Complete Protocol

Complete Protocol
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Technical Manual
GenePrint ® Fluorescent
STR Systems
(For use with the Hitachi FMBIO® and FMBIO® II
Fluorescence Imaging Systems, ABI PRISM® 377 DNA
Sequencer and ABI PRISM® 310 and 3100 Genetic
Analyzers.)
INSTRUCTIONS FOR USE OF PRODUCTS DC5171, DC6070, DC6071,
DC6171, DC6300, DC6301, DC6310, DC6311, DG2121, DG2131 AND
DG3291.
PRINTED IN USA
Revised 8/12
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GenePrint ® Fluorescent STR Systems
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 ....................................................................4
A. GenePrint ® Fluorescent STR Multiplex Systems..........................................................4
B. Allelic Ladders and Size Markers..................................................................................5
C. GenePrint ® Fluorescent Sex Identification Systems ....................................................5
D. GenePrint ® Fluorescent Monoplex Systems .................................................................5
3.
Before You Begin .......................................................................................................................6
A. Precautions ........................................................................................................................6
B. Matrix Standardization or Spectral Calibration ..........................................................6
4.
Amplification .............................................................................................................................7
A. Choice of Thermal Cycling Protocol .............................................................................8
B. Amplification Setup.......................................................................................................12
C. Amplification Thermal Cycling ...................................................................................14
5.
Polyacrylamide Gel Preparation...........................................................................................15
A. Gel Preparation for the Hitachi FMBIO® and FMBIO® II
Fluorescence Imaging Systems ....................................................................................15
B. Gel Preparation for the ABI PRISM® 377 DNA Sequencer .....................................18
6.
Polyacrylamide Gel Electrophoresis and Detection.........................................................19
7.
Sample Preparation, Gel Electrophoresis and Detection on the
Hitachi FMBIO® and FMBIO® II Fluorescence Imaging Systems ................................20
A. Gel Pre-Run.....................................................................................................................20
B. Sample Preparation, Loading and Electrophoresis ..................................................21
C. Detection..........................................................................................................................22
D. Data Analysis..................................................................................................................22
E. Reuse of Glass Plates .....................................................................................................23
8.
Sample Preparation, Gel Electrophoresis and Detection on the
ABI PRISM® 377 DNA Sequencer and ABI PRISM® 310 Genetic Analyzer ..............24
A. Instrument Preparation .................................................................................................24
B. Sample Preparation and Loading................................................................................25
C. Electrophoresis and Detection .....................................................................................26
D. Data Analysis..................................................................................................................27
E. Reuse of Glass Plates .....................................................................................................27
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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9.
Detection of Amplified Fragments Using the ABI PRISM® 3100
Genetic Analyzer and Data Collection Software, Version 1.1 .......................................28
A. Sample Preparation........................................................................................................28
B. Instrument Preparation .................................................................................................29
C. Sample Detection............................................................................................................30
D. Data Analysis..................................................................................................................31
10.
Representative STR Data .......................................................................................................31
11.
Troubleshooting.......................................................................................................................34
12.
References .................................................................................................................................36
A. Cited References.............................................................................................................36
B. Additional STR References ...........................................................................................38
13.
Appendix ...................................................................................................................................38
A. Advantages of STR Typing...........................................................................................38
B. Advantages of Using the Loci in the GenePrint ® Fluorescent STR Systems .........39
C. Power of Discrimination ...............................................................................................42
D. DNA Extraction and Quantitation Methods..............................................................43
E. Agarose Gel Electrophoresis of Amplification Products .........................................44
F.
Composition of Buffers and Solutions........................................................................45
G. Related Products ............................................................................................................46
Description
STR(a) (short tandem repeat) loci consist of short, repetitive sequence elements of
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 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 radioactive, silver stain or fluorescence
detection following electrophoretic separation.
The GenePrint ® Fluorescent STR Systems contain all materials, except for Taq DNA
polymerase and sample DNA, required to perform 100 or 400 amplification
reactions. Accessory components are available to simplify many of the procedures
related to STR analysis (Section 13.G).
All GenePrint ® Fluorescent STR Systems contain 10X primer pairs. In each pair, one
primer is labeled with fluorescein (FL), and the matched primer is unlabeled. In the
GenePrint ® Fluorescent Identification System—TMR, the Amelogenin-specific primer
is labeled with tetramethylrhodamine (TMR). STR 10X Buffer, loading solution, the
appropriate allelic ladder and K562 DNA (positive control template) are also
provided.
The GenePrint ® Fluorescent STR Systems can be detected using any of the following
instruments: Hitachi FMBIO® and Hitachi FMBIO® II Fluorescence Imaging Systems,
ABI PRISM® 377 DNA Sequencer and ABI PRISM® 310 and 3100 Genetic Analyzers.
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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This manual describes methods that we have evaluated and recommend to prepare
samples, amplify samples, separate amplified products and detect separated
material. Instructions to operate fluorescence-detecting instrumentation should be
obtained from the instrument manufacturer.
The GenePrint ® Fluorescent STR Multiplex Systems CSF1PO, TPOX, TH01, vWA
(CTTv); F13A01, FESFPS, F13B and LPL (FFFL); and D16S539, D7S820, D13S317 and
D5S818 (GammaSTR®) are currently quality certified on the Hitachi FMBIO® II
Fluorescence Imaging System. The CTTv Multiplex System and GenePrint ®
Fluorescent Sex Identification System—Amelogenin are quality certified for
amplification using the PerkinElmer Model 480 thermal cycler, while the
GammaSTR® Multiplex System is quality certified for amplification using the
PerkinElmer GeneAmp® PCR System 9600 thermal cycler. The FFFL Multiplex is
optimized for use on the GeneAmp® PCR System 9700 thermal cycler.
All GenePrint ® Fluorescent Systems can be amplified on the PerkinElmer Model 480
or GeneAmp® PCR System 9600 or 9700 System thermal cycler, but slight differences
in yield or balance between loci might be observed if the system was not optimized
on that particular thermal cycler. This manual provides a number of cycling protocol
options so that excellent results can be obtained regardless of the thermal cycler
used.
Allele frequencies for African-Americans, Caucasian-Americans and HispanicAmericans for all currently available STR systems can be found at:
www.promega.com/techserv/apps/hmnid/referenceinformation/popstat/
custstat_Allelefreq.htm. Additional population data for STR loci can be found in
references 3 and 9–13. Additional STR references are listed in Section 12.B.
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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Revised 8/12
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Product Components and Storage Conditions
2.A. GenePrint ® Fluorescent STR Multiplex Systems
Below is a description of the components of the GenePrint ® Fluorescent STR
Multiplex Systems. All GenePrint ® STR Multiplex Systems include the required
fluorescein-labeled 10X primer pairs as a mixture for simultaneous
amplification of more than one locus and a mixture of the fluorescein-labeled
allelic ladders for the same set of loci. Additional components include STR 10X
Buffer, K562 DNA, loading solutions and Gel Tracking Dye.
Product
GenePrint ® Fluorescent STR Multiplex—
CSF1PO, TPOX, TH01, vWA (Fluorescein)(a,b)
GenePrint ® Fluorescent STR Multiplex—
GammaSTR® (Fluorescein)(a,c)
D16S539, D7S820, D13S317, D5S818
Size
Cat.#
100 reactions
400 reactions
DC6301
DC6300
100 reactions
400 reactions
DC6071
DC6070
GenePrint ® Fluorescent STR Multiplex—
F13A01, FESFPS, F13B, LPL (Fluorescein)(a,d)
100 reactions
DC6311
400 reactions
DC6310
Not for Medical Diagnostic Use. Cat.# DC6300, DC6070 and DC6310 contain
sufficient reagents for 400 reactions of 25µl each. Each system includes:
•
•
•
•
•
•
•
•
•
•
4 × 250µl
4 × 150µl
4 × 300µl
3µg
2 × 1ml
2 × 1ml
250µl
1 × 100µl
1 × 1.2ml
1
FFFL, CTTv or GammaSTR® 10X Primer Pair Mix (Fluorescein)
FFFL, CTTv or GammaSTR® Allelic Ladder Mix (Fluorescein)
STR 10X Buffer
K562 DNA High Molecular Weight (10ng/µl)
Bromophenol Blue Loading Solution
Blue Dextran Loading Solution
Gel Tracking Dye
TH01 Allele 9.3 (Fluorescein), 200 lanes (CTTv system only)
Gold ST*R 10X Buffer (FFFL system only)
Protocol
Storage Conditions: Store all components at –20°C. The fluorescent 10X Primer
Pair Mix and fluorescent Allelic Ladder Mix are light-sensitive; therefore,
minimize light exposure, and store in the dark. The post-amplification
components (allelic ladder, loading solutions and Gel Tracking Dye) are sealed in
separate packages to prevent cross-contamination. We strongly recommend that
pre-amplification and post-amplification reagents be stored and used separately
with different pipettes, tube racks, etc. Store amplified material at –20°C.
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2.B. Allelic Ladders and Size Markers
Product
Fluorescent Ladder (CXR), 60–400 Bases
Size
65µl
Cat.#
DG6221
Size
150µl
150µl
150µl
Cat.#
DG2121
DG2131
DG3291
Allelic Ladders
Product
CTTv Allelic Ladder Mix (Fluorescein)(a)
FFFL Allelic Ladder Mix (Fluorescein)(a)
GammaSTR® Allelic Ladder Mix (Fluorescein)(a)
The Fluorescent Ladder (CXR), 60–400 Bases, is a size marker composed of
16 evenly spaced DNA fragments labeled with carboxy-X-rhodamine. When
this marker is included in each gel lane, the instruments recommended for
fluorescence detection are capable of monitoring and correcting lane-to-lane
sample migration differences. The Internal Lane Standard 600 contains the
same DNA fragments found in the Fluorescent Ladder (CXR) with additional
fragments in the range of 425–600 bases.
2.C. GenePrint ® Fluorescent Sex Identification Systems
Product
GenePrint ® Fluorescent Sex Identification
System—Amelogenin (Fluorescein)
GenePrint ® Fluorescent Sex Identification
System—Amelogenin (TMR)
Not for Medical Diagnostic Use.
Size
Cat.#
100 reactions
DC5171
100 reactions
DC6171
The GenePrint ® Fluorescent Sex Identification System—Amelogenin (Fluorescein)
can be amplified independently or simultaneously with the CTTv Multiplex.
X-specific and Y-specific chromosome bands will fall between the TPOX and
TH01 loci if amplified along with the CTTv Multiplex.
In the GenePrint ® Fluorescent Sex Identification System—Amelogenin (TMR), the
Amelogenin-specific primer is labeled with tetramethylrhodamine (TMR) and can
be co-amplified with the PowerPlex® 1.1 System (compatible with the Hitachi
FMBIO® Fluorescence Imaging Systems). The CTTv component of the PowerPlex®
System is labeled with TMR, so the TMR-labeled Amelogenin is required.
2.D. GenePrint ® Fluorescent Monoplex Systems
GenePrint ® Fluorescent Monoplex Systems containing the specific primer and
allelic ladder plus other components sufficient to perform 100 reactions are
available by custom order. Contact your local Promega Branch Office or
Distributor for ordering information.
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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Before You Begin
3.A. Precautions
The application of PCR-based typing for forensic or paternity casework requires
validation studies and quality control measures that are not contained in this manual
(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 PCR amplification. We suggest strict adherence
to recommended procedures for amplification, as well as for denaturing gel
electrophoresis and fluorescence detection.
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 (e.g., STR 10X Buffer, K562 control DNA and fluorescein-labeled 10X
primer pairs) should be stored separately from those used following amplification
(e.g., fluorescein-labeled allelic ladders, loading solutions and Gel Tracking Dye).
Always include a negative control reaction (i.e., no template) to ensure reagent
purity. We highly recommend the use of gloves and aerosol-resistant pipette tips
(e.g., ART® tips, Section 13.G).
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 (STR 2X Loading Solution)
bind silane (methacryloxypropyltrimethoxysilane)
TEMED
urea
xylene cyanol FF (STR 2X Loading Solution)
suspected carcinogen, toxic
oxidizer, corrosive
toxic, irritant
irritant, teratogen
toxic, moisture sensitive
corrosive, flammable
irritant
irritant
3.B. Matrix Standardization or Spectral Calibration
Proper generation of a matrix file is critical to evaluate multicolor systems with
the ABI PRISM® 310 and 3100 Genetic Analyzers. A matrix must be generated
for each individual instrument.
The PowerPlex® Matrix Standards, 310 (Cat.# DG4640), is required for matrix
standardization for the ABI PRISM® 310 Genetic Analyzer and ABI PRISM® 377
DNA Sequencer for all GenePrint ® Fluorescent STR Systems, except the
GenePrint ® Fluorescent Sex Identification System—Amelogenin, which requires
the PowerPlex® Matrix Standards, 310/377 (Cat.# DG3640). For best results, the
PowerPlex® Matrix Standards, 3100/3130 (Cat.# DG4650), should not be used to
generate a matrix on the ABI PRISM® 310 Genetic Analyzer.
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The PowerPlex® Matrix Standards, 3100/3130 (Cat.# DG4650), is required for
spectral calibration on the ABI PRISM® 3100 Genetic Analyzer for all GenePrint ®
Fluorescent STR Systems, except the GenePrint ® Fluorescent Sex Identification
System—Amelogenin, which requires the PowerPlex® Matrix Standards, 3100—
Custom (Cat.# X3121). The PowerPlex® Matrix Standards, 310 (Cat.# DG4640),
or PowerPlex® Matrix Standards, 310/377 (Cat.# DG3640), cannot be used to
generate a matrix on this instrument.
For protocols and additional information on matrix standardization, see the
PowerPlex ® Matrix Standards, 310, Technical Bulletin #TBD021, which is supplied
with Cat.# DG4640. For protocols and additional information on spectral
calibration, see the PowerPlex ® Matrix Standards, 3100/3130, Technical Bulletin
#TBD022, which is supplied with Cat.# DG4650. These manuals are available
upon request from Promega or online at: www.promega.com/tbs/
4.
Amplification
The GenePrint ® Fluorescent 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, we recommend using the Gold ST*R 10X
Buffer (Cat.# DM2411), instead of the STR 10X Buffer. Currently, the STR 10X Buffer
(pH 9.0) is not compatible with AmpliTaq Gold® DNA polymerase because the
optimal pH for the modified Taq DNA polymerase is pH 8.3. Also, when using
AmpliTaq Gold® DNA polymerase, an additional incubation at 95°C for 11 minutes
must be incorporated prior to initiating the thermal cycling program.
The following section gives detailed amplification protocols for the GenePrint ®
Fluorescent STR Systems. Thermal cycling protocols for the PerkinElmer Model 480
and GeneAmp® System 9600 and 9700 thermal cyclers are given for each GenePrint ®
System.
Note: Protocol 12 (Table 6) has been developed for optimal performance of the
GenePrint ® Fluorescent STR System—F13A01, FESFPS, F13B, LPL (Fluorescein) with
the GeneAmp® PCR System 9600 thermal cycler (16). This protocol uses Gold ST*R
10X Buffer and AmpliTaq Gold® DNA polymerase.
Materials to Be Supplied by the User
(Solution compositions are provided in Section 13.F.)
• thermal cycler, Model 480 or GeneAmp® PCR System 9600 or 9700 (PerkinElmer)
• microcentrifuge
• Taq DNA polymerase
• Nuclease-Free Water (Cat.# P1193 or equivalent)
• Mineral Oil (Cat.# DY1151 or equivalent)
• 0.5ml microcentrifuge tubes
• 1.5ml microcentrifuge tubes
• aerosol-resistant pipette tips
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4.A. Choice of Thermal Cycling Protocol
The CTTv Multiplex and GenePrint ® Fluorescent Sex Identification System—
Amelogenin (Fluorescein) are optimized for use with PerkinElmer GeneAmp®
reaction tubes and PerkinElmer Model 480 thermal cycler. The GammaSTR®
Multiplex and GenePrint ® Fluorescent Sex Identification System—Amelogenin
(TMR) are optimized for use with the GeneAmp® PCR System 9600 thermal
cycler. The FFFL Multiplex optimized for use with the GeneAmp® PCR System
9700 thermal cycler. However, each system may be used with any of these
thermal cyclers.
Please refer to Tables 2, 3 and 4 for recommended and alternative protocols for
each system and thermal cycler. Table 5 describes the special template
requirement of each multiplex system for use with various detection
instruments when using protocol #10. Many customers prefer protocol #10
because it uses the GeneAmp® PCR System 9600 thermal cycler with a heated
thermal cycler lid, MicroAmp® reaction tubes and no mineral oil. Specific
details for each protocol, including number of cycles, incubation temperatures
and times, and ramp times, are provided in Table 6.
When using a thermal cycler for 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.
Table 2. Protocol Options for the Model 480 Thermal Cycler.
GenePrint ® STR System
CTTv Multiplex
CTTv Multiplex with Amelogenin3
FFFL Multiplex
GammaSTR® III Multiplex
Amelogenin, CSF1PO, F13A01, TH01 or TPOX
D16S539, D7S820, D13S317 or D5S818
F13B or FESFPS
LPL or vWA
Recommended
Protocol1
Alternative
Protocol2
7
7
7
7
2
7
1
7
1
1
1
NA
1
NA
NA
1
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Table 3. Protocol Options for the GeneAmp® PCR System 9600 Thermal Cycler.
GenePrint ® STR System
Recommended
Protocols1
Alternative
Protocols2
8,9 or 104
8,9 or 104
8,9 or 104
3,4,11
3,4,11
3,4,11
12
104
NA
9
3,4
9 or 104
8,9
NA
NA
3,4
CTTv Multiplex
CTTv Multiplex with Amelogenin3
FFFL Multiplex (using AmpliTaq® DNA polymerase)
FFFL Multiplex (using AmpliTaq Gold® DNA
polymerase)
GammaSTR® III Multiplex
Amelogenin, CSF1PO, F13A01, F13B,
FESFPS, TH01 or TPOX
D16S539, D7S820, D13S317 or D5S818
LPL or vWA
Table 4. Protocol Options for the GeneAmp® PCR System 9700 Thermal Cycler.
Recommended
Protocol
GenePrint ® STR System
CTTv Multiplex
CTTv Multiplex with Amelogenin3
FFFL Multiplex
GammaSTR® III Multiplex
Amelogenin, CSF1PO, F13A01, F13B, FESFPS, TH01 or TPOX
D16S539, D7S820, D13S317 or D5S818
LPL or vWA
13
13
13
13
13
13
13
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.
3The amplification of 25ng or more of K562 DNA using the CTTv system with
Amelogenin may result in extra bands at 338, 254 and 161 bases.
4Special template requirements for use of protocol #10 with certain multiplex system
and detection instrument combinations are described in Table 5.
Table 5. Recommended Amounts of Template For Various Instruments
Using Protocol #10.
Fluorescent STR System
Hitachi FMBIO® and FMBIO® II Fluorescence
Imaging Systems
ABI PRISM® 377 DNA Sequencer and
ABI PRISM® 310 and 3100 Genetic Analyzers
CTTv
FFFL
GammaSTR®
5ng
2–5ng
1ng
1ng
1ng
1ng
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Table 6. Amplification Protocols.
Protocol
Number
Thermal
Initial
Cycler1 Incubation2
Programmed
Ramp Times
Cycling for First
10 Cycles
Programmed
Ramp Times
Cycling for Last Extension
20 Cycles
Step
Hold
Step
1
(Refer to
Note 1)
480
96°C for
2 minutes
None
94°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
None
4°C
2
(Refer to
Note 1)
480
96°C for
2 minutes
None
94°C, 1 minute
64°C, 1 minute
70°C, 1.5 minutes
None
90°C, 1 minute
64°C, 1 minute
70°C, 1.5 minutes
None
4°C
3
(Refer to
Note 2)
9600
96°C for
2 minutes
None
94°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
None
4°C
4
(Refer to
Note 3)
9600
96°C for
2 minutes
None
4°C
50 seconds to 94°C, 1 minute
34 seconds to 60°C, 1 minute
25 seconds to 70°C, 1.5 minutes
5
Protocol 5 is no longer recommended.
6
Protocol 6 is no longer recommended.
45 seconds to 90°C, 1 minute
30 seconds to 60°C, 1 minute
25 seconds to 70°C, 1.5 minutes
7
(Refer to
Note 1)
9600
96°C for
2 minutes
None
94°C, 1 minute
60°C, 1 minute
70°C, 1.5 minutes
None
90°C, 1 minute
60°C, 1 minute
60°C for
70°C, 1.5 minutes 30 minutes
4°C
8
(Refer to
Note 2)
9600
96°C for
2 minutes
None
94°C, 1minute
60°C, 1 minute
70°C, 1.5 minutes
None
90°C, 1 minute
60°C, 1 minute
60°C for
70°C, 1.5 minutes 30 minutes
4°C
9
(Refer to
Note 3)
9600
96°C for
2 minutes
50 seconds to 94°C, 30 seconds
34 seconds to 60°C, 1 minute
25 seconds to 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
60°C for
30 minutes
4°C
10
(Refer to
Note 4)
9600
96°C for
1 minute
Default ramp to 94°C, 30 seconds
68 seconds to 60°C, 30 seconds
50 seconds to 70°C, 45 seconds
Default ramp to 90°C, 30 seconds
60 seconds to 60°C, 30 seconds
50 seconds to 70°C, 45 seconds
60°C for
30 minutes
4°C
11
(Refer to
Note 4)
9600
96°C for
2 minutes
50 seconds to 94°C, 1 minute
34 seconds to 60°C, 1 minute
25 seconds to 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
60°C for
30 minutes
4°C
Protocol
Number
Initial
Thermal
Cycler1 Incubation2
Programmed
Ramp Times
Cycling for First
10 Cycles
Programmed
Ramp Times
Cycling for Last Extension
Step
22 Cycles3
12
(Refer to
Note 4)
9600
96°C for
2 minutes
50 seconds to 94°C, 1 minute
30 seconds to 64°C, 1 minute
15 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
13
(Refer to
Notes 4 and 5)
9700
96°C for
1 minute
ramp 100% to 94°C, 30 seconds
ramp 29% to 60°C, 30 seconds
ramp 23% to 70°C, 45 seconds
ramp 100% to 90°C, 30 seconds
ramp 29% to 60°C, 30 seconds
ramp 23% to 70°C, 45 seconds
Hold
Step
None
4°C
60°C for
30 minutes
4°C
1480
refers to the PerkinElmer model 480 thermal cycler, 9600 refers to the GeneAmp® PCR System 9600 thermal cycler and 9700
refers to the GeneAmp® PCR System 9700 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 with any protocol. Also when using
AmpliTaq® DNA polymerase, be sure to use the Gold ST*R 10X Buffer.
3Please note that Protocols 12 and 13 incorporates two additional cycles in the last portion of the cycling protocol.
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Notes for Table 6:
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.
Do not cover the reactions with the system 9600 thermal cycler lid. Cover
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. We recommend
Sigma BSA (Cat.# A2153). Performance may vary depending on the source
of this component.
4.
See Table 5 for recommended amounts of template.
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 thermal cycler lid.
Optional: Add BSA Fraction V (final concentration 60µg/ml) to all
reactions. This may result in a slight increase in yield. We recommend
Sigma BSA (Cat.# A2153). Performance may vary depending on the source
of this component.
5.
When using the GeneAmp® 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.
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4.B. Amplification Setup
The use of gloves and aerosol-resistant pipette tips (see Section 13.G) is highly
recommended to prevent cross-contamination.
Alternative steps are included in the following procedure for laboratories using
the Hitachi FMBIO® and FMBIO® II Fluorescence Imaging Systems and
ABI PRISM® 377 DNA Sequencer, and ABI PRISM® 310 and 3100 Genetic
Analyzers.
1.
Thaw the STR 10X Buffer and 10X Primer Pair(s), and place on ice.
Note: Mix the STR 10X Buffer and 10X Primer Pair by vortexing each tube
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.
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 6 for tube selection.
3.
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.
4.
Calculate the required amount of each PCR master mix component
(Table 7). Multiply the volume (µl) per sample by the total number of
reactions (from Step 3) to obtain the final volume (µl).
Note: The CTTv Multiplex and Amelogenin locus can be amplified
simultaneously.
5.
In the order listed in Table 7, add the final volume of each reagent to a
sterile tube. Mix well, and place on ice.
Note: 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
first 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.
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Table 7. PCR Amplification Reaction Setup.
Multiplex Reactions Containing Four Loci
PCR Master Mix Component
Volume Per
Sample (µl)
sterile water
17.30
STR 10X Buffer1
2.50
Multiplex 10X Primer Pair Mix
Taq DNA polymerase (at 5u/µl)1
total volume
Number of
Final
× Reactions = Volume (µl)
2.50
0.2 (1.0u)
22.50
Combined CTTv Multiplex and Amelogenin Reactions
PCR Master Mix Component
sterile water
STR 10X
Volume Per
Sample (µl)
×
Number of
Final
Reactions = Volume (µl)
16.25
Buffer1
2.50
CTTv 10X Primer Pair Mix
Amelogenin (Fluorescein)
10X Primer Pair2
Taq DNA polymerase (at 5u/µl)1
total volume
2.50
1.0
0.25 (1.25u)
22.50
Monoplex or Amelogenin-Only Reactions
PCR Master Mix Component
sterile water
STR 10X
Number of
Reactions
Final
= Volume (µl)
2.50
locus-specific 10X primer pair
total volume
×
17.45
Buffer1
Taq DNA polymerase (at
Volume Per
Sample (µl)
5u/µl)1
2.50
0.05 (0.25u)
22.50
1The 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 using AmpliTaq Gold® DNA polymerase, use the Gold ST*R 10X Buffer
(instead of the STR 10X Buffer).
2Use
of more Amelogenin primer has produced extra bands below the expected 212- and
218-base fragments with some samples using protocol #7, especially when 25ng or more
of template are used. Amelogenin (TMR) is only for use with the PowerPlex® Systems
and should not be used with the CTTv Multiplex System.
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4.B. Amplification Setup (continued)
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.
Hitachi FMBIO® Users: Pipet 2.5µl (1–25ng template DNA) of each
sample into a tube containing 22.5µl of PCR master mix.
ABI PRISM® 377 DNA Sequencer, ABI PRISM® 310 Genetic Analyzer,
and ABI PRISM® 3100 Genetic Analyzer Users: Use only 1–2ng template
DNA.
Protocol #10 Users: See Table 5 for the amount of template DNA to use
with each instrument and GenePrint ® system.
Note: 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 greatly altered 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 but may contain other PCR inhibitors at low concentrations.
8.
Hitachi FMBIO® Users: Pipet 2.5µl (25ng) of K562 DNA into a 0.5ml
microcentrifuge tube containing 22.5µl of PCR master mix as a positive
amplification control.
ABI PRISM® 377 DNA Sequencer, ABI PRISM® 310 Genetic Analyzer,
and ABI PRISM® 3100 Genetic Analyzer Users: Use only 1–2ng of K562
template DNA as a positive amplification control.
9.
Pipet 2.5µl of sterile water (instead of template DNA) into a 0.5ml
microcentrifuge tube containing 22.5µl of PCR master mix as a negative
amplification control.
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 aqueous 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, 3 or 4 (Section 4.A).
3.
After completion of the thermal cycling protocol, store the samples at –20°C
in a light-protected box.
Note: Storage of amplified samples at 4°C or above may produce
degradation products.
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Polyacrylamide Gel Preparation
Gel preparation depends on the type of instrument used for fluorescent detection.
Outlined below are procedures to prepare gels for the Hitachi FMBIO® and FMBIO® II
Fluorescence Imaging Systems and ABI PRISM® 377 DNA Sequencer. If you are
using a different instrument, please refer to the manufacturer’s recommendations.
New glass plates should be soaked in 10% NaOH for 1 hour, then rinsed thoroughly
with deionized water before use. New plates should also 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.A. Gel Preparation for the Hitachi FMBIO® and FMBIO® II Fluorescence
Imaging Systems
There are two size options for gels on the Hitachi FMBIO® and FMBIO® II
Fluorescence Imaging Systems, either 32cm × 19cm × 0.4mm (h × w × thickness)
or 43cm × 19cm × 0.4mm. The 43cm × 19cm × 0.4mm low-fluorescence glass
plates are strongly recommended for better separation. The use of the longer
glass plates enables the instrument to distinguish one-base-pair differences and
differences in alleles over 300bp more easily. If the Hitachi STaRCall™ Software
is used to identify alleles, use square-tooth combs to maximize software
performance. Square-tooth combs provide better separation between the lanes.
If allelic ladders are used to make allele determinations visually, use either a
sharkstooth or a square-tooth comb.
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) if square-tooth combs
are to be used
•
0.5% acetic acid in 95% ethanol
•
Nalgene® tissue culture filter (0.2 micron)
•
32cm × 19cm × 0.4mm (h × w × thickness) low fluorescence glass plates
(MiraiBio)
•
spacers for SA-32 low fluorescence glass plates
•
43cm × 19cm × 0.4mm (h × w × thickness) low-fluorescence glass plates
(Whatman Biometra)
•
SA-43 Spacer Set (Whatman Biometra)
•
SA-43 Extension (Whatman Biometra) for use with SA-43 glass plates
•
power supply
•
polyacrylamide gel electrophoresis apparatus for gels ≥30cm
•
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)
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5.A. Gel Preparation for the Hitachi FMBIO® and FMBIO® II Fluorescence
Imaging Systems (continued)
Materials to Be Supplied by the User (continued)
•
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
!
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.
Hitachi FMBIO® Fluorescence Imaging Systems
1.
Thoroughly clean the shorter and longer glass plates twice with 95% ethanol
and Kimwipes® tissues.
Note: The plates require bind silane treatment if using a square-tooth comb
(see below). The plates do not require a special bind silane treatment when
using a sharkstooth comb.
Bind Silane Treatment of Glass Plate
Prepare fresh binding solution in a chemical fume hood. Add 1.5µl 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 the 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 the excess binding solution.
2.
Assemble the glass plates by placing 0.4mm side spacers between the
plates and 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 4% or 6% acrylamide solution (total of 30ml for a 32cm plate or
Table 8. Preparation of 4% and 6% Polyacrylamide Gels.
4% Gel
(32cm)
4% Gel
(43cm)
6% Gel
(32cm)
6% Gel
(43cm)
Final
Concentration
urea
12.6g
18.9g
12.6g
18.9g
7M
Component
deionized water
16.0ml
24.0ml
14.5ml
21.75ml
–
10X TBE buffer
40% acrylamide:bis
(19:1)
1.5ml
2.25ml
1.5ml
2.25ml
0.5X
3.0ml
4.5ml
4.5ml
6.75ml
4% or 6%
total volume
30.0ml
45.0ml
30.0ml
45.0ml
45ml for a 43cm plate) by combining the ingredients listed in Table 8.
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Note: If preparing multiple gels on a daily basis, a larger 4% or 6% stock
solution may be prepared, filtered as in Step 4 below and stored at 4°C in
the dark for up to one month. To prepare a single gel, remove either 30ml
or 45ml of the stock solution, and continue with Step 6.
4.
Filter the acrylamide solution through a 0.2 micron filter (e.g., Nalgene®
Component
32cm gel (30ml)
43cm gel (45ml)
TEMED
20µl
30µl
10% ammonium persulfate
200µl
300µl
tissue culture filter).
5.
Slowly pour the filtered acrylamide solution into a squeeze bottle.
6.
Add the following amounts of TEMED and 10% ammonium persulfate,
and mix gently.
7.
Pour the gel by starting at the well end of the plates. Carefully pour the
acrylamide between the horizontal glass plates. Allow the solution to fill
the top width of the plates. Slightly tilt the plates to assist the movement
of the solution to the bottom of the plates while maintaining a constant
flow of the solution. When the solution begins to flow out from the
bottom, position the plates horizontally.
8.
Insert the straight side of a 14cm doublefine (49 point) sharkstooth comb
(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.
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 (Step 10) to be sure that polymerization has
occurred.
Note: The gel may be stored overnight if a paper towel saturated with
deionized water and plastic wrap are placed around the top and bottom to
prevent the gel from drying out (crystallization of the urea will destroy the
gel).
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5.B. Gel Preparation for the ABI PRISM® 377 DNA Sequencer
When working with the glass plates for the ABI PRISM® 377 DNA Sequencer, it
is extremely important to avoid contact between the gel side of the plates and
paper towels. Rinse the plates extremely well with deionized water, and allow
to air-dry in a dust-free environment before use.
Materials to Be Supplied by the User
(Solution compositions are provided in Section 13.F.)
• Long Ranger® gel solution (Cambrex Cat.# 50611)
• 10X TBE Buffer (Cat.# V4251)
• 10% Ammonium Persulfate (Cat.# V3131)
• TEMED
• Urea (Cat.# V3171)
• Nalgene® tissue culture filter (0.2 micron)
• 36cm front and rear glass plates (refer to the instrument manual for
recommendations)
• 36cm gel spacers (0.2mm thick)
• 36-well sharkstooth comb or 34-well square-tooth comb (0.2mm thick)
• clamps
• Liqui-Nox® detergent (Use of Liqui-Nox® detergent is extremely
important, as other kinds of detergent can build up on the glass plates.)
The following protocol is for the preparation of a 36cm denaturing polyacrylamide
gel for use with the ABI PRISM® 377 DNA Sequencer. Low-fluorescence glass
plates are recommended and may be obtained from the instrument manufacturer.
1.
Thoroughly clean the glass plates with hot water and a 1% Liqui-Nox®
solution. Rinse extremely well using deionized water. Allow the glass
plates to air-dry.
2.
Assemble the glass plates by placing 0.2mm side gel spacers between the
front and rear glass plates. Hold the plates together using binder clamps
(4 clamps on each side). Place the assembly horizontally on a test tube rack
or similar support.
3.
Prepare a 5% Long Ranger® acrylamide gel (total of 50ml) by combining the
ingredients listed in Table 9. Stir the solution until the urea has dissolved.
Table 9. Preparation of a 5% Long Ranger® Polyacrylamide Gel.
Component
urea
deionized water
10X TBE buffer
50% Long Ranger® gel solution
total volume
5% Gel
18g
26ml
5.0ml
5.0ml
50.0ml
Final Concentration
6M
–
0.5X
5%
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4.
Filter the acrylamide solution through a 0.2 micron filter (e.g., Nalgene®
tissue culture filter), and de-gas for an additional 5 minutes.
5.
Add 35µl of TEMED and 250µl of 10% ammonium persulfate to 50ml of
acrylamide solution, and mix gently.
6.
Using a disposable 30cc syringe, pour the gel by starting at the well end of
the plates and carefully injecting the acrylamide solution between the
horizontal glass plates. Allow the solution to fill the top width of the
plates. While maintaining a constant flow of solution, gently tap the glass
plates to assist the movement of solution to the bottom of the plates.
7.
Insert the straight edge of one 36-well sharkstooth comb, or insert a 34-well
square-tooth comb between the glass plates.
8.
Secure the comb with three evenly spaced clamps.
9.
Pour the remaining acrylamide solution into a disposable conical tube as a
polymerization control.
10. Allow polymerization to proceed for at least 2 hours. Check the
polymerization control (Step 9) to ensure that polymerization has
occurred.
Note: The gel may be stored overnight if a paper towel saturated with
1X TBE 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).
6.
Polyacrylamide Gel Electrophoresis and Detection
Electrophoresis protocols depend on the type of instrument used for fluorescence
detection. The following sections provide procedures for loading and running gels
on the Hitachi FMBIO® and FMBIO® II Fluorescence Imaging Systems (Section 7),
ABI PRISM® 377 DNA Sequencer and ABI PRISM® 310 Genetic Analyzer, (Section 8)
and ABI PRISM® 3100 Genetic Analyzer (Section 9). If a different instrument is used
for detection, please refer to the manufacturer's recommendations for that particular
instrument.
The Fluorescent Ladder
The Fluorescent Ladder (CXR), 60–400 Bases, contains 16 evenly spaced DNA
fragments of 60, 80, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375 and
400 bases in length. This ladder may be used as an internal size standard in each lane
to increase precision in analyses. Inclusion of the Fluorescent Ladder (CXR) in each
lane reduces the number of allelic ladder lanes needed per gel and, therefore,
increases the number of lanes available for samples. Allelic ladders still need to be run
on one or two lanes of every gel as a control to verify that the gel ran correctly and
that the allele sizes are correct. Be aware that the sizes determined by the software
may not exactly correspond to the sequenced sizes given in Table 13 because samples
migrate on a gel according to length, sequence and dye label.
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Polyacrylamide Gel Electrophoresis and Detection (continued)
The Fluorescent Ladder (CXR), 60–400 Bases, is required when using the ABI PRISM®
377 DNA Sequencer, ABI PRISM® 310 Genetic Analyzer or ABI PRISM® 3100 Genetic
Analyzer. The Genescan® software provided with these instruments requires use of a
sizing ladder. The Fluorescent Ladder (CXR) must be included in all lanes to account
for lane-to-lane or run-to-run variability. Adjustments in the allele sizes will be made
from lane to lane when the allele size is compared to the Fluorescent Ladder (CXR)
contained within each lane.
When using the Hitachi FMBIO® and FMBIO® II Fluorescence Imaging Systems,
inclusion of the Fluorescent Ladder (CXR) is optional. If alleles are called visually
by comparing the sample alleles directly against the allelic ladders, the Fluorescent
Ladder (CXR) is not needed. However, if the Hitachi STaRCall™ Software is used
for identifying alleles, we recommend including the Fluorescent Ladder (CXR).
Note: The Internal Lane Standard 600 (Cat.# DG1071) contains the same DNA
fragments as the Fluorescent Ladder (CXR), 60–400 Bases, with additional DNA
fragments of 425, 450, 475, 500, 550 and 600 bases.
7.
Sample Preparation, Gel Electrophoresis and Detection on the Hitachi FMBIO®
and FMBIO® II Fluorescence Imaging Systems
7.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, and remove the
comb.
3.
Add 0.5X TBE Buffer to the bottom chamber of the electrophoresis apparatus.
4.
Gently lower the gel (glass plates) into the bottom chamber 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 Buffer to the top chamber of the electrophoresis apparatus.
7.
Use a 50–100cc syringe filled with buffer to remove any air bubbles on 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
air bubbles from the bottom of the gel.
8.
Pre-run the gel to achieve a surface temperature of approximately 50°C.
Consult the manufacturer’s instruction manual for recommended
electrophoresis conditions.
Note: As a reference, we generally use 40–45 watts for 30 minutes for a
32cm gel or 60–65 watts for 30 minutes for a 43cm gel. The gel running
conditions may have to be adjusted to reach a temperature of 50°C.
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7.B. Sample Preparation, Loading and Electrophoresis
Use of the Fluorescent Ladder (CXR) is optional with the Hitachi instruments. If
alleles are called visually, we recommend running an allelic ladder in every
third lane so that each sample is next to a ladder.
1.
If the Fluorescent Ladder (CXR) is not used, prepare the PCR samples or
allelic ladders by combining 2.5µl of Bromophenol Blue Loading Solution
with 2.5µl of PCR sample or allelic ladder.
If the Fluorescent Ladder (CXR) is used, prepare PCR samples and allelic
ladders by combining 1µl of Fluorescent Ladder (CXR), 3µl of
Bromophenol Blue Loading Solution and 2µl of sample or allelic ladder
[we recommend running two lanes of allelic ladder per gel when using the
Fluorescent Ladder (CXR)].
Note: The Bromophenol Blue Loading Solution does not contain xylene
cyanol because it fluoresces and is detected by the FMBIO® instruments.
2.
After samples are prepared, centrifuge the tubes briefly to bring the
contents to the bottom of the tube.
3.
Optional: Place 6µl of Gel Tracking Dye in one tube. The Gel Tracking
Dye contains both bromophenol blue and xylene cyanol. This dye may be
loaded in the outermost lane of the gel and used as a visual indicator of
migration. We recommend leaving two empty lanes between the gel
tracking dye and sample lanes so that the xylene cyanol fluorescence does
not interfere with sample interpretation.
Notes:
4.
1.
To analyze the CTTv Multiplex with Amelogenin reactions, mix the
corresponding ladders 1:1 before mixing with loading solution. The
number of ladder lanes depends on personal preference and the
number of samples analyzed.
2.
To use the TH01 allele 9.3 alone, mix 0.5µl of allele 9.3 with 1.5µl of
1X STR Buffer before mixing with the loading solution. To use in
combination with the TH01 monoplex or CTTv Allelic Ladder, mix
0.5µl of allele 9.3 with 2.0µl of allelic ladder before mixing with the
appropriate loading dye.
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. Sample DNA will re-anneal
if denatured hours before loading. This may produce fragments of
indeterminate migration.
5.
If using a sharkstooth comb, flush the urea from the well area with a
50–100cc syringe filled with buffer. Carefully insert the comb teeth into the
gel approximately 1–2mm. Leave the comb inserted in the gel during gel
loading and electrophoresis. If a square-tooth comb is used, clean the
individual wells with buffer using a 50–100cc syringe, and do not reinsert
the comb. The samples will be loaded directly into the wells.
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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Revised 8/12
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7.B. Sample Preparation, Loading and Electrophoresis (continued)
6.
Load 3µl of each sample into the respective wells. We recommend using
gel loading tips to load the wells formed by the square-tooth combs. The
loading process should take no longer than 20 minutes to prevent the gel
from cooling.
7.
When loading is complete, run the gel using the same conditions as in
Section 7.A (gel pre-run).
Note: In 6% gels, bromophenol blue migrates at approximately 25 bases,
and xylene cyanol migrates at approximately 105 bases. In 4% gels,
bromophenol blue migrates at approximately 40 bases, and xylene cyanol
migrates at approximately 170 bases.
8.
Use the size ranges for each locus (see Table 13, Section 13.B) and the
migration characteristics of the dyes (see Step 7) to 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.
7.C. 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 very clean for scanning. Clean both sides of the
gel/glass plate unit with deionized water and paper towels. Do not use
ethanol to clean the plates. The ethanol fluoresces and is detected by the
FMBIO® instruments.
3.
Scan the gel according to the parameters listed in Table 10. Use the 505nm
filter to detect fluorescein-labeled fragments and the 650nm filter to detect
the Fluorescent Ladder (CXR), 60–400 Bases. Different laboratories may
wish to modify these parameters according to their specific preferences.
Note: If the signal is too intense, dilute samples in 1X STR Buffer before
mixing with loading solution or use less DNA template in the amplification
reactions.
7.D. Data Analysis
Controls
Observe the lanes containing negative controls. They should be devoid of
amplification products.
Observe the lanes containing K562 DNA positive controls. Compare the K562
alleles with the locus-specific ladder. The expected K562 alleles for each locus
are listed in Table 13. Figure 2 (Section 10) shows an example of results
obtained after amplification of the positive control K562 DNA using the
GenePrint ® Multiplex CTTv, FFFL and GammaSTR® Systems. The K562 DNA
contains imbalanced alleles at several loci. This result is due to the unusual
chromosome content of this cell line and is not a function of the GenePrint ®
Fluorescent STR System performance.
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Table 10. Instrument Parameters for the Hitachi FMBIO® and FMBIO® II
Fluorescence Imaging Systems.
Parameter
FMBIO® Fluorescence
Imaging System
FMBIO® II Fluorescence
Imaging System
Acrylamide gel
Acrylamide gel
150dpi
150dpi
0.1024s/line
1 time
Range
150dpi
150dpi
NA
256 times
Range
50%
1%
50%
1%
100% (505nm channel)
100% (650nm channel)
0mm
Material Type
Resolution:
Horizontal
Vertical
Rate
Repeat
Gray Level Correction Type
Cutoff Threshold:
Low (Background)
High (signal)
Reading Sensitivity
Focusing Point
80%
NA
NA = Not applicable.
Representative STR Data
Representative STR data obtained using the Hitachi FMBIO® II Fluorescent
Scanner and GenePrint ® Fluorescent STR Multiplex—GammaSTR® are shown in
Figure 3 (Section 10).
Allelic and Fluorescent Ladders
In general, allelic ladders contain fragments of the same lengths as either
several or all known alleles for the locus. The allelic ladder sizes and repeat
units are listed in Table 13. Visual comparison between the 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 size standard, the calculated lengths of allelic ladder
components will differ from those listed in Table 13. This is due to differences
in migration resulting from sequence differences between the allelic ladder
fragments and those of the internal size standard.
7.E. Reuse of Glass Plates
Separate the glass plates, and discard the gel. Clean the glass plates with
deionized water and a 1% solution of Liqui-Nox® detergent. The use of
Liqui-Nox® detergent is extremely important, as other kinds of soap can build
up on the glass plates. Buildup will result in low signal and high background on
the gels. If the glass plates have a soap residue buildup 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) before
cleaning the glass plates.
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Revised 8/12
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Sample Preparation, Gel Electrophoresis and Detection on the ABI PRISM® 377
DNA Sequencer and ABI PRISM® 310 Genetic Analyzer
Instructions for use with the ABI PRISM® 377 DNA Sequencer and ABI PRISM® 310
Genetic Analyzer are given below. Please refer to the user’s manuals provided with
these instruments for specific operating instructions.
8.A. Instrument Preparation
ABI PRISM® 377 DNA Sequencer
1.
Open the GeneScan® data collection software.
2.
Prepare a GeneScan® sample sheet as described in the GeneScan® analysis
software user’s manual.
3.
Create a new GeneScan® run using the following settings:
Plate Check Module:
PreRun Module:
Run Module:
Collect Time:
Well-to-Read Distance:
Plate Check A
PR GS 36A-2400
GS 36A-2400
2.25 hours
36cm
4.
Select the appropriate sample sheet and comb selection by using the pulldown menus.
5.
Select the appropriate gel matrix file created in Section 3.B.
ABI PRISM® 310 Genetic Analyzer
Refer to the ABI PRISM® 310 Genetic Analyzer user’s manual for instructions
on cleaning the pump block, installing the capillary, calibrating the autosampler
and adding polymer to the syringe.
1.
Open the ABI PRISM® 310 data collection software.
2.
Prepare a GeneScan® sample sheet as described in the ABI PRISM® 310
Genetic Analyzer user’s manual. Enter the appropriate sample information
in the “sample info” column.
3.
Create a new GeneScan® injection list. Select the appropriate sample sheet
by using the pull-down menu.
4.
Select the “GS STR POP4 (1ml) F” Module using the pull-down menu. Use
the settings shown below.
Inj. Secs:
Inj. kV:
Run kV:
Run °C:
Run Time (min):
!
5
15.0
15.0
60
24
You may need to optimize the injection time for individual instruments.
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5.
Select the appropriate gel matrix file created in Section 3.B.
6.
To automatically analyze data, select the Auto Analyze checkbox and the
appropriate analysis parameters and size standard. Refer to the
ABI PRISM® 310 Genetic Analyzer user’s manual for specific information
on these options.
8.B. Sample Preparation and Loading
The Fluorescent Ladder (CXR), 60–400 Bases, is available as the internal size
marker for two-color detection and analysis of amplified samples. With this
approach, only 2–3 lanes of allelic ladder are required per gel.
1.
Prepare samples according to the instructions given in Table 11.
2.
Briefly centrifuge samples to bring the contents to the bottom of the tubes.
3.
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. Sample DNA will partially
reanneal if denatured hours before loading.
Table 11. Instrument-Specific Instructions for Sample Preparation Using the
ABI PRISM® 377 DNA Sequencer and ABI PRISM® 310 Genetic Analyzer.
ABI PRISM® 377 DNA Sequencer
For PCR-amplified samples, combine 1µl of sample with 1.5µl of Blue Dextran Loading
Solution and 0.5µl of Fluorescent Ladder (CXR).
For allelic ladders, dilute the ladder 1:10 in 1X STR Buffer, then combine 1µl of diluted
ladder with 1.5µl of Blue Dextran Loading Solution and 0.5µl of Fluorescent Ladder (CXR).
ABI PRISM® 310 Genetic Analyzer
For PCR-amplified samples, combine 1µl of sample with 24.5µl of formamide (deionized)
and 0.5µl of Fluorescent Ladder (CXR).
For allelic ladders, dilute the ladder 1:10 in 1X STR Buffer, then combine 1µl of diluted
ladder with 24.5µl of formamide (deionized) and 0.5µl of Fluorescent Ladder (CXR).
Notes:
1.
Samples amplified with the GammaSTR® Multiplex (1–2ng of template)
will need to be diluted 1:5 (i.e., 1 part sample:4 parts 1X STR Buffer) before
mixing with the loading solution.
2.
To analyze the CTTv Multiplex with Amelogenin reactions, mix the
corresponding ladders 1:1 before mixing with loading solution. The
number of ladder lanes depends on personal preference and the number
of samples analyzed.
3.
To use the TH01 allele 9.3 alone, mix 0.5µl of allele 9.3 with 1.5µl of 1X STR
Buffer before mixing with the loading solution. To use in combination with
the TH01 monoplex or CTTv Allelic Ladder, mix 0.5µl of allele 9.3 with
2.0µl of allelic ladder before mixing with the appropriate loading dye.
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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Revised 8/12
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8.B. Sample Preparation and Loading (continued)
ABI PRISM® 377 DNA Sequencer
1.
Pre-run the gel to achieve a surface temperature of approximately 50°C.
After the 15–20 minute pre-run, pause the instrument by clicking on Pause.
2.
Use a 30cc syringe filled with buffer to flush the urea from the well area.
3.
Load 1.5µl of each denatured sample into the respective wells.
4.
Place the lid on the upper buffer chamber, and close the instrument door.
ABI PRISM® 310 Genetic Analyzer
1.
Assemble the tubes in the appropriate autosampler tray (48-tube or 96-tube).
2.
Place the autosampler tray in the instrument, and close the instrument doors.
8.C. Electrophoresis and Detection
ABI PRISM® 377 DNA Sequencer
1.
After loading, select Cancel to stop the pre-run. Select Run to begin
electrophoresis.
2.
Monitor electrophoresis by observing the gel image and status windows.
3.
Allow electrophoresis to proceed for 2.25 hours. At this point, the 400-base
fragment will have migrated past the laser.
4.
Analyze the gel according to the GeneScan® analysis software user’s
manual.
Note: If the signal is too intense (a peak height greater than 3,000RFU),
dilute the sample in 1X STR Buffer before mixing with loading solution or
use less DNA template in the amplification reaction.
ABI PRISM® 310 Genetic Analyzer
1.
After loading the sample tray and closing the doors, select Run to start the
capillary electrophoresis system.
2.
Monitor electrophoresis by observing the raw data and status windows.
3.
Each sample will take approximately 30 minutes for syringe pumping,
sample injection and sample electrophoresis.
4.
Analyze data according to the GeneScan® analysis software user’s manual.
Note: Peak heights outside the linear range of the instrument may
generate artifact peaks due to instrument saturation (i.e., overloading the
sample). Bleedthrough (pull-ups) from one color to another may be
observed. Saturated signal may also appear as two peaks (split peak).
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8.D. Data Analysis
Representative STR data obtained using the ABI PRISM® 377 DNA Sequencer,
GenePrint ® Fluorescent STR Multiplex—GammaSTR® and Fluorescent Ladder
(CXR), 60–400 Bases, are shown in Figure 4 (Section 10).
Controls
Observe the lanes containing negative controls. They should be devoid of
amplification products.
Observe the lanes containing K562 DNA positive control reactions. Compare the
K562 alleles with the locus-specific allelic ladder. The expected K562 alleles for
each locus are listed in Table 13, Section 13.B.
The K562 DNA contains imbalanced alleles at several loci. This result is due to
the unusual chromosome content of the K562 cell line and is not a function of
the GenePrint ® Fluorescent STR Systems.
Allelic Ladders
In general, allelic ladders contain fragments of the same lengths as either
several or all known alleles for the locus. The allelic ladder sizes and repeat
units are listed in Table 13, Section 13.B. Analysis using GeneScan® analysis
software allows allele determination by comparing amplified sample fragments
with either allelic ladders, internal size standards or both. When using an
internal size standard, the calculated lengths of allelic ladder components will
differ from those listed in Table 13. This is due to differences in migration
resulting from sequence differences between the allelic ladder fragments and
those of the internal size standard.
8.E. Reuse of Glass Plates
For the ABI PRISM® 377 DNA Sequencer, separate the glass plates, and discard
the gel. Clean the plates in the following manner: rinse with hot water, wash
with 1% Liqui-Nox® solution, rinse well with hot water, wash with 1N NaOH,
rinse extremely well with deionized water and allow the plates to air-dry.
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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Revised 8/12
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Detection of Amplified Fragments Using the ABI PRISM® 3100 Genetic Analyzer
and Data Collection Software, Version 1.1
9.
Materials to Be Supplied by the User
• dry heating block, water bath or thermal cycler
• crushed ice or ice-water bath
• aerosol-resistant pipette tips
• 3100 capillary array, 36cm
• performance optimized polymer 4 (POP-4™) for the 3100
• 10X genetic analyzer buffer with EDTA
• sample tubes and septa for the 3100
• Hi-Di™ formamide (Applied Biosystems Cat.# 4311320)
• PowerPlex® Matrix Standards, 3100/3130 (Cat.# DG4650)
!
Formamide quality is critical. Use deionized formamide with a conductivity
<100µS/cm. Formamide can be dispensed into aliquots and frozen at –20°C. Multiple
freeze-thaw cycles or long-term storage at 4°C may cause breakdown of formamide.
Formamide with a conductivity greater than 100µS/cm may contain ions that compete
with DNA during injection, resulting in lower peak heights and reduced sensitivity. A
longer injection time may not increase the signal.
9.A. Sample Preparation
The Fluorescent Ladder (CXR), 60–400 Bases (Cat.# DG6221), is available as the
internal lane standard for 4-color detection and analysis of amplified sample.
1.
Prepare a loading cocktail by combining and mixing the Fluorescent
Ladder (CXR), 60–400 Bases, and deionized formamide as follows:
[(1µl Fluorescent Ladder × (# injections)] + [(9µl deionized formamide)
× (# injections)]
2.
Pipet 10µl of formamide/fluorescent ladder loading cocktail into each well.
3.
Add 1µl of amplified sample.
Notes:
1. Samples (1–2ng of template) amplified with the FFFL, CTTv and
GammaSTR® Systems will need to be diluted 1:5 (i.e., 1 part sample:
4 parts 1X STR Buffer) before mixing with the loading solution.
2. To analyze the CTTv Multiplex with Amelogenin reactions, mix the
corresponding ladders 1:1 before mixing with loading solution. The
number of ladder lanes depends on personal preference and the
number of samples analyzed.
3. To use the TH01 allele 9.3 alone, mix 0.5µl of allele 9.3 with 1.5µl of
1X STR Buffer before mixing with the loading solution. To use in
combination with the TH01 monoplex or CTTv Allelic Ladder, mix
0.5µl of allele 9.3 with 2.0µl of allelic ladder before mixing with the
appropriate loading dye.
4. Instrument detection limits vary; therefore, injection time or the
amount of product mixed with loading cocktail may need to be
increased or decreased. If peak heights are too high (>4,000RFU), the
samples can be diluted in the reaction buffer used for amplification
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(Gold ST*R 1X Buffer or 1X STR Buffer) before mixing with loading
cocktail. This may result in uneven allele peak heights across loci. For
best results, use less DNA template in the amplification reactions or
reduce the number of cycles in the amplification program by
2–4 cycles.
4.
For allelic ladders, dilute the ladder 1:5 (i.e., 1 part sample:4 parts 1X STR
Buffer), then combine 1µl of diluted ladder with 9.0µl of deionized
formamide and 1.0µl of Fluorescent Ladder (CXR). Cover wells with
appropriate septa.
5.
Denature samples at 95°C for 3 minutes, then immediately chill on
crushed ice or in an ice-water bath for 3 minutes. Denature the samples
just prior to loading into the ABI PRISM® 3100 Genetic Analyzer.
Note: Brief centrifugation of prepared samples will remove bubbles that
may affect analysis.
9.B. Instrument Preparation
Refer to the ABI PRISM® 3100 Genetic Analyzer user’s manual for instructions
on cleaning the pump blocks, installing the capillary array, performing a spatial
calibration, and adding polymer to the reserve syringe.
1.
Open the ABI PRISM® 3100 data collection software.
2.
Open a new plate record. Name the plate, and select “GeneScan”. Select
the plate size (96-well or 384-well). Select “Finish”.
3.
Complete the plate record spreadsheet for the wells you have loaded.
4.
In the “BioLIMS Project” column, select “3100_Project1” from the pulldown menu.
5.
In the “Dye Set” column, select “Z” from the pull-down menu.
6.
In the “Run Module 1” column, select “GeneScan36_POP4DefaultModule”
from the pull-down menu.
7.
To collect data without autoanalyzing, select “No Selection” in the
“Analysis Module 1” column. Analysis parameters can be applied after
data collection and during data analysis using the GeneScan® analysis
software.
To analyze data during data collection, an appropriate analysis module
must be selected in the “Analysis Module 1” column. Refer to the
ABI PRISM® 3100 Genetic Analyzer user’s manual for specific instructions
on creating analysis modules.
8.
Select “OK”. This new plate record will appear in the pending plate records
table on the plate setup page of the data collection software.
9.
Place samples in instrument, and close the instrument doors.
10. In the pending plate records table, click once on the name of the plate
record you just created.
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9.B. Instrument Preparation (continued)
11. Once the plate record is highlighted, click the plate graphic that corresponds
to the plate on the autosampler that contains your amplified samples to
link the plate to the plate record.
12. When the plate record is linked to the plate, the plate graphic will change
from yellow to green, the plate record moves from the pending plate
records table to the linked plate records table, and the “Run Instrument”
button becomes enabled.
13. Select “Run Instrument” on the toolbar to start the sample run.
14. Monitor electrophoresis by observing the run, status, array and capillary
views windows in the collection software. Each run (16 samples/
capillaries) will take approximately 45 minutes.
9.C. Sample Detection
1.
Analyze data using the GeneScan® analysis software.
2.
Review the raw data for one or more sample runs. Highlight the sample
file name, then under the sample menu, select “raw data”. Move the
cursor so the crosshair is on the baseline to the right of the large primer
peak (before the first internal lane standard peak [red]). Use the X-value
number shown at the bottom left of the window for the start position in
the analysis parameters.
The recommended analysis parameters are:
Analysis Range
Data Processing
Peak Detection
Size Call Range
Size Calling Method
Split Peak Correction
Start: Defined in Step 2
Stop: 10,000
Baseline: Checked
Multicomponent: Checked
Smooth Options: Light1
Peak Amplitude Thresholds2:
B:
Y:
G:
R:
Min. Peak Half Width: 2pts
Min: 60
Max: 600
Local Southern Method
None
1Smoothing
options should be determined by individual laboratories. Occasionally
the separation control alleles and the TH01 alleles 9.3 and 10 will not be
distinguished using heavy smoothing.
2The
peak amplitude thresholds are the minimum peak height that the software
will call as a peak. Values for the peak amplitude thresholds are usually 50–200RFU
and should be determined by individual laboratories.
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3.
The analysis parameters can be saved in the “Params” folder.
4.
Apply the stored analysis parameters file to the samples.
5.
Assign a new size standard. Select a sample file, and highlight the arrow next
to size standard, then select “define new.” Assign size standard peaks as
shown in Figure 2. Store the size standard in the “SizeStandards” folder.
6.
Apply the size standard file to the samples, then analyze the sample files.
7.
See Section 9.D for further data analysis.
For additional information regarding the GeneScan® analysis software, refer to
the GeneScan® analysis software user’s manual.
Notes:
1.
Peak heights outside the linear range of the instrument may generate
artifact peaks due to instrument saturation (i.e., overloading the sample).
2.
If the sample peak heights are not within the linear detection of the
instrument, the ratio of stutter peaks to real allele peaks increases and
allele designations become difficult to interpret. The balance of the peak
heights may also appear less uniform.
3.
There may be variation between instruments regarding their relative
fluorescent units detected using the same sample.
9.D. Data Analysis
Controls
Observe the lanes containing negative controls. They should be devoid of
amplification products.
Observe the lanes containing K562 DNA positive control reactions. Compare
the K562 alleles with the locus-specific allelic ladder. The expected K562 alleles
for each locus are listed in Table 13, Section 13.B.
The K562 DNA contains imbalanced alleles at several loci. This result is due to
the unusual chromosome content of the K562 cell line and is not a function of
the GenePrint ® Fluorescent STR Systems performance.
Allelic Ladders
In general, allelic ladders contain fragments of the same lengths as either
several or all known alleles for the locus. The allelic ladder sizes and repeat
units are listed in Table 13, Section 13.B. Analysis using GeneScan® analysis
software allows allele determination by comparing amplified sample fragments
with either allelic ladders, internal size standards or both. When using an
internal size standard, the calculated lengths of allelic ladder components will
differ from those listed in Table 13. This is due to differences in migration
resulting from sequence differences between the allelic ladder fragments and
those of the internal size standard.
10.
Representative STR Data
Representative data are shown in Figures 2, 3 and 4.
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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Revised 8/12
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CTTv
Lane Trace
GammaSTR®
Lane Trace
FFFL
Lane Trace
CSF1PO
D16S539
F13A01
FESFPS
D7S820
TPOX
TH01
F13B
D13S317
vWA
D5S818
LPL
Allele Height
(Shorter Peak/
Higher Peak)
(Shorter Peak/
Higher Peak)
Locus
Allele Height
Locus
(Shorter Peak/
Higher Peak)
CSF1PO
51%
F13A01
38%
D16S539
42%
TPOX
53%
FESFPS
45%
D7S820
83%
TH01
NA
F13B
NA
D13S317
NA
vWA
NA
LPL
50%
D5S818
59%
5857TA
Locus
Allele Height
Figure 2. K562 DNA amplified using the GenePrint ® Fluorescent STR Systems. Five nanograms
of K562 DNA were amplified using the GenePrint ® CTTv and FFFL Multiplex Systems, and one
nanogram of K562 DNA was amplified using the GammaSTR® Multiplex System. The amplified
DNA was separated on a 43cm, 4% denaturing polyacrylamide gel for 1 hour at 65 watts using
the Hitachi FMBIO® Fluorescence Imaging System. Lane traces for K562 DNA amplified using
each of the three GenePrint ® Systems are shown. The table below each lane trace indicates the
percent differences in allele height (i.e., peak heights minus typical interlocus background value)
occurring as a result of the unusual chromosome content of the K562 cell line. These variations
are not a consequence of primer imbalance in amplification.
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CTTv
Multiplex
Page 33
GammaSTR®
Multiplex
FFFL
Multiplex
L 12 L3 4L56L
CSF1P0
L1 2L3 4L56L
L 1 2 L3 4L 5 6L
– 15
–7
– 15
– 16
D16S539
F13A01
–5
– 14
–4
– 13
TPOX
– 14
D7S820
FESFPS
–6
–6
–7
– 15
– 11
D13S317
TH01
– 11
–5
F13B
–7
–6
– 20
– 15
vWA
5858TA
– 13
D5S818
– 14
–7
LPL
–7
Figure 3. STR analyses performed using the fluorescein-labeled GenePrint ® STR Multiplex
Systems and Hitachi FMBIO® Fluorescence Imaging System. DNA samples amplified using the
CTTv, FFFL and GammaSTR® Multiplex Systems are shown. For each system, six DNA samples
were amplified (lanes 1–6) and are shown with allelic ladders for the corresponding loci (lanes L).
Each allelic ladder is labeled to its right with the number of copies of the repeat sequence contained
within the corresponding largest and smallest alleles of each locus. All materials were separated
using 4% denaturing polyacrylamide gels. The CTTv, FFFL and GammaSTR® Multiplex Systems
were detected using the Hitachi FMBIO® II Fluorescence Imaging System.
D5S818
D13S317
100
D7S820
D16S539
200
225
250
275
400
325 350 375
5860TA
120 140 160 180
300
Figure 4. STR analyses performed using the GammaSTR® Multiplex System, Fluorescent Ladder
(CXR), 60–400 Bases, and ABI PRISM® 310 Genetic Analyzer. The upper panel shows the alleles of
loci D5S818, D13S317, D7S820 and D16S539 amplified using the GammaSTR® System and 1ng DNA
template. The sample was diluted 1:5 (1 part sample:4 parts STR 1X Buffer) prior to loading. The
bottom panel shows the relevant portion of the Fluorescent Ladder (CXR), 60–400 Bases. The
numbers above the peaks indicate the sizes of the Fluorescent Ladder (CXR) fragments.
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Representative STR Data (continued)
Artifacts and Stutter
Stutter bands are a common amplification artifact associated with STR analysis.
Stutter products are often observed one repeat unit below the true allele peak and,
occasionally, two repeat units smaller or one repeat unit larger than the true allele
peak. Frequently, alleles with a greater number of repeat units will exhibit a higher
percent stutter. The pattern and intensity of stutter may differ slightly between
primer sets for the same loci.
In addition to stutter peaks, other artifact peaks can be observed. There is an artifact
noted in the D5S818 typing region when amplifying the GenePrint ® Fluorescent STR
Multiplex—GammaSTR® System and using ABI PRISM® 3130 Genetic Analyzer for
detection. This artifact has been characterized as secondary structure associated with
the D16S539 primer set and migrates between 80 and 110bp, depending upon
instrumentation. The artifact has broad peak morphology and should not fall
on-ladder. In addition, samples are diluted as recommended in Section 7.B, 8.C or
9.B, the peak should fall below 300RFU.
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/allele peaks
Causes and Comments
Impure template DNA. 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
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.
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Symptoms
Faint or no bands/allele peaks
(continued)
Extra bands visible in
one or all lanes
Bands are fuzzy
throughout the lanes
Extra peaks visible in one
or all color channels
(ABI PRISM® 310 or 3100
Genetic Analyzer)
Allelic ladder not running the
same as the sample
Uneven peak heights
between loci
High background with low
signal
11:29 AM
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Causes and Comments
Samples were not denatured before loading in the gel. Be sure
the samples are heated at 95°C for 2 minutes and cooled on
crushed ice or in an ice-water bath immediately prior to
loading.
Poor CE injection. Re-inject the sample.
Poor-quality formamide. Be sure that high-quality formamide
is used when running samples on the ABI PRISM® 310 or
3100 Genetic Analyzer.
Contamination with another template DNA or previously
amplified DNA. Cross-contamination can be a problem. Use
aerosol-resistant pipette 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.
Samples were not completely denatured. Heat denature
samples at 95°C for 2 minutes immediately prior to loading
the gel.
Insufficient pre-run of gel. Pre-run gels until a temperature of
50°C is reached before loading samples
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 a lower
temperature (40–60°C).
CE-related artifacts. Minor voltage changes or urea crystals
passing by the laser may cause “spikes” or unexpected peaks.
Spikes sometimes appear in one color but often are easily
identified by their presence in more than one color. Re-inject
the samples to confirm.
Wrong allelic ladder or primer pair mix was used. Be sure
that the allelic ladder is from the same kit as the 10X Primer
Pair Mix.
Poor-quality formamide was used. Be sure that high-quality
formamide is used when running samples on the ABI PRISM®
310 or 3100 Genetic Analyzer.
Thermal cycler problems. Review the thermal cycling protocols
in Section 4. We have not tested other thermal cyclers.
Excessive amount of DNA. Use the recommended amount of
template. See Table 5, Section 4.A, for recommendations.
Use of FTA® paper. Results may be similar to use of excess
amounts of DNA template. Reduce the number of cycles in the
amplification program by 2–4 cycles (10/18 or 10/16 cycling)
to improve the locus-to-locus balance.
Degraded DNA sample. DNA template is degraded into
smaller fragments, with the larger loci showing diminished
yield.
Part of the spacers were scanned. Re-scan the gel being careful
not to scan any portion of the spacers.
Plates were improperly washed. Improper washing of the
plates can cause a soap residue to build up on the plates. This
can cause background fluorescence.
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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References
12.A. Cited 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. Nucleic 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.
Micka, K.A. et al. (1999) TWGDAM validation of a nine-locus and a four-locus fluorescent STR
multiplex system. J. Forensic Sci. 44, 1243–57.
17.
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.
18.
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.
19.
Nakamura, Y. et al. (1987) Variable number of tandem repeat (VNTR) markers for human gene
mapping. Science 235, 1616–22.
20.
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.
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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21.
Levinson, G. and Gutman, G.A. (1987) Slipped-strand mispairing: A major mechanism for DNA
sequence evolution. Mol. Biol. Evol. 4, 203–21.
22.
Schlotterer, C. and Tautz, D. (1992) Slippage synthesis of simple sequence DNA. Nucleic Acids Res. 20,
211–5.
23.
Smith, J.R. et al. (1995) Approach to genotyping errors caused by nontemplated nucleotide addition by
Taq DNA polymerase. Genome Res. 5, 312–7.
24.
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.
25.
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.
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.
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.
29.
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.
30.
Gill, P. et al. (1997) Considerations from the European DNA Profiling Group (EDNAP) concerning
STR nomenclature. Forensic Sci. Int. 87, 185–92.
31.
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.
32.
Mandrekar, P.V., Krenke, B.E. and Tereba, A. (2001) DNA IQ™: The intelligent way to purify DNA.
Profiles in DNA 4(3), 16.
33.
Krenke, B.E. et al. (2005) Development of a novel, fluorescent, two-primer approach to quantitative
PCR. Profiles in DNA 8(1), 3–5.
34.
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.
35.
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), 11–3.
36.
Cowan, C. (2006) The DNA IQ™ System on the Tecan Freedom EVO® 100. Profiles in DNA 9(1), 8–10.
37.
Bjerke, M. et al. (2006) Forensic application of the Maxwell™ 16 Instrument. Profiles in DNA 9(1), 3–5.
38.
Mandrekar, P. et al. (2007) Introduction to Maxwell® 16 low elution volume configuration for forensic
casework. Profiles in DNA 10(2), 10–2.
39.
Grimberg, J. et al. (1989) A simple and efficient non-organic procedure for the isolation of genomic
DNA from blood. Nucleic Acids Res. 17, 8390.
40.
Miller, S., Dykes, D. and Polesky, H. (1988) A simple salting out procedure for extracting DNA from
human nucleated cells. Nucleic Acids Res. 16, 1215.
41.
Comey, C. et al. (1994) DNA extraction strategies for amplified fragment length polymorphism
analysis. J. Forensic Sci. 39, 1254–69.
42.
Walsh, P.S., Metzger, D.A. and Higuchi, R. (1991) Chelex® 100 as a medium for simple extraction of
DNA for PCR-based typing from forensic material. BioTechniques 10, 506–13.
43.
Higuchi, R. (1989) Rapid, efficient DNA extraction for PCR from cells or blood. In: Amplifications: A
Forum for PCR Users (May 1989) PerkinElmer, Norwalk, CT, Issue 2.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
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12.B.Additional STR References
A substantial reference list of publications describing STRs and related
information can be found at a web site created by the National Institutes of
Standards and Technology (NIST) Biotechnology Division. This web site
(www.cstl.nist.gov/div831/strbase/) is occasionally updated and has numerous
links to many other useful sites.
The references below provide an overview of the listed topics and are available
at: www.promega.com/profiles/. The spreadsheet for the PowerStats
application is available at: www.promega.com/geneticidtools/powerstats/.
PowerStats is a Microsoft® Excel workbook spreadsheet that allows genotype
data from STaRCall™ or Genotyper® software to be pasted directly into the
workbook to obtain standard population statistics on the distribution of alleles
within particular population subsets. If you cannot access the web site, please
contact your local Promega branch office or distributor.
1.
The Short Tandem Repeat DNA Database Web Site:
Butler, J.M. and Reeder, D.J. (1997) STRBase: A short tandem repeat DNA database. Profiles in
DNA 1(2), 10.
2.
PowerStats Analysis of Population Data:
Tereba, A. (1999) Tools for analysis of population statistics. Profiles in DNA 2(3), 14–6.
13.
Appendix
13.A. Advantages of STR Typing
The GenePrint ® Fluorescent STR Systems provide a rapid, non-radioactive
method to evaluate small amounts (e.g., 1ng) of human DNA. The protocols in
this manual describe the use of a fluorescein label to detect amplified STR
products following their separation by denaturing polyacrylamide gel
electrophoresis. For information on multicolor fluorescent STR systems, refer to
the PowerPlex ® 16 System Technical Manual #TMD012, PowerPlex ® 16 BIO System
Technical Manual #TMD016, PowerPlex ® ES System Technical Manual #TMD017,
PowerPlex ® 1.1 System Technical Manual #TMD008, PowerPlex ® 1.2 System
Technical Manual #TMD009 or PowerPlex ® 2.1 System Technical Manual
#TMD011. Refer to the GenePrint ® STR Systems Technical Manual #TMD004 for
information about detecting STR products using silver staining (17). These
Technical Manuals are available at: www.promega.com/tbs/
STR typing is more tolerant of degraded DNA templates than other methods of
individual identification because amplification products are less than 400bp
long, much smaller than the material detected with AMP-FLP (18) or VNTR (19)
analysis. This format is also amenable to a variety of rapid DNA purification
techniques.
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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 ® Fluorescent 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 (20).
13.B. Advantages of Using the Loci in the GenePrint ® Fluorescent STR Systems
The STR loci and primers contained in the GenePrint ® Fluorescent STR Systems
(Tables 12 and 13) 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 (21,22), 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 forensics community.
Terminal nucleotide addition (23,24) occurs when Taq DNA polymerase adds a
nucleotide, generally adenine, to the 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. Redefinition of
the primer sequences and/or addition of a final extension step of 60°C for
30 minutes to the amplification protocol can lead to essentially full terminal
nucleotide addition (25).
Notes for Table 13:
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 containing
tandem repeats of short sequences (21,22). 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 four copies of the repeat
but has a two-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 in October 1993.
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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Table 12. Locus-Specific Information.
STR Locus
Chromosomal Location
Amelogenin1
CSF1PO
Xp22.1–22.3 and Y
GenBank® Locus and
Locus Definition
HUMAMEL, Human Y
chromosomal gene for
amelogenin-like protein
Repeat Sequence2
5´→ 3´
NA
5q33.3–34
HUMCSF1PO, Human c-fms
proto-oncogene for CSF-1
receptor gene
AGAT
D16S539
16q24–qter
NA
AGAT
D7S820
7q11.21–q22
NA
AGAT
D13S317
13q22–q31
NA
AGAT
D5S818
5q23.3–32
NA
AGAT
F13A01
6p24.3–p25.1
HUMF13A01, Human
coagulation factor XIII a
subunit gene
AAAG
F13B
1q31–q32.1
AAAT
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
TH01
11p15.5
TPOX
2p24–pter
vWA
12p13.31
HUMLIPOL, Human
lipoprotein lipase gene
HUMTH01, Human tyrosine
hydroxylase gene
HUMTPOX, Human thyroid
peroxidase gene
HUMVWFA31, Human von
Willebrand factor gene
AAAT
AGAT
AAAT
AATG
AATG
TCTA Complex (28)
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.
2The
August 1997 report (29,30) 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”.
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Table 13. Additional Locus-Specific Information.
Allelic Ladder
Size Range1
(bases)
STR Ladder
Alleles
(# of repeats)
CSF1PO
212–218
295–327
D16S539
264–304
D7S820
215–247
D13S317
165–197
D5S818
119–151
F13A01
283–331
F13B
FESFPS
HPRTB
169–189
222–250
259–303
LPL
TH01
TPOX
vWA
105–133
179–203
224–252
139–167
NA
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
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
7,8,9,10,11,12,13,14
6,7,8,9,10,11,
12,13,14,15,16,17
7,9,10,11,12,13,14
5,6,7,8,9,10,11
6,7,8,9,10,11,12,13
13,14,15,16,
17,18,19,20
STR Locus
Amelogenin3
Other Known K562 DNA
Alleles2
Allele Sizes
(# of repeats) (# of repeats)
Comments
None
6
212,212
10,9
1,2
1
None
12,11
1
None
11,9
1
None
8,8
1
16
12,11
1
3.2,104
5,45
1,3
12
None
None
10,10
12,10
13,13
1
1
1
8
9.3
None
10,11,
21,22
12,10
9.3,9.3
9,8
16,16
1
1,4
1
1
NA = not applicable.
Alleles in bold are present in greater amounts than other alleles. This simplifies interpretation.
1Lengths
2Alleles
of each allele in the allelic ladders have been confirmed by sequence analyses.
that represent <0.2% of the population may not be listed in this table.
3Amelogenin
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.
4Allele
10 (307 bases) is not included because it is rare and its exclusion creates a gap that simplifies
interpretation of the allelic ladder (27,31).
5F13A01
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.
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 8/12
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13.C. Power of Discrimination
The GenePrint ® Fluorescent STR Systems provide extremely powerful
discrimination. The combined matching probability of the CTTv, FFFL and
GammaSTR® quadriplexes range from 1 in 303,000,000,000 in CaucasianAmericans to 1 in 4,610,000,000,000 in African-Americans (Table 14).
Typical paternity indices for the GenePrint ® Fluorescent STR Systems are shown
in Table 15. An alternative calculation in paternity analyses is the power of
exclusion. Table 16 gives typical values for the power of exclusion for the
GenePrint ® Fluorescent STR Systems in various populations.
Table 14. Matching Probability of Various Populations.
STR System
CTTv quadriplex
(CSF1PO, TPOX, TH01, vWA)
FFFL quadriplex (F13A01,
FESFPS, F13B, LPL)
GammaSTR® III quadriplex
(D16S539, D7S820, D13S317,
D5S818)
All 3 quadriplexes (12 loci)
African-American
Matching Probability
Caucasian-American Hispanic-American
1 in 25,236
1 in 6,796
1 in 7,219
1 in 16,802
1 in 2,658
1 in 3,276
1 in 10,872
1 in 4.61 × 1012
1 in 16,790
1 in 3.03 × 1011
1 in 20,106
1 in 4.75 × 1011
Table 15. Typical Paternity Indices of the Multiplex GenePrint ® STR Systems in
Various Populations.
STR System
CTTv quadriplex
(CSF1PO, TPOX, TH01, vWA)
FFFL quadriplex (F13A01,
FESFPS, F13B, LPL)
GammaSTR® III quadriplex
(D16S539, D7S820, D13S317,
D5S818)
All 3 quadriplexes (12 loci)
Typical Paternity Index
African-American Caucasian-American Hispanic-American
29.4
19.26
10.51
16.83
15.28
8.23
16.93
8,373
13.51
3,976
30.40
2,627
Table 16. Power of Exclusion of the GenePrint ® STR Systems in Various Populations.
STR System
CTTv quadriplex
(CSF1PO, TPOX, TH01, vWA)
FFFL quadriplex (F13A01,
FESFPS, F13B, LPL)
GammaSTR® III quadriplex
(D16S539, D7S820, D13S317,
D5S818)
All 3 quadriplexes (12 loci)
African-American
Power of Exclusion
Caucasian-American
Hispanic-American
0.967
0.953
0.918
0.946
0.941
0.902
0.946
0.99990
0.934
0.99981
0.967
0.99974
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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Printed in USA.
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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 (32). This novel technology
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. For 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. See Section 13.G for additional information.
For applications requiring human-specific DNA quantification, the Plexor® HY
System (Cat.# DC1000) has been developed (33). See Section 13.G for ordering
information.
The DNA IQ™ System has been fully automated on the Beckman Coulter Biomek®
2000 Laboratory Automation Workstation (34), Biomek® 3000 Laboratory Automation
Workstation (35) and Tecan Freedom EVO® Liquid Handler (36). In addition, the
DNA IQ™ Reference Sample Kit for Maxwell® 16 (Cat.# AS1040) and DNA IQ™
Casework Sample Kit for Maxwell® 16 (Cat.# AS1210) are available (37,38). For
information on 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]
Traditional DNA extraction methods, such as phenol:chloroform extraction methods
(32) or inorganic methods (39,40), can be used for STR analysis. For stains from
blood and saliva, scientists at the FBI Academy have suggested an alternative method
for DNA extraction (see reference 41). DNA isolation methods such as Chelex® 100
(42) and cell lysis followed by proteinase K digestion (43) are capable of extracting
DNA from bloodstains or from as little as 3µl of whole blood. Because both of these
methods produce single-stranded DNA, they should not be used for VNTR (variable
number tandem repeat) polymorphism analysis.
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 8/12
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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.
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 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 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 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# TMD006
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Printed in USA.
Revised 8/12
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13.F. Composition of Buffers and Solutions
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
Add 0.5g of ammonium persulfate
to 5ml of deionized water. Use 200µl
for 30ml of acrylamide gel solution.
Store the remaining volume in 200µl
aliquots at –20°C.
Blue Dextran Loading Solution
4.1mM
88.25%
15mg/ml
EDTA
formamide, ACS grade
blue dextran
Bromophenol Blue Loading
Solution
10mM
95%
0.05%
NaOH
formamide
bromophenol blue
0.5M EDTA (pH 8.0)
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 final
volume to 1 liter. Dispense into
aliquots, and sterilize by autoclaving.
ethidium bromide solution
(10mg/ml)
1.0g
ethidium bromide
Dissolve in 100ml of deionized
water. Wrap in aluminum foil or
transfer to a dark bottle, and store at
room temperature.
!
Caution: Ethidium bromide is a
powerful mutagen. Wear gloves at
all times, and use a mask when
weighing out ethidium bromide
powder.
Gel Tracking Dye
10mM
95%
0.05%
0.05%
NaOH
formamide
bromophenol blue
xylene cyanol FF
Gold ST*R 10X Buffer
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)
STR 2X Loading Solution
10mM
95%
0.05%
0.05%
NaOH
formamide
bromophenol blue
xylene cyanol FF
STR 10X Buffer
500mM
100mM
15mM
1%
2mM
KCl
Tris-HCl (pH 9.0)
MgCl2
Triton® X-100
each dNTP
50X TAE buffer (pH 7.2)
242g Tris base
57.1ml glacial acetic acid
100ml 0.5M EDTA (pH 8.0)
Add Tris base and EDTA to 500ml of
deionized water. Add the glacial
acetic acid. Bring the 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 8/12
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13.F. Composition of Buffers and Solutions (continued)
1X TAE buffer (pH 7.2)
0.5X TBE buffer
Add 20ml of 50X TAE buffer to
980ml of deionized water.
Add 50ml of 10X TBE buffer to
950ml of deionized water.
10X TBE 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 volume to 1 liter
with deionized water.
13.G. Related Products
Fluorescent STR Multiplex Systems
Product
PowerPlex® 1.1 System
PowerPlex® 2.1 System
PowerPlex® 1.2 System
PowerPlex® 16 System
PowerPlex® 16 BIO System
PowerPlex® ES System
Size
100 reactions
400 reactions
100 reactions
400 reactions
100 reactions
100 reactions
400 reactions
100 reactions
400 reactions
100 reactions
400 reactions
Cat.#
DC6091
DC6090
DC6471
DC6470
DC6101
DC6531
DC6530
DC6541
DC6540
DC6731
DC6730
Not for Medical Diagnostic Use.
The PowerPlex® 1.1, 2.1, and 16 BIO Systems are compatible with the Hitachi FMBIO®
Fluorescence Imaging Systems.
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# TMD006
Page 46
Printed in USA.
Revised 8/12
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Accessory Components
Product
Acrylamide
Ammonium Persulfate
Bisacrylamide
Blue Dextran Loading Solution
Bromophenol Blue Loading Solution
Gel Tracking Dye
Gold ST*R 10X Buffer
K562 DNA High Molecular Weight
Mineral Oil
Nuclease-Free Water
PowerPlex® Matrix Standards, 310**
PowerPlex® Matrix Standards, 3100/3130**
STR 10X Buffer
STR 2X Loading Solution
TBE Buffer, 10X
Urea
Size
100g
25g
25g
3ml
3ml
1ml
1.2ml
30µg
12ml
50ml
150ml
50µl (each dye)
25µl (each dye)
1.2ml
3ml
1L
1kg
Cat.#
V3111
V3131
V3141
DV4351
DV4371
DV4361
DM2411
DD2011
DY1151
P1193
P1195
DG4640
DG4650
DM2211
DV4331
V4251
V3171
Size
150µl
65µl
Cat.#
DG1071
DG6221
Size
200 reactions
800 reactions
100 reactions
400 reactions
48 preps
48 preps
50 samples
200 samples
10 pack
Cat.#
DC1001
DC1000
DC6701
DC6700
AS1210
AS1040
DC6801
DC6800
V1391
**Not for Medical Diagnostic Use.
Internal Lane Standards
Product
Internal Lane Standard 600
Fluorescent Ladder CXR, 60–400 Bases
Sample Preparation Systems
Product
Plexor® HY System*
DNA IQ™ System
DNA IQ™ Casework Sample Kit for Maxwell® 16
DNA IQ™ Reference Sample Kit for Maxwell® 16
Differex™ System*
Slicprep™ 96 Device
*Not for Medical Diagnostic Use.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 8/12
Part# TMD006
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13.G. Related Products (continued)
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
0.5–20µl
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
Part# TMD006
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Printed in USA.
Revised 8/12
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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. 5,843,660 and 6,221,598, Australian Pat. No. 724531, Canadian Pat. No. 2,118,048,
Korean Pat. No. 290332, Singapore Pat. No. 57050, Japanese Pat. No. 3602142 and other patents
pending.
(c)U.S.
Pat. No. 5,843,660, Australian Pat. No. 724531, Korean Pat. No. 290332, Singapore Pat. No.
57050, Japanese Pat. No. 3602142 and other patents pending.
(d)U.S.
Pat. No. 6,221,598, Canadian Pat. No. 2,118,048, Korean Pat. No. 290332 and other patents
pending.
© 2012 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.
ABI PRISM, GeneScan, Genotyper and MicroAmp are registered trademarks of Applera Corporation.
AmpliTaq, AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems,
Inc. ART is a registered trademark of Molecular BioProducts, Inc. Biomek is a registered trademark
of Beckman Coulter, Inc. Chelex is a registered trademark of Bio-Rad Laboratories, 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. Hi-Di
and POP-4 are trademarks of Applera Corporation. Kimwipes is a registered trademark of KimberlyClark. Liqui-Nox is a registered trademark of Alconox, Inc. Long Ranger is a registered trademark of
Cambrex Corporation. Microsoft is a registered trademark of Microsoft Corporation. Nalgene is a
registered trademark of Nalge Nunc International. Polaroid is a registered trademark of Polaroid
Corporation. STaRCall is a trademark of Hitachi Software Engineering Company, Ltd. Triton is a
registered trademark of Union Carbide Chemicals and Plastics Technology Corporation.
Products may be covered by pending or issued patents or may have certain limitations. Please visit
our Web site for more information.
All prices and specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the
Promega online catalog for the most up-to-date information on Promega products.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 8/12
Part# TMD006
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