly to developer solution to stop developing
reaction.
Position the gel and shorter plate upright, and allow it to dry overnight.
For best results, the gel should be completely dried before APC Film
development. Alternatively, to create film prints of the gel immediately,
cover the gel with plastic wrap, and proceed to Section 8.
Figure 1, Section 10 shows typical results for the multiplex GenePrint ® STR
Systems. Figure 3, Section 10, shows typical results for the Amelogenin
locus and the STR loci, F13B, HPRTB and LPL using a dilution series of
250ng to 0.5ng of template DNA.
7.B. Reuse of Glass Plates
1.
For disposal, immerse the plate and affixed gel in a 10% NaOH solution
for 1 hour to overnight. Discard the gel, and clean the glass plate with
deionized water and a detergent such as Liqui-Nox® detergent. The 10%
NaOH solution may be reused for additional gels.
2.
All cleaning utensils and sponges for the longer glass plates should be
kept separate from those for the shorter glass plates to prevent crosscontamination of the binding solution.
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8.
Exposure of Film
A direct image may be produced using Automatic Processor Compatible (APC) Film.
The image produced on APC Film is the mirror image of the gel. Use of film allows
the generation of multiple permanent images with more control over band and
background intensity than does development of the gel alone. Handle all plates with
gloved hands to avoid fingerprints.
Materials to Be Supplied by the User
(Solution compositions are provided in Section 13.F.)
• white light box
• automatic film processor or film developing tanks
• Automatic Processor Compatible (APC) Film (Cat.# Q4411)
1.
In the darkroom with a safelight on, place the dry, stained gel attached to the
shorter plate (gel side up) on a white fluorescent light box.
Note: For best results, the gel should be completely dry before the image is
captured with APC film. If capturing an image from a gel that has not been
dried, cover the gel with plastic wrap.
2.
Position the APC Film, emulsion side down, over the gel to be copied.
Note: The emulsion side of the film can be identified as the glossy white surface;
the nonemulsion side has a gray tint.
3.
Place a clean glass plate on top of the film to maintain contact between the gel
and film. Turn on the white light box, and expose the film for 1–2 minutes,
depending on the gel background level and the intensity of the white light.
(This step must be optimized for individual light boxes.)
4.
Develop the film as recommended by the manufacturer. APC film may be
processed manually or with an automatic film processor. For automatic film
processors, follow the manufacturer’s instructions.
Note: The image produced on APC Film is the mirror image of the gel.
5.
If there is very little signal, decrease the exposure time used in Step 3. If the film
appears brown or black, increase the exposure time.
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9.
Data Analysis
For ease of interpretation, allelic ladders should be run in lanes adjacent to each
sample. Direct comparison between the allelic ladders and amplified samples of the
same locus allows for assignment of alleles. Note that microvariant alleles, such as
the TH01 allele 9.3 and the F13A01 allele 3.2, do not co-migrate with allelic ladder
fragments. In addition, mutations or rare alleles may be seen occasionally. The
migration of such “off-ladder” alleles cannot be predicted.
With silver stain detection, both DNA strands are detected. For some loci, such as
TH01, FESFPS and vWA, the difference in the sequence of the opposing strands causes
them to migrate at different rates. This results in doublets for each allele (Figure 1).
This strand separation may be more pronounced with longer electrophoresis of gels as
seen for the sequential loading of TH01 amplification products in Figure 4 (Section 10).
Note that in the case of locus F13A01, more pronounced separation of opposing
strands is observed with the larger alleles (see Figure 1).
Artifact bands also may be detected with these systems. Shadow banding (16–18) or
repeat slippage appears as faint bands one repeat unit (i.e., 4 bases) below the true
alleles. This is most pronounced with the vWA locus (Figure 1).
Terminal nucleotide addition occurs when Taq DNA polymerase catalyzes
nontemplated addition of a nucleotide to the 3´-termini of amplified DNA fragments
(18–20). A band that is one base shorter than the expected allele may result from the
inefficiency of the terminal nucleotide addition. An artifact band is generated when
this terminal addition does not occur with 100% efficiency. This may be visualized as
an extra band, as seen in Figure 3 with the LPL and F13B loci. The addition of a final
extension step (amplification protocols 7,8,9,10,11; Table 4) increases the amount of
product that contains the added terminal nucleotide, thus minimizing the shorter
artifact band (18).
For the GenePrint ® SilverSTR® III System, performance variation of thermal cyclers
may cause extraneous bands to be generated above the allele range. The use of
AmpliTaq Gold® DNA polymerase with this system may minimize or eliminate
these extra bands. Alternatively, raising the annealing temperature to 62°C can also
minimize or eliminate these bands. At annealing temperatures higher than 62°C, the
amplification of D7S820 alleles may be compromised.
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9.A. pGEM® DNA Markers
The pGEM® DNA Markers are visual standards used to confirm allelic size
ranges for the loci. The markers consist of fifteen DNA fragments with the
following sizes (in base pairs):
2,645
1,605
1,198
676
517
460
396
350
222
179
126
75
65
51
36
9.B Controls
Observe the lanes containing the negative controls. They should be devoid of
amplification products.
Observe the lanes containing the positive K562 DNA positive controls.
Compare the K562 DNA allelic repeat sizes with the locus-specific allelic ladder.
The expected K562 DNA allele size(s) for each locus are listed in Tables 7 and 8,
Section 13.B.
9.C. STR Ladders
Each locus or multiplex has a characteristic allelic ladder. Please refer to Section
13.B for locus-specific allelic ladder information. In general, the allelic ladders
contain fragments of the same lengths as either several or all known alleles for
the locus. Visual comparison between the allelic ladder and amplified samples
of the same locus allows precise assignment of alleles.
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10.
Representative STR Data
CTT
Multiplex
FFv
Multiplex
L 1 2 L 3 4 L
SilverSTR® III
Multiplex
L1 2L 3 4 L
– 15
CSF1PO
L
– 16
F13A01
–7
1
2
L
3
4
L
– 15
D16S539
–4
–5
– 14
– 13 FESFPS
– 14
–7
D7S820
TPOX
–6
–6
– 15
– 11
– 20
TH01
vWA
– 13
–7
Figure 1. GenePrint ® STR Multiplex Systems. Individual genomic DNA samples (lanes 1–4) were
amplified using GenePrint ® STR Systems as indicated and detected using silver staining as described
in this manual. The amplification products using the CTT (CSF1PO, TPOX, TH01) Multiplex and the
FFv (F13A01, FESFPS, vWA) Multiplex were separated using a 4% denaturing polyacrylamide gel.
Amplification products using the SilverSTR® III (D16S539, D7S820, D13S317) System were separated
in a 6% denaturing polyacrylamide gel. Lanes labeled (L) contain allelic ladders for the respective
loci. Numbers to the right of each image indicate the smallest and largest number of repeat units
present in corresponding fragments of each allelic ladder.
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5808TA
–5
D13S317
6% Denaturing Gel
4% Denaturing Gel
L 1
2
L
3
4
L
5
6
L 1
L
15
2 L
3
4 L
5
6
L
15
D16S539
D16S539
5
5
14
14
D7S820
D7S820
6
6
D13S317
D13S317
7
5809TA
15
15
7
Figure 2. GenePrint ® SilverSTR® III System. Lanes 1–6 show amplification of 1ng of human DNA
using the GenePrint ® SilverSTR® III System. Amplification products were separated using a 4%
denaturing polyacrylamide gel and a 6% denaturing polyacrylamide gel and were detected by silver
stain analysis. Lanes labeled L contain the SilverSTR® Allelic Ladder Mix. Numbers to the right of
each image indicate the smallest and largest number of tandem repeat units present in corresponding
fragments of each allelic ladder. Note that for the locus D16S539 the strands of the individual alleles
separate forming doublets. This results from sequence differences between the two complementary
strands, which affect their relative migration. In a 6% gel, the doublets are closely spaced and do not
interfere with interpretation. In a 4% gel, however, these doublets separate such that the top strand
from one allele overlaps with the bottom strand of the next larger allele, requiring greater care during
interpretation.
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A.
B.
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
17
16
15
14
13
12
11
10
9
8
7
6
218bp (Y)
212bp (X)
Amelogenin
C.
1 2 3 4 5 6 7 8
HPRTB
14
13
D.
1 2 3 4 5 6 7 8
12
11
10
10
9
9
8
6
7
LPL
F13B
0753TA08_4A
0753TA08_4A
7
Figure 3. Amplification of varying concentrations of K562 template DNA at different STR
loci and the Amelogenin locus. K562 DNA was amplified at the Amelogenin locus and various
STR loci. The Perkin-Elmer model 480 thermal cycler was used with protocols 1 or 2 (Table 4).
The use of protocol 7 does not produce the lowest fragment of each trio seen in the F13B and
LPL products (data not shown). For each panel, lanes 1 and 8 contain the locus-specific allelic
ladder; lanes 2–6 contain amplified K562 DNA using 250, 25, 5, 1 and 0.5ng of starting template,
respectively; lane 7 contains a negative control amplification reaction (i.e., no template DNA).
Note: The F13B Allelic Ladder has been updated to include alleles 5 through 11.
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1 L 2 3 L 4 5 L 6 7 L 8
0097TA07_3A
11
10
9
8
7
6
5
Load #3
Total time = 50 Minutes
11
10
9
8
7
6
5
Load #2
Total time = 80 Minutes
11
10
9
8
7
6
5
Load #1
Total time = 110 Minutes
Figure 4. Sequential loading of STR locus TH01. For more efficient use of one 4% denaturing
polyacrylamide gel, samples may be loaded at 30-minute intervals to obtain three times the
amount of information when analyzing an individual STR system. Randomly selected DNA
samples were amplified using the GenePrint ® STR System—TH01 as described in this manual.
Samples and ladders were loaded at three different times: load #1 (time 0), load #2 (30 minutes
after load #1) and load #3 (60 minutes after load #1). Following load #3, samples were run for
an additional 50 minutes. Separated products were detected by silver stain analysis. Lanes L,
TH01 Allelic Ladder; lanes 1–8, amplified DNA from several individuals.
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11.
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 no bands
Bands are fuzzy
throughout the lanes
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, inhibitors may exist in the
DNA sample.
Insufficient template DNA. Use the recommended amount of
template DNA.
Insufficient enzyme activity. Use the recommended amount of
Taq DNA polymerase. Check the expiration date on the tube
label.
Wrong amplification program. Choose the correct amplification
program for each locus.
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. We have not tested other reaction tubes
or thermal cyclers. Calibration of the thermal cycler heating
block may be required.
Primer concentration was too low. Use the recommended
primer concentration. Mix well before use.
Ice was not used to set up reactions. Set up the reactions on
crushed ice. Very light allele intensity is obtained with some
loci if ice is not used when setting up the reactions. The use of
AmpliTaq Gold® DNA polymerase will also remedy this
problem.
Samples were not denatured before loading onto the gel. Be
sure the samples are heated at 95°C for 2 minutes immediately
prior to loading.
Improper rinsing following staining. The rinse step was
performed for more than 20 seconds. Longer rinses remove the
silver deposited on the DNA. Rinse for a shorter time.
Poor-quality water was used. Use ultrapure water (e.g.,
NANOpure®- or Milli-Q®-purified water) or double-distilled
water.
Poor-quality polyacrylamide gel. Prepare acrylamide and
buffer solutions using high-quality reagents. Store acrylamide
solutions in the dark.
Electrophoresis temperature was too high. Run gel at lower
temperature (40–60°C).
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11.
Troubleshooting (continued)
Symptoms
Background smearing
within lanes
Extra bands visible in
one or all of the lanes
Gel looks yellow after
silver staining
High background on gel
Gel adheres to both plates
Causes and Comments
Poor-quality polyacrylamide gel. The amount of smearing may
be reduced with high-quality polyacrylamide.
Note: The allelic ladder for the D16S539 locus in the
SilverSTR® III System shows some smearing.
Sample overloading. Reduce the amount of template DNA
used, or dilute the amplification reaction before mixing with
loading buffer.
BSA was used in amplification. The use of BSA is optional in
the amplifications. BSA will stain with silver, resulting in a
high-molecular-weight smear within the sample lane.
Contamination with another template DNA or previously
amplified DNA. Cross-contamination can be a problem. Use
aerosol-resistant pipet tips, and change gloves regularly.
Artifacts of STR amplification. PCR amplification sometimes
generates artifacts that appear as faint bands one or four bases
below an allele. Refer to Section 13.B for locus-specific
information regarding this event.
Artifacts of amplification with the GenePrint ® SilverSTR® III
System. Performance variation of thermal cyclers may cause
extraneous bands to be generated outside of the allele range.
The use of AmpliTaq Gold® DNA polymerase with this system
may minimize or eliminate these extra bands. Alternatively,
raising the annealing temperature to 62°C can also minimize
or eliminate these bands. At annealing temperatures higher
than 62°C, the amplification of D7S820 alleles may be
compromised.
Samples were not completely denatured. Heat denature the
samples at 95°C for 2 minutes immediately prior to loading
the gel.
Gel was left in the developer solution too long. Do not leave
gel in developer solution for more than 5 minutes. Often,
2 minutes is sufficient.
Excess silver nitrate was present. Rinse the gel for 10 seconds
in deionized water before adding developer solution.
Development was too long. Stop the development reaction
after the appearance of the alleles and ladders.
Longer glass plate was contaminated with binding solution, or
treatment of the longer glass plate with Gel Slick® solution was
inadequate. Wipe excessive binding solution from the short
glass plate. Exercise care to avoid contaminating the longer
glass plate with binding solution. Ensure uniform coverage of
the longer plate with Gel Slick® 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
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12.
References
1.
Edwards, A. et al. (1991) DNA typing with trimeric and tetrameric tandem repeats: Polymorphic loci,
detection systems, and population genetics. In: Proceedings from 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, 74–56.
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 beta-actin related
pseudogene 2 (ACTBP2) detected using the polymerase chain reaction. Nucl. Acids Res. 19, 6980.
5.
Ausubel, F.M. et al. (1993) Unit 15: The polymerase chain reaction. In: Current Protocols in Molecular
Biology, Greene Publishing Associates and Wiley-Interscience, NY.
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, Cold Spring Harbor, NY.
7.
PCR Technology: Principles and Applications for DNA Amplification (1989) ed., Erlich, H.A., Stockton
Press, NY.
8.
PCR Protocols: A Guide to Methods and Applications (1990) eds., Innis, M.A. et al., Academic Press, San
Diego, CA.
9.
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.
10.
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.
11.
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.
12.
Sprecher, C.J. et al. (1996) General approach to analysis of polymorphic short tandem repeat loci.
BioTechniques 20, 266–76.
13.
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.
14.
Presley, L.A. et al. (1992) The implementation of the polymerase chain reaction (PCR) HLA DQ alpha
typing by the FBI laboratory. In: Proceedings from the Third International Symposium on Human
Identification 1992, Promega Corporation, 245–69.
15.
Hartmann, J.M. et al. (1991) Guidelines for a quality assurance program for DNA analysis. Crime
Laboratory Digest 18, 44–75.
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. Nucl. Acids Res. 20,
211–5.
18.
Walsh, P.S., Fildes, N.J. and Reynolds, R. (1996) Sequence analysis and characterization of stutter
products at the tetranucleotide repeat locus vWA. Nucl. Acids Res. 24, 2807–12.
19.
Smith, J.R. et al. (1995) Approach to genotyping errors caused by nontemplated nucleotide addition by
Taq DNA polymerase. Genome Res. 5, 312–7.
20.
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.
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# TMD004
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Printed in USA.
Revised 10/15
12.
References (continued)
21.
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.
22.
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.
23.
Nakamura, Y. et al. (1987) Variable number of tandem repeat (VNTR) markers for human gene
mapping. Science 235, 1616–22.
24.
Budowle, B. and Monson, K.L. (1989) In: Proceedings of an International Symposium on the Forensic
Aspects of DNA Analysis, Government Printing Office, Washington, D.C.
25.
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. Leg. Med. 110, 175–6.
26.
Puers, C. et al. (1994) Analysis of polymorphic STR loci using well-characterized allelic ladders. In:
Proceedings from the Fourth International Symposium on Human Identification 1993, Promega Corporation,
161–72.
27.
Puers, C. et al. (1994) Allelic ladder characterization of the short tandem repeat polymorphism located
in the 5´ flanking region to the human coagulation factor XIII A subunit gene. Genomics 23, 260–4.
28.
Jones, D.A. (1972). Blood samples: Probability of discrimination. J. Forensic Sci. Soc. 12, 355–9.
29.
Brenner, C. and Morris, J.W. (1990) In: Proceedings from the International Symposium on Human
Identification 1989, Promega Corporation, 21–53.
30.
Mandrekar, P.V., Krenke, B.E. and Tereba, A. (2001) DNA IQ™: The intelligent way to purify DNA.
Profiles in DNA 4(3), 16.
31.
Mandrekar, M.N. et al. (2001) Development of a human DNA quantitation system. Profiles in DNA
4(3), 9–12.
32.
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.
33.
Comey, C. et al. (1994) DNA extraction strategies for amplified fragment length polymorphism
analysis. J. Forensic Sci. 39, 1254–69.
34.
Lins, A. 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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13.
Appendix
13.A.Advantages of STR Typing
The GenePrint ® STR Systems provide a rapid, non-radioactive method, which
can be used to evaluate very small amounts (e.g., 1ng) of human DNA. The
protocols detailed in this manual describe the use of silver staining (21) to detect
the presence of amplified STR products following their separation by denaturing
polyacrylamide gel electrophoresis. Information on detecting STR products by
fluorescence methods is available at: www.promega.com
STR typing is more tolerant of the use of degraded DNA templates than other
methods of individual identification because the amplification products are less
than 400bp long, much smaller than the material detected with AMP-FLP (22)
or VNTR (23) analysis. This format is also amenable to a variety of rapid DNA
purification techniques.
In addition to these advantages, the STR loci chosen for inclusion in the
GenePrint ® Systems contain alleles of discrete and separable lengths. This allows
the construction of allelic ladders, which contain fragments of the same lengths
as several or all known alleles for the locus. Visual comparison between the
allelic ladder and amplified samples of the same locus allows rapid and precise
assignment of alleles. Results obtained using the GenePrint ® STR Systems 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 (24).
13.B. Advantages of Using the Loci in the GenePrint ® STR Systems
The STR loci and primers contained in the GenePrint ® STR Systems (Tables 6 and
7) have been carefully selected to 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
(16–18), sometimes called “n–4 bands”, “stutter” or “shadow bands”, is due to
the loss of a repeat unit during DNA amplification. The amount of this artifact
observed depends primarily on the locus and the DNA sequence being replicated.
We have chosen loci that exhibit little or no repeat slippage. The vWA locus is an
exception, revealing as much as 10% stutter. This locus has been included
primarily for its popularity in the forensic DNA-testing community.
Terminal nucleotide addition occurs when Taq DNA polymerase adds a
nucleotide, generally adenine, to the ends of amplified DNA fragments in a
template-independent manner (19,20). 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.
Redefinition of the primer sequences and/or the addition of a final extension
step of 60°C for 30 minutes to the amplification protocol can lead to essentially
full terminal nucleotide addition (18).
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Table 6. Locus-Specific Information.
STR Locus
Amelogenin1
Chromosomal Location
Xp22.1–22.3 and Y
GenBank® Locus and
Locus Definition
HUMAMEL, Human Y
chromosomal gene for
amelogenin-like protein
Repeat Sequence
5´→ 3´
NA
CSF1PO
5q33.3–34
HUMCSF1PO, Human c-fms
proto-oncogene for CSF-1
receptor gene
AGAT2
D16S539
16q24–qter
NA
AGAT2
D7S820
7q11.21–22
NA
AGAT2
D13S317
13q22–q31
NA
AGAT2
F13A01
6p24.3–p25.1
HUMF13A01, Human
coagulation factor XIII a
subunit gene
AAAG2
F13B
1q31–q32.1
AAAT2
FESFPS
15q25–qter
HPRTB
Xq26
HUMBFXIII, Human factor XIII
b subunit gene
HUMFESFPS, Human c-fes/fps
proto-oncogene
HUMHPRTB, Human
hypoxanthine phosphoribosyltransferase gene
LPL
8p22
AAAT2
TH01
11p15.5
TPOX
2p25.1–pter
vWA
(formerly vWF)
12p12–pter
HUMLIPOL, Human
lipoprotein lipase gene
HUMTH01, Human tyrosine
hydroxylase gene
HUMTPOX, Human thyroid
peroxidase gene
HUMVWFA31, Human von
Willebrand factor gene
AAAT2
AGAT2
AATG2
AATG2
AGAT2
NA = not applicable.
1Amelogenin
is not an STR, but displays a 212-base, X-specific band and a 218-base, Y-specific band.
K562 DNA (female) displays only the 212-base, X-specific band.
2Repeat
sequences represent all four possible permutations (e.g., AGAT is used for AGAT, GATA,
ATAG or TAGA). The first alphabetic representation of the repeat (e.g., AGAT) is used according to
the precedent of Edwards et al. (2). The published article, “DNA Guidelines: Further Report of the
DNA Commission of the ISFH Regarding the use of Short Tandem Repeat Systems” (25) describes
different rules for STR allele nomenclature. Allele designations for all listed loci are identical using
both methods except for the locus F13B. In this case, alleles are one repeat unit larger when using the
method described by the ISFH. For this locus, the community will have to decide whether to follow
the new nomenclature or maintain the Edwards nomenclature to avoid confusion. The DNA
Commission of the ISFH states “If a repeat designation of a commonly used STR system does not
follow these guidelines, the established nomenclature for the sequence can continue to be used to
avoid new confusion”.
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Table 7. Additional Locus-Specific Information.
Allelic Ladder
Size Range1
(bases)
212–218
STR Ladder
Alleles
(# of repeats)2
NA
CSF1PO
295–327
D16S539
264–304
D7S820
215–247
D13S317
165–197
F13A01
283–331
F13B
169–189
7,8,9,10,11,
12,13,14,15
5,8,9,10,11,
12,13,14,15
6,7,8,9,10,
11,12,13,14
7,8,9,10,11,
12,13,14,15
4,5,6,7,8,9,11,
12,13,14,15,16
6,7,8,9,10,11
FESFPS
222–250
HPRTB
259–303
LPL
STR Locus
Amelogenin4
Other Known K562 DNA
Alleles3
Allele Sizes
(# of repeats) (# of repeats)
None
212,212
Comments
1,2
6
10,9
1
None
12,11
1
None
11,9
1
None
8,8
1
3.2,105
5,46
1,3
12
10,10
1
7,8,9,10,11,12,13,14
None
12,10
1
None
13,13
1
105–133
6,7,8,9,10,11,
12,13,14,15,16,17
7,9,10,11,12,13,14
8
12,10
1
TH01
179–203
5,6,7,8,9,10,11
9.3
9.3,9.3
1,4
TPOX
224–252
6,7,8,9,10,11,12,13
None
9,8
1
vWA
(formerly vWF)
139–167
13,14,15,16,
17,18,19,20
11,21
16,16
1
NA = not applicable.
1Lengths
of each allele in the allelic ladders have been confirmed by sequence analyses.
2Alleles
in bold are present in greater amounts than other alleles. This simplifies interpretation.
3Alleles
that represent <0.2% of the population may not be listed in this table.
4Amelogenin
is not an STR, but displays a 212 base X-specific band and a 218 base Y-specific band.
K562 DNA (female) displays only the 212 base X-specific band.
5Allele
10 (307 bases) is not included because it is rare and its exclusion creates a gap that simplifies
interpretation of the allelic ladder (26,27).
6F13A01
allele 5 appears more intense than allele 4 in the K562 control sample. The K562 strain
contains an unusual number of chromosomes, and some are represented more than twice per cell. It
is hypothesized that in this strain the allele 5 version of chromosome 6 is present twice, while the
allele 4 version of chromosome 6 is present only once.
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Table 8. Multiplex System Information.
Allelic
STR Ladder
GenePrint ® STR Component Ladder Size Alleles (number
Range (bases)
of repeats)
Multiplex Cat.#
Loci
2
CSF1PO
295–327
7,8,9,10,11,
“CTT triplex”
12,13,14,15
(DC6000 and
TPOX
224–252
6,7,8,9,10,11,12,13
DC6001)
TH01
179–203
5,6,7,8,9,10,11
“FFv triplex”
F13A01
283–331
(DC6030 and
DC6031)
FESFPS
222–250
vWA
139–167
“Silver-STR® III
triplex”
D16S539
264–304
D7S820
215–247
D13S317
165–197
(DC6450 and
DC6451)
1Alleles
Other K562 DNA
Known
Allele
Alleles1
Sizes
Comments
6
10,9
1,5
None
9,8
1,5
9.3
9.3,9.3
1,4,5
4,5,6,7,8,9,10,
11,12,13,14,15,16
7,8,9,10,11,12,13,14
3.2,10
53,4
1,3,5
None
12,10
1,5
13,14,15,16,
17,18,19,20
5,8,9,10,11,
12,13,14,15
6,7,8,9,10,
11,12,13,14,
7,8,9,10,11,
12,13,14,15
11,21
16,16
1,5
None
12,11
1,5
None
11,9
1,5
None
8,8
1,5
that represent <0.2% of the population may not be listed in this table.
2The
GenePrint ® Sex Identification System—Amelogenin primers may be combined with the CTT
primers to allow simultaneous amplification of all four loci as described in Section 4.B. The results
provide information regarding the gender of the individual who contributed the DNA sample, as well
as the STR information. Ordering information for the Amelogenin system may be found in Section 13.I.
3F13A01
allele 5 appears more intense than allele 4 in the K562 control sample. The K562 strain is
known to contain an unusual number of chromosomes, and some are represented more than twice
per cell. It is hypothesized that in this strain the allele 5 version of chromosome 6 is present twice
while the allele 4 version of chromosome 6 is present only once.
Comments on Tables 7 and 8
1.
PCR amplification sometimes generates artifacts that appear as faint bands below
the alleles. These products probably result from a process known as slippage,
commonly observed in PCR amplification of regions that contain tandem repeats
of short sequences (16–18). This characteristic is most pronounced with the vWA
locus.
2.
A strong extra band may be observed below the 212bp Amelogenin allele when
more than 25ng of template DNA is amplified.
3.
Locus F13A01 has a common allele 3.2. It contains 4 copies of the repeat but has
a 2-base deletion in the region flanking the repeat (26,27).
4.
Locus TH01 has a common 9.3 allele (9). A one-base deletion is present in the
allele that contains 10 repeats. Note that reference 9 refers to this allele as 10–1
rather than 9.3. This allele was renamed 9.3 at the ISFH Conference in Venice,
Italy, in October of 1993.
5.
A background haze of silver stain is sometimes seen in the region in and above
the allelic ladder.
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13.C. Power of Discrimination
Table 9 shows the matching probability (28) for the multiplex GenePrint ® STR
Systems in various populations. When taken together, the triplexes described in
this manual produce matching probabilities ranging from 1 in 1,030,000,000 in
Caucasian-Americans to 1 in 5,180,000,000 in African-Americans.
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 a tested man (29). The typical PIs for
the multiplex GenePrint ® STR Systems are shown in Table 10. The three triplexes
together give typical paternity indices exceeding 500 in each group, enough to
satisfy routine requirements for paternity determination.
An alternative calculation used in paternity analyses is the power of exclusion
(29). This value, calculated for the combined triplexes, exceeds 0.9985 in all
populations tested (Table 11).
Table 9. Matching Probability of Various Populations.
Matching Probability
STR System
CTT triplex
(CSF1PO, TPOX, TH01)
FFv triplex (F13A01,
FESFPS, vWA)
African-American
Caucasian-American
Hispanic-American
1 in 1,590
1 in 435
1 in 549
1 in 2,828
1 in 927
1 in 1,343
1 in 1,152
1 in 2,552
1 in 2,493
1 in 5.18 × 109
1 in 1.03 × 109
1 in 1.84 × 109
SilverSTR®
III triplex
(D16S539, D7S820,
D13S317)
All 3 triplexes
(9 loci)
Table 10. Typical Paternity Indices of the Multiplex GenePrint ® STR Systems in
Various Populations.
Typical Paternity Index
STR System
African-American
CTT triplex
(CSF1PO, TPOX, TH01)
10.2
FFv triplex (F13A01,
FESFPS, vWA)
16.0
Caucasian-American
Hispanic-American
6.8
5.2
9.8
7.8
7.6
7.7
14.1
1233
521
563
SilverSTR®
III triplex
(D16S539, D7S820,
D13S317)
All 3 triplexes
(9 loci)
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Table 11. Power of Exclusion of the GenePrint ® STR Systems in Various Populations.
Power of Exclusion
STR System
African-American
CTT triplex
(CSF1PO, TPOX, TH01)
0.906
FFv triplex (F13A01,
FESFPS, vWA)
0.938
SilverSTR® III triplex
(D16S539, D7S820,
D13S317)
All 3 triplexes
(9 loci)
Caucasian-American
Hispanic-American
0.869
0.830
0.904
0.881
0.877
0.880
0.929
0.9993
0.9985
0.9986
13.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 (30). 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.
For applications requiring human-specific DNA quantification, the AluQuant®
Human DNA Quantitation System (Cat.# DC1010) has been developed (31).
The DNA IQ™ System and AluQuant® Human DNA Quantitation System have
been fully automated on the Beckman Coulter Biomek® 2000 Laboratory
Automation Workstation (32). 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]
Note: For stains from blood and saliva, scientists at the FBI Academy have
suggested an alternative method for DNA extraction (see reference 33).
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 10/15
Part# TMD004
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13.E. 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 electrophoresis.
Materials to Be Supplied by the User
(Solution compositions are provided in Section 13.F.)
• TAE 1X buffer
• agarose
• 5X loading solution
• ethidium bromide solution, 0.5µg/ml
!
Ethidium bromide is a powerful mutagen. Wear gloves at all times, and use a
mask when weighing out ethidium bromide powder.
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 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.
Using a UV transilluminator (302nm), photograph the gel (e.g., with
Polaroid® 667 film).
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.
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# TMD004
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Printed in USA.
Revised 10/15
13.F. Composition of Buffers and Solutions
0.5% acetic acid in 95% ethanol
ethidium bromide stock solution
Add 1ml of glacial acetic acid to
199ml of 95% ethanol.
Add 1g of ethidium bromide to
100ml of deionized water. Stir on a
magnetic stirrer until dye is
dissolved. Wrap the container in
aluminum foil, or transfer to a dark
bottle. Store at room temperature.
40% acrylamide: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
GoldST*R 10X Buffer
500mM
100mM
KCl
Tris-HCl (pH 8.3
at 25°C)
MgCl2
Triton® X-100
each dNTP
BSA
Add 0.5g of ammonium persulfate
to 5ml of deionized water. Use
500µl for one acrylamide gel
solution (75ml). Store the remaining
volume in 500µl aliquots at –20°C.
15mM
1%
2mM
1.6mg/ml
developer solution
5X loading solution
3ml
400µl
2L
60g
37% formaldehyde
(H2CO)
10mg/ml sodium
thiosulfate
(Na2S2O3 • 5H2O)
deionized water
sodium carbonate
(Na2CO3)
5%
0.1%
0.1%
100mM
10mM
Ficoll® 400
bromophenol blue
xylene cyanol
EDTA
(Na2EDTA • 2H2O)
Tris-HCl (pH 7.5)
fix/stop solution (10% acetic acid)
200ml
1,800ml
glacial acetic acid
deionized water
Sodium carbonate must be ACS
grade. We have confirmed quality
with material from Fisher Scientific
(Fisher Scientific Cat.# S263-500).
Results may vary depending on
source.
sodium thiosulfate solution
(10mg/ml)
Prepare the solution, and chill to
10°C before use. Use only highquality deionized water and
sodium carbonate. Prepare fresh
before each use.
staining solution
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). Adjust the
final volume to 1 liter. Dispense
into aliquots, and sterilize by
autoclaving.
Add 5g of sodium thiosulfate
(Na2S2O3 • 5H2O) to 500ml of
deionized water.
2g
3ml
2,000ml
silver nitrate (AgNO3)
37% formaldehyde
(H2CO)
deionized water
STR 2X Loading Solution
10mM
95%
0.05%
0.05%
NaOH
formamide
bromophenol blue
xylene cyanol FF
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 10/15
Part# TMD004
Page 37
13.F. Composition of Buffers and Solutions (continued)
STR 10X Buffer
500mM
100mM
15mM
1%
2mM
KCl
Tris-HCl (pH 9.0)
at 25°C
MgCl2
Triton® X-100
each dNTP
50X TAE 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 to
1 liter with deionized water.
0.5X TBE buffer
Add 50ml of 10X TBE to 950ml of
deionized water.
10X TBE buffer
107.8g
7.44g
~55.0g
Tris base
EDTA
(Na2EDTA • 2H2O)
boric acid
Dissolve the Tris base and EDTA in
800ml of deionized water. Add
slightly less than the total amount
of boric acid. Mix until completely
dissolved, check the pH, and adjust
to 8.3 with boric acid. Bring the
volume to 1 liter with deionized
water.
TE–4 buffer (10mM Tris-HCl,
0.1mM EDTA [pH 7.5])
1.21g
0.037g
2H2O)
Tris base
EDTA (Na2EDTA •
Dissolve the Tris base and EDTA in
900ml of deionized water. Adjust to
pH 7.5 with HCl. Increase volume
to 1 liter with deionized water.
13.G. Population Data
Allele frequencies for African-Americans, Caucasian-Americans and HispanicAmericans were generated as part of a collaborative effort between Genetic
Design, Inc. (Greensboro, NC), and Promega Corporation (34). This population
data can be found at: www.promega.com/techserv/apps/hmnid/
referenceinformation/popstat/custstat_Allelefreq.htm
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# TMD004
Page 38
Printed in USA.
Revised 10/15
13.H. Organizational Sheets
Sample Preparation
Tube Number
Sample ID
negative
control
Sample Conc. (ng/µl)
–
Sample (µl)/reaction
0
Sterile Water (µl)
2.5
Tube Number
Sample ID
Sample Conc. (ng/µl)
Sample (µl)/reaction
Sterile Water (µl)
Tube Number
Sample ID
Sample Conc. (ng/µl)
Sample (µl)/reaction
Sterile Water (µl)
Tube Number
Sample ID
Sample Conc. (ng/µl)
Sample (µl)/reaction
Sterile Water (µl)
Tube Number
Sample ID
Sample Conc. (ng/µl)
Sample (µl)/reaction
Sterile Water (µl)
Tube Number
Sample ID
Sample Conc. (ng/µl)
Sample (µl)/reaction
Sterile Water (µl)
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 10/15
Part# TMD004
Page 39
Master Mix Preparation
Date:
Name:
GenePrint ® STR Systems Locus =
Reaction volume (sample + master mix)
=
Number of reactions
=
Master Mix
Component
Lot
Number
25µl
Volume Per
×
Sample (µl)
17.45 for monoplex
×
17.35 for quadriplex
sterile water
=
Final
Volume (µl)
=
2.50
×
=
2.50
0.05 for monoplex
0.15 for quadriplex
×
=
×
=
STR 10X Buffer1
10X Primer Pair
Taq DNA polymerase
(5u/µl)2
Number of
Reactions
total volume
=
1If
using AmpliTaq Gold® DNA polymerase, use the GoldST*R 10X Buffer (Cat.# DM2411,
available separately) instead of the STR 10X Buffer.
2The
volume given assumes a Taq DNA polymerase concentration of 5u/µl. For different
enzyme concentrations, the volume of enzyme added must be adjusted accordingly.
To assemble reactions, add 2.5µl DNA to each tube containing 22.5µl of master mix.
Thermal Cycling Profile
Perkin-Elmer Thermal Cycler Model Number:
Annealing Temperature:
File Number:
Full Program Description:
______cycles:
_______°C _______ minutes
_______°C _______ minutes
_______°C _______ minutes
______cycles:
_______°C _______ minutes
_______°C _______ minutes
_______°C _______ minutes
Hold:
4°C
indefinitely
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# TMD004
Page 40
Printed in USA.
Revised 10/15
Experiment
Date:
Name:
Electrophoresis
Pre-run:
minutes
Starting time:
Stopping time:
Watts:
Watts:
Milliamps:
Milliamps:
Voltage:
Voltage:
Notes
Gel Number:
Lane
Sample #
Description
Lane
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
21
21
22
22
23
23
24
24
25
25
Sample #
Description
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 10/15
Part# TMD004
Page 41
13.I. Related Products
Fluorescent STR Multiplex Systems
Product
PowerPlex® 16 System
GenePrint® GammaSTR® Multiplex (Fluorescein)
D16S539, D7S820, D13S317, D5S818
GenePrint® Fluorescent STR CSF1PO,
TPOX, TH01, vWA Multiplex (Fluorescein)
Size
100 reactions
400 reactions
Cat.#
DC6531
DC6530
100 reactions
DC6071
100 reactions
DC6301
Size
1.2ml
12ml
50ml (2 × 25ml)
Cat.#
DM2411
DY1151
P1193
Size
100 reactions
400 reactions
50 samples
200 samples
10 pack
Cat.#
DC6701
DC6700
DC6801
DC6800
V1391
Size
25g
1L
1kg
3ml
Cat.#
V3131
V4251
V3171
DV4351
Not for Medical Diagnostic Use.
Accessory Components
Product
GoldST*R 10X Buffer
Mineral Oil
Nuclease-Free Water
Sample Preparation Systems
Product
DNA IQ™ System
Differex™ System*
Slicprep™ 96 Device
*Not for Medical Diagnostic Use.
Polyacrylamide Gel Electrophoresis Reagents
Product
Ammonium Persulfate
TBE Buffer, 10X
Urea
Blue Dextran Loading Solution
14. Summary of Changes
The 10/15 version of this document has been updated to remove expired patent and
disclaimer statements and update the related products listing.
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# TMD004
Page 42
Printed in USA.
Revised 10/15
(a).Australian
Pat. No. 724531, Korean Pat. No. 290332, Singapore Pat. No. 57050, Japanese Pat.
No. 3602142 and other patents pending.
© 2010–2015 Promega Corporation. All Rights Reserved.
AluQuant, GammaSTR, GenePrint, pGEM, PowerPlex and SilverSTR are registered trademarks
of Promega Corporation. Differex, DNA IQ and Slicprep are trademarks of Promega Corporation.
AmpliTaq, AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular
Systems, Inc. Biomek is a registered trademark of Beckman Coulter, Inc. Costar is a registered
trademark of Corning, Inc. Ficoll is a registered trademark of GE Healthcare Bio-sciences. FTA
is a registered trademark of Flinders Technologies, Pty, Ltd., and is licensed to Whatman. Gel
Slick is a registered trademark of BioWhittaker. GenBank is a registered trademark of the U.S.
Dept. of Health and Human Services. Kimwipes is a registered trademark of Kimberly-Clark.
Liqui-Nox is a registered trademark of Alconox. MicroAmp is a registered trademark of
Applera Corporation. Milli-Q is a registered trademark of Millipore Corporation. Nalgene is a
registered trademark of Nalge Nunc International. NANOpure is a registered trademark of
Barnstead/Thermolyne Corporation. Nonidet is a registered trademark of Shell International
Petroleum Company, Ltd. Parafilm is a registered trademark of American National Can
Company. Polaroid is a registered trademark of Polaroid Corporation. Spin-X is a registered
trademark of Costar Corporation. Triton is a registered trademark of Union Carbide Chemicals
and Plastics Technology Corporation. Tween is a registered trademark of ICI Americas, Inc.
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 10/15
Part# TMD004
Page 43
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