Thermo Fisher Scientific VeriFiler Express PCR Amplification Kit User Guide
The VeriFiler Express PCR Amplification Kit is a 6-dye, short tandem repeat (STR) multiplex assay for the amplification of single-source human genomic DNA. It is optimized for paternity and single-source samples and is part of a fully integrated and validated forensic DNA workflow.
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VeriFiler ™ Express PCR Amplification Kit
USER GUIDE
Catalog Numbers A32014, A32070, A33032
Publication Number 100043588
Revision C
For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.
Manufacturer: Thermo Fisher Scientific | 7 Kingsland Grange | Warrington, Cheshire WA1 4SR | United Kingdom
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,
INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR
USE OF IT.
Revision history : Pub. No. 100043588
Revision
C
Date
12 September 2017
Description
• Add 4N6FLOQSwabs ™ as a validated collection method.
• Add DNA-dependent artifact VIC110-120 (extra peaks in the electropherogram).
B
A
14 November 2016
04 October 2016
Add representative electropherograms for several types of substrate.
New document
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. 4N6FLOQSwabs, COPAN, and NUCLEIC-CARD are trademarks of Copan Italia S.P.A., and used by Life Technologies under their permission. Adobe, Acrobat, and Reader are trademarks of Adobe Systems Incorporated. Agilent is a trademark of Agilent Technologies, Inc. Bode Buccal DNA Collector is a trademark of Bode
Technology Group, Inc. EasiCollect, FTA, and Whatman are trademarks of Whatman Limited. Harris Micro-Punch is a trademark of Harris, Joel S. TA
Shunderson Communications. Robbins Scientific is a trademark of Molecular Bioproducts, Inc. VWR Scientific is a trademark of VWR International,
Inc. Windows and Windows Vista are trademarks of Microsoft Corporation.
©2017 Thermo Fisher Scientific Inc. All rights reserved.
Contents
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CHAPTER 1 Product information ....................................... 8
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CHAPTER 2 Perform PCR ............................................. 16
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Contents
4
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CHAPTER 3 Perform electrophoresis ................................. 27
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) . . . . 28
Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) . . . . . . . 30
Set up the 3130/3130 xl instruments for electrophoresis (before first use of the kit) . . . . . 31
Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) . . . . . . . 33
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CHAPTER 4 Analyze data with GeneMapper
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™
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ID ‑X Software for analysis (before first use of the kit) . . . . . . . . 40
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ID ‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Download newer versions of panel bin and stutter files if needed . . . . . . . . . . . . . . . . 40
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Express PCR Amplification Kit User Guide
Contents
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polymer on a 3730 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Analyze and edit sample files with GeneMapper
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ID ‑X Software . . . . . . . . . . . . . . . . . . . . . 55
For more information on using the GeneMapper
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ID ‑X Software . . . . . . . . . . . . . . . . . . . . . 56
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CHAPTER 5 Experiments and results ................................. 57
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Contents
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APPENDIX A Troubleshooting ........................................ 111
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APPENDIX B Materials required but not supplied .................. 114
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APPENDIX C Plate layouts ............................................ 120
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Express PCR Amplification Kit User Guide
Contents
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APPENDIX D PCR work areas ........................................ 122
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Safety ..................................................................... 124
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APPENDIX E Documentation and support .......................... 127
References
Index ..................................................................... 135
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8
1
Product information
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IMPORTANT! Before using this product, read and understand the information in the
“Safety” appendix in this document.
Product description
Kit overview
The VeriFiler ™ Express PCR Amplification Kit is a 6-dye, short tandem repeat (STR) multiplex assay for the amplification of single-source human genomic DNA.
The kit is part of a fully integrated and validated forensic DNA workflow backed by best-in-class global training, service, and support. This kit is optimized for paternity and single-source samples.
The kit amplifies the following loci within the read region of 75–465 nt:
• 23 autosomal STR loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179,
D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317,
D7S820, D10S1248, D1S1656, D12S391, D2S1338, D6S1043, Penta D, Penta E)
• 1 insertion/deletion polymorphic marker on the Y chromosome (Y indel)
• Amelogenin (sex-determining marker)
The VeriFiler ™ Express kit delivers a 25-locus multiplex with the highest discrimination power of any Thermo Fisher Scientific Human Identification Kit. The highly optimized buffer formulation allows completion of amplification in
~45 minutes.
The VeriFiler ™ Express kit uses the same improved process for synthesis and purification of the amplification primers developed for other next-generation Thermo
Fisher Scientific STR chemistries. The improved amplification primers deliver clean electrophoretic backgrounds that assist interpretation.
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Express PCR Amplification Kit User Guide
Chapter 1 Product information
Product description
1
Single-source sample types supported
Substrate examples
The VeriFiler ™ Express kit is optimized to allow direct amplification from the following types of single-source samples without the need for sample purification:
• Blood and buccal samples on treated paper substrates.
• Blood and buccal samples collected on untreated paper substrates and treated with Prep-n-Go ™ Buffer.
• Buccal samples collected on swab substrates and treated with Prep-n-Go ™ Buffer.
• Treated paper: COPAN NUCLEIC-CARD ™ system or Whatman FTA ™ cards
• Untreated paper: Bode Buccal DNA Collector ™ or 903 paper
• Swab: COPAN 4N6FLOQSwabs ™ reference collection devices or cotton swabs
Note: Our testing does not include blood samples on swab substrates. This sample type is not typically used for the collection of reference samples.
About the primers
The VeriFiler ™ Express kit primers are manufactured using the same synthesis and purification improvements as the primers in the NGM SElect ™ , the Sinofiler ™ , and the
Identifiler ™ Plus kits. These improvements enhance the assay signal-to-noise ratio and simplify the interpretation of results.
The VeriFiler ™ Express kit includes the same primers as the GlobalFiler ™ Express PCR
Amplification Kit with the following primer additions and modifications:
• Addition of new primers for Penta D, Penta E, and D6S1043.
• Addition of 1 new SNP-specific primer for D19S433 loci to address a SNP in the primer binding site.
Non-nucleotide linkers are used in primer synthesis for the following loci: D19S433, vWA, CSF1PO, D2S441, TH01, FGA, and D12S391. For these primers, non-nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis (Butler 2005, Grossman et al., 1994). Non-nucleotide linkers enable reproducible positioning of the alleles to facilitate interlocus spacing. The combination of a 6-dye fluorescent system and the use of non-nucleotide linkers allows simultaneous amplification and efficient separation of all 25 markers during automated DNA fragment analysis.
Dyes used in the kit
Label
Samples, allelic ladders, and controls
Dye
6 ‑FAM ™
VIC ™
NED ™
TAZ ™
SID ™
LIZ ™
Color
Blue
Green
Yellow
Red
Purple
Orange GeneScan ™ 600 LIZ ™ Size Standard v2.0
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Express PCR Amplification Kit User Guide 9
1 Chapter 1 Product information
Product description
Loci amplified by the kit
Table 1 VeriFiler ™ Express kit loci and alleles
Locus designation
D3S1358 vWA
D16S539
CSF1PO
TPOX
Y indel Yq11.221
1, 2
Amelogenin X: p22.1-22.3 Y: p11.2
X, Y
D8S1179 8q24.13
5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
D21S11
D18S51
Penta E
D2S441
D19S433
21q11.2-q21 24, 24.2, 25, 26, 27, 28, 28.2, 29, 29.2, 30, 30.2, 31,
31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36, 37,
38
18q21.33
15q26.2
2p14
19q12
7, 9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26
8, 9, 10, 11, 11.3, 12, 13, 14, 15, 16, 17
6, 7, 8, 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15,
15.2, 16, 16.2, 17, 17.2, 18.2, 19.2
TH01
FGA
D22S1045
D5S818
D13S317
D7S820
D6S1043
D10S1248
D1S1656
Chromosome location
3p21.31
Alleles included in Allelic Ladder
12p13.31
16q24.1
5q33.3-34
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24
5, 8, 9, 10, 11, 12,13, 14, 15
6, 7, 8, 9, 10, 11, 12, 13, 14, 15
2p23-2per 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
11p15.5
4q28
22q12.3
5q21-31 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18
13q22-31 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
7q11.21-22 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
6q16.1
10q26.3
1q42.2
4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
26.2, 27, 28, 29, 30, 30.2, 31.2, 32.2, 33.2, 42.2,
43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
9, 10, 11, 12, 13, 14, 14.3, 15, 15.3, 16, 16.3, 17,
17.3, 18.3, 19.3, 20.3
Dye label
6-FAM ™
VIC ™
NED ™
TAZ ™
SID ™
DNA Control
007
15, 16
14, 16
9, 10
11, 12
8, 8
2
X, Y
12, 13
28, 31
11, 16
11, 11
11, 11
7, 12
12, 14
12, 15
13, 16
12, 15
7, 12
14, 15
14, 15
7, 9.3
24, 26
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Express PCR Amplification Kit User Guide
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
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1 1 1
1
Chapter 1 Product information
Product description
1
Locus designation
D12S391
D2S1338
Penta D
Standards and controls that are required
Chromosome location
12p13.2
2q35
21q22.3
Alleles included in Allelic Ladder
14, 15, 16, 17, 18, 19, 19.3, 20, 21, 22, 23, 24, 25,
26, 27
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28
2.2, 3.2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17
Dye label
SID ™
DNA Control
007
18, 19
20, 23
11, 12
For the VeriFiler ™ Express kit, the panel of standards needed for PCR amplification,
PCR product sizing, and genotyping are:
• DNA Control 007—A positive control for evaluating the efficiency of the amplification step and STR genotyping using the VeriFiler ™ Express kit Allelic
Ladder. DNA Control 007 is present in the kit. See “DNA Control 007 profile“ on page 12.
• GeneScan ™ 600 LIZ ™ Size Standard v2.0—Used for obtaining sizing results. This standard, which has been evaluated as an internal size standard, yields precise sizing results for PCR products. Order the GeneScan ™ 600 LIZ ™ Size Standard v2.0 (Cat. No. 4408399) separately.
• VeriFiler ™ Express Allelic Ladder—Developed for accurate characterization of the alleles amplified by the kit. The Allelic Ladder is present in the kit, and allows automatic genotyping of most of the reported alleles for the loci in the kit. See
“Allelic ladder profile“ on page 11.
Allelic ladder profile
Figure 1 GeneMapper ™ ID ‑X Software plot of the VeriFiler ™ Express Allelic Ladder
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Express PCR Amplification Kit User Guide 11
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
1 1 1
1
1 Chapter 1 Product information
Product description
DNA Control 007 profile
Figure 2 DNA Control 007 (2 ng) amplified at 27 PCR cycles with the VeriFiler ™
Applied Biosystems ™ 3500xL Genetic Analyzer (Y-axis scale 0 to 14,000 RFU).
Express kit and analyzed on an
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Express PCR Amplification Kit User Guide
Chapter 1 Product information
Contents and storage
1
Contents and storage
Table 2 VeriFiler ™ Express PCR Amplification Kit (Cat. No. A32014; 200 reactions)
Contents
VeriFiler ™ Express Master Mix
Contains MgCl
2
, dATP, dGTP, dCTP, and dTTP, bovine serum albumin, enzyme, and 0.05% sodium azide in buffer and salt.
VeriFiler ™ Express Primer Set
Contains locus-specific 6-FAM ™ , VIC ™ , NED ™ ,TAZ ™ , and SID ™ dye-labeled and unlabeled primers in buffer. The primers amplify the STR loci D3S1358, vWA, D16S539, CSF1PO,TPOX, D8S1179,
D21S11, D18S51, Penta E, D2S441, D19S433, TH01, FGA,
D22S1045, D5S818, D13S317, D7S820, D6S1043, D10S1248,
D1S1656, D12S391, D2S1338, Penta D, and the sex-determining markers, Y indel and Amelogenin.
VeriFiler ™ Express Allelic Ladder
Contains the following amplified alleles:
• 6-FAM ™ dye (blue): D3S1358 9–20; vWA 11–24; D16S539 5,
8–15; CSF1PO 6–15; TPOX 5–15.
• VIC ™ dye (green): Y indel 1, 2; Amelogenin X, Y; D8S1179
5-19; D21S11 24, 24.2, 25–28, 28.2, 29, 29.2, 30, 30.2, 31,
31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36–38; D18S51 7, 9,
10, 10.2, 11–13, 13.2, 14, 14.2, 15–27; DYS391 7–13; Penta E
5–26.
• NED ™ dye (yellow): D2S441 8–11, 11.3, 12–17; D19S433 6–
12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2, 18.2,
19.2; TH01 4–9, 9.3, 10, 11, 13.3; FGA 13–26, 26.2, 27–30,
30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2,
50.2, 51.2.
• TAZ ™ dye (red): D22S1045 8–19; D5S818 7–18; D13S317 5–
16; D7S820 6–15; D6S1043 9–25.
• SID ™ dye (purple): D10S1248 8–19; D1S1656 9–14, 14.3, 15,
15.3, 16, 16.3, 17, 17.3, 18.3, 19.3, 20.3; D12S391 14–19, 19.3,
20–27; D2S1338 11–28; Penta D 2.2, 3.2, 5–17.
Amount Storage
2 tubes, 1.0 mL –25°C to –15°C on receipt.
2°C to 8°C after first use, for up to 6 months or up to the expiration date stated on the kit (whichever comes first).
2 tubes, 1.0 mL –25°C to –15°C on receipt.
2°C to 8°C after first use, for up to 6 months or up to the expiration date stated on the kit (whichever comes first).
Store protected from light.
1 tube, 0.05 mL –25°C to –15°C on receipt.
2°C to 8°C after first use, up to the expiration date stated on the kit.
Store protected from light.
IMPORTANT! The allelic ladder contains PCR products. Do not amplify. To avoid contamination, store the allelic ladder separate from the other kit components and unamplified DNA.
DNA Control 007
Contains 2.0 ng/μL human male genomic DNA in 0.05% sodium azide and buffer.
The profile of this male DNA is D3S1358 15,16; vWA 14, 16;
D16S539 9, 10; CSF1PO 11,12; TPOX 8; D8S1179 12, 13; D21S11
28, 31; D18S51 12, 15; Penta E 7, 12; D2S441 14,15; D19S433
14,15; TH01 7, 9.3; FGA 24, 26; D22S1045 11, 16; D5S818 11;
D13S317 11; D7S820 7,12; D6S1043 12, 14; D10S1248 12, 15;
D1S1656 13, 16; D12S391 18, 19; D2S1338 20, 23; Penta D 11, 12.
1 tube, 0.05 mL –25°C to –15°C on receipt.
2°C to 8°C after first use, up to the expiration date stated on the kit.
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1 Chapter 1 Product information
Required materials not supplied
Required materials not supplied
See Appendix B, “Materials required but not supplied“.
Instrument and software compatibility
Instrument type
Thermal cyclers
Validated models
• ProFlex ™ 96 ‑well PCR System (Cat. No. 4484075)
• GeneAmp ™ PCR System 9700, 96-Well Silver (Cat. No. N8050001)
• GeneAmp ™ PCR System 9700, 96-Well Gold-Plated (Cat. No. 4314878)
IMPORTANT! VeriFiler ™ Express kit is NOT validated for use with:
· Veriti ™ 96 ‑Well Thermal Cycler (Cat. No. 4479071)
· Veriti ™ 96 ‑Well Fast Thermal Cycler (Cat. No. 4375305)
· GeneAmp ™ PCR System 9700, 96-Well Aluminum (Cat. No. 4314879)
Genetic analyzers [1]
• 3500/3500xL Genetic Analyzer with any of the following:
– 3500 Data Collection Software v1 (Windows ™ Vista operating system) and HID Updater
3500 Data Collection Software v2 (Cat. No. 4480670)
– 3500 Data Collection v2 Software (Windows ™ 7 operating system) and HID Updater 3500
Data Collection Software v2 (Cat. No. 4480670)
– 3500 Data Collection v3 Software (Windows ™ 7 operating system)
• 3130/3130 xl Genetic Analyzer with:
– Data Collection Software v4 (Windows ™ 7 operating system)
– 3130/3730 Data Collection v4 6-Dye Module v1
• 3730 Genetic Analyzer
– Data Collection Software v4 (Windows ™ 7 operating system)
– 3130/3730 Data Collection v4 6-Dye Module v1
Analysis software
GeneMapper ™ ID ‑X Software v1.4 or later
Windows ™ XP or Windows ™ 7 operating system
[1] We conducted validation studies using the 3130 xl, 3500, and 3500xL configurations.
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Chapter 1 Product information
Workflow
1
Workflow
Perform PCR on treated or untreated paper substrates
Perform PCR on swab substrates
“Prepare the reactions: treated paper substrate“ on page 19 or
“Prepare the reactions: untreated paper substrate“ on page 21
▼
Obtain punch with Harris Manual Punch or BSD
Semi-Automated Dried Sample Punch Instrument
“Prepare the reactions: swab substrate“ on page 24
▼
▼
Untreated paper only: Process with Prep ‑n‑Go ™
Buffer
▼
Process with VeriFiler ™ Express kit
Lyse in Prep ‑n‑Go ™ Buffer
▼
Process with VeriFiler ™ Express kit
▼ ▼
Amplify with the GeneAmp ™ PCR System 9700 or ProFlex ™ 96 ‑well PCR System
▼
Perform electrophoresis
“Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)“ on page 28 or
“Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)“ on page 31 or
“Set up the 3730 instruments for electrophoresis (before first use of the kit)“ on page 34
▼
“Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)“ on page 30 or
“Prepare samples for electrophoresis (3730 instruments)“ on page 36
▼
Analyze data
“Set up the GeneMapper ™ ID ‑X Software for analysis (before first use of the kit)“ on page 40
“Create an analysis method“ on page 45
“Create a size standard definition file“ on page 53
“Analyze and edit sample files with GeneMapper ™ ID ‑X Software“ on page 55
“Examine or edit a project“ on page 56
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Express PCR Amplification Kit User Guide 15
16
2
Perform PCR
■
Optimize PCR cycle number (before first use of the kit) . . . . . . . . . . . . . . . . . . . . 16
■
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Treated paper substrates: prepare the amplification kit reactions . . . . . . . . . . . . 18
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Untreated paper substrates: prepare the amplification kit reactions . . . . . . . . . 20
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Swab substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . 23
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Optimize PCR cycle number (before first use of the kit)
Before using the VeriFiler ™ Express kit for the first time, perform a single initial sensitivity experiment to determine the appropriate cycle number to use during internal validation studies and operational use of the kit. This experiment accounts for instrument-to-instrument and sample-to-sample variations. If you are processing multiple sample type and substrate combinations (for example, buccal samples on treated paper and buccal samples on swabs), perform separate sensitivity experiments for each sample type and substrate to be used for testing.
Procedural guidelines
Optimize PCR cycle number
Select samples and prepare plates
• Use 19 samples so that you can complete electrophoresis using a single 96-well plate. This minimizes the impact of run-to-run variation on the results.
Additional samples may be used for a more comprehensive sensitivity experiment. Examples of PCR and electrophoresis plate layouts are provided on
page “Example PCR plate layout“ on page 120.
• To maximize result quality, prepare and amplify Plate 1, then repeat for Plates 2,
3, and 4. Do not prepare all 4 plates before amplification.
• To minimize the effect of instrument-to-instrument variation, use the same thermal cycler to amplify all 4 plates.
1. Select 19 of each sample+substrate type. Ensure that the selected samples represent a "typical" range of samples analyzed in your laboratory.
2. Prepare the samples and the reactions as described in the appropriate protocols later in this chapter. Prepare sufficient PCR reagents to complete amplification of three replicate plates.
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Express PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Before you begin
2
Determine optimum PCR conditions
4. Amplify each plate using a different cycle number to determine the optimum conditions for use in your laboratory.
Suggested cycle numbers for different sample type and substrate combinations are listed in the following table.
Sample type
Blood
Buccal
Substrate
Treated paper Untreated paper
25, 26, 27, 28 cycles 25, 26, 27, 28 cycles
Swab
N/A
25, 26, 27, 28 cycles 25, 26, 27, 28 cycles 25, 26, 27. 28 cycles
1. Run the PCR products on the appropriate CE platform using the recommended
protocol that is described in Chapter 3, “Perform electrophoresis“.
2. Based on the results of the sensitivity study, select the appropriate PCR cycle number for future experiments.
Our studies indicate the optimum PCR cycle number should generate profiles with the following heterozygote peak heights, with no instances of allelic dropout and minimal occurrence of off-scale allele peaks:
Instrument
3500 Series
3130 Series
3730
Heterozygous peak height
3,000–12,000 RFU
1,000–3,000 RFU
3,000–12,000 RFU
When amplifying single-source, unpurified samples, you will see greater sample-to-sample variation in peak height than you see with purified samples.
Careful optimization of the cycle number helps to minimize this variation.
Before you begin
Thaw reagents
(before first use of the kit)
Thaw the Master Mix and Primer Set.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.
IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2°C to 8°C and do not require subsequent thawing. Do not refreeze the reagents.
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Express PCR Amplification Kit User Guide 17
2 Chapter 2 Perform PCR
Treated paper substrates: prepare the amplification kit reactions
Treated paper substrates: prepare the amplification kit reactions
Sample preparation guidelines: treated paper substrate
Prepare low-TE buffer
• Do not add water to the wells on the reaction plate before adding the punches. If you observe static issues with the paper discs, you can prepare and dispense the
25-µL reaction mix into the wells of the reaction plate before adding the punches.
Alternatively, dispense 3 µL of low-TE Buffer into each sample and negative amplification control well (not the positive amplification control wells) before adding the punches.
• Make the punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification.
• For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card, hold the barrel of the Harris Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction plate.
• For automated punching: See the User Guide of your automated or semiautomated disc punch instrument for proper guidance.
For optimal results, we recommend using low-TE buffer for sample preparation.
Prepare it as described in this procedure or buy it from Teknova (Cat. No. T0223).
1. Mix together:
• 10 mL of 1 M Tris-HCl, pH 8.0
• 0.2 mL of 0.5 M EDTA, pH 8.0
• 990 mL glass-distilled or deionized water
Note: Adjust the volumes accordingly for specific needs.
2. Aliquot, then autoclave the solutions.
3. Store the aliquots at room temperature.
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Chapter 2 Perform PCR
Treated paper substrates: prepare the amplification kit reactions
2
Prepare the reactions: treated paper substrate
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.
If this is the first time you are using the kit, follow the instructions in “Thaw reagents
(before first use of the kit)“ on page 17 before proceeding.
1. Add samples to the MicroAmp ™ Optical 96-well Reaction Plate:
To these wells ...
Negative control
Test samples
Positive control
IMPORTANT! Do not add a blank disc to the positive control well.
Add...
1.2 mm blank disc
1.2 mm sample disc
For 25 and 26 cycles 3 μL of Control DNA 007
For 27 cycles 2 μL of Control DNA 007
For 28 cycles 1 μL of Control DNA 007
Note: The volumes of positive control are suggested amounts and can be adjusted if peak heights are too high or too low for your optimized cycle number.
2. Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.
3. Pipet the required volumes of components into an appropriately sized polypropylene tube.
Reaction component
Master Mix
Primer Set
Low-TE buffer
Volume per reaction
10.0 μL
10.0 μL
5.0 μL
Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! This kit is optimized for a 25-µL PCR volume to overcome the
PCR inhibition that is expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles.
4. Vortex the reaction mix for 3 seconds, then centrifuge briefly.
5. Dispense 25 µL of the reaction mix into each reaction well of a MicroAmp ™
Optical 96-Well Reaction Plate.
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Express PCR Amplification Kit User Guide 19
2 Chapter 2 Perform PCR
Untreated paper substrates: prepare the amplification kit reactions
6. Seal the plate with MicroAmp ™ Clear Adhesive Film (Cat. No. 4306311) or
MicroAmp ™ Optical Adhesive Film (Cat. No. 4311971).
IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across all wells.
IMPORTANT! If you are using the GeneAmp ™ PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp ™ Optical Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. Other validated thermal cyclers do not require a compression pad.
7. Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.
8. Amplify the samples as described in Chapter 2, “Perform PCR“.
IMPORTANT! This kit is not validated for use with the GeneAmp ™ PCR System
9700 with the aluminum 96-well block. Use of this thermal cycling platform may adversely affect performance of this kit.
Untreated paper substrates: prepare the amplification kit reactions
Sample preparation guidelines: untreated paper substrate
• Make a 1.2 mm punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification.
• If you are using a Bode
Buccal DNA Collector ™ , make a 1.2 mm punch as close as possible to the tip of the DNA collector to ensure optimum peak intensity. A larger punch may cause inhibition during PCR amplification.
1
1 Location of punch with a Bode Buccal DNA Collector ™
• For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card, hold the barrel of the Harris Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction plate.
• For automated punching: See the User Guide of your automated or semiautomated disc punch instrument for proper guidance.
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Chapter 2 Perform PCR
Untreated paper substrates: prepare the amplification kit reactions
2
Prepare the reactions: untreated paper substrate
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.
If this is the first time you are using the kit, follow the instructions in “Thaw reagents
(before first use of the kit)“ on page 17 before proceeding.
1. Add Prep-n-Go ™ Buffer (Cat. No. 4467079) to the MicroAmp ™ Optical 96-Well
Reaction Plate:
To these wells ...
Negative control
Test samples
Positive control
For 27 cycles
For 28 cycles
Add...
5 μL of Prep ‑n‑Go ™ Buffer
5 μL of Prep ‑n‑Go ™ Buffer
For 25 and 26 cycles
2 μL of Prep ‑n‑Go ™ Buffer
3 μL of Prep ‑n‑Go ™ Buffer
4 μL of Prep ‑n‑Go ™ Buffer
2. Add samples to the reaction plate:
To these wells ...
Negative control
Test samples
Positive control
IMPORTANT! Do not add a blank disc to the positive control well.
Add...
1.2 mm blank disc
1.2 mm sample disc
For 25 and 26 cycles 3 μL of Control DNA 007
For 27 cycles 2 μL of Control DNA 007
For 28 cycles 1 μL of Control DNA 007
Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number.
3. Centrifuge the plate to ensure that the punches are immersed in the Prep-n-Go ™
Buffer.
4. Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.
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2 Chapter 2 Perform PCR
Untreated paper substrates: prepare the amplification kit reactions
5. Pipet the required volumes of components into an appropriately sized polypropylene tube.
Reaction component
Master Mix
Primer Set
Volume per reaction
10.0 μL
10.0 μL
Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! This kit is optimized for a 25-µL PCR volume to overcome the
PCR inhibition that is expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles.
6. Vortex the reaction mix for 3 seconds, then centrifuge briefly.
7. Dispense 20 µL of the reaction mix into each reaction well of a MicroAmp ™
Optical 96-Well Reaction Plate.
The final volume in each well is 25 µL (reaction mix plus Prep-n-Go ™ Buffer and sample or positive control).
8. Seal the plate with MicroAmp ™ Clear Adhesive Film (Cat. No. 4306311) or
MicroAmp ™ Optical Adhesive Film (Cat. No. 4311971).
IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across all wells.
IMPORTANT! If you are using the GeneAmp ™ PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp ™ Optical Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. Other validated thermal cyclers do not require a compression pad.
9. Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.
10. Amplify the samples as described in Chapter 2, “Perform PCR“.
IMPORTANT! This kit is not validated for use with the GeneAmp ™ PCR System
9700 with the aluminum 96-well block. Use of this thermal cycling platform may adversely affect performance of this kit.
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Chapter 2 Perform PCR
Swab substrates: prepare the amplification kit reactions
2
Swab substrates: prepare the amplification kit reactions
Sample preparation guidelines: swab substrate
Prepare the sample lysate: room temperature
Prepare the sample lysate: heat protocol
• Detach each buccal swab head from the swab shaft before lysis.
• If you are using the heated lysis protocol, perform lysis in either of the following formats:
– 1.5-mL tubes with a heat block (VWR ™ Scientific Select dry heat block or similar)
– PrepFiler ™ 96-Well Processing Plates (Cat. No. 4392904)
– Robbins Scientific ™ Model 400 Hybridization Incubator or similar
– Agilent ™ Benchtop Rack for 200 µL Tubes/V Bottom Plates (metal) or similar
(Cat. No. 410094)
IMPORTANT! Do not use a plastic plate adaptor.
• For optimum performance, lyse the entire swab. If you need to preserve the sample, use half of the lysate prepared from the entire swab.
This protocol may improve the performance for challenging or aged samples.
1. Add 400 µL Prep-n-Go ™ Buffer (Cat. No. 4471406) to 1.5-mL tubes or the appropriate wells of a PrepFiler ™ 96-Well Processing Plate (Cat. No. 4392904).
2. Into each tube or well, put the entire head of each swab, then let stand for
20 minutes at room temperature (20°C to 25°C) to lyse the sample.
3. After 20 minutes, transfer the sample lysate out of the sample plate into tubes or plates for storage, then discard the deep-well plate containing the swab heads.
Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate.
4. Go to “Prepare the reactions: swab substrate“ on page 24 or “Store the sample
This protocol may improve the performance for challenging or aged samples.
1. Preheat the heat block to 90°C or the oven with metal plate adaptor to 99°C.
2. Add 400 µL Prep-n-Go ™ Buffer (for buccal swabs, Cat. No. 4471406) to 1.5-mL tubes or the appropriate wells of a PrepFiler ™ 96-Well Processing Plate
(Cat. No. 4392904).
3. Into each tube or well, put the entire head of each swab. If you are using tubes, cap the tubes. Let the tubes or plate stand for 20 minutes in the preheated heat block or oven to lyse the sample.
4. After 20 minutes, remove the tubes or the deep-well plate from the heat block or oven.
5. Let the lysate stand at room temperature for at least 15 minutes to cool the lysate
(for accurate pipetting).
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2 Chapter 2 Perform PCR
Swab substrates: prepare the amplification kit reactions
Prepare the reactions: swab substrate
6. Transfer the sample lysate out of the 1.5-mL tubes or sample plate into tubes or plates for storage. Discard the 1.5-mL tubes or deep-well plate containing the swab heads.
Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate.
7. Go to “Prepare the reactions: swab substrate“ on page 24 or “Store the sample
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.
If this is the first time you are using the kit, follow the instructions in “Thaw reagents
(before first use of the kit)“ on page 17 before proceeding.
1. Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.
2. Pipet the required volumes of components into an appropriately sized polypropylene tube.
Reaction component
Master Mix
Primer Set
Low-TE buffer
Volume per reaction
10.0 μL
10.0 μL
3.0 μL
Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! This kit is optimized for a 25-µL PCR volume to overcome the
PCR inhibition that is expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles.
3. Vortex the reaction mix for 3 seconds, then centrifuge briefly.
4. Dispense 23 µL of the reaction mix into each reaction well of a MicroAmp ™
Optical 96-Well Reaction Plate.
The final volume in each well is 25 µL (reaction mix plus Prep-n-Go ™ Buffer or sample lysate).
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Express PCR Amplification Kit User Guide
Store the sample lysate
Chapter 2 Perform PCR
Swab substrates: prepare the amplification kit reactions
2
5. Add samples to the MicroAmp ™ Optical 96-Well Reaction Plate:
To these wells ...
Test samples
Positive control
Add...
2 μL of sample lysate
For 25 and 26 cycles
For 27 cycles
3 μL of Control
DNA 007
2 μL of Control
DNA 007
For 28 cycles 1 μL of Control
DNA 007
Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number.
6. Seal the plate with MicroAmp ™ Clear Adhesive Film (Cat. No. 4306311) or
MicroAmp ™ Optical Adhesive Film (Cat. No. 4311971).
IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across all wells.
IMPORTANT! If you are using the GeneAmp ™ PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp ™ Optical Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. Other validated thermal cyclers do not require a compression pad.
7. Vortex the reaction mix at medium speed for 3 seconds.
8. Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.
9. Amplify the samples as described in Chapter 2, “Perform PCR“.
IMPORTANT! This kit is not validated for use with the GeneAmp ™ PCR System
9700 with the aluminum 96-well block or the Veriti ™ Thermal Cyclers. Use of these thermal cycling platforms may adversely affect performance of this kit.
1. Cap the sample lysate storage tubes or seal the sample lysate storage plate with
MicroAmp ™ Clear Adhesive Film.
2. Store the sample lysate as needed:
If you are storing the sample lysate...
<2 weeks
>2 weeks
Then place at...
2°C to 8°C
–25°C to –15°C
Note: The effects of multiple freeze/thaw cycles on the lysate have not been fully evaluated. Therefore, multiple freeze/thaw cycles are not recommended.
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2 Chapter 2 Perform PCR
Perform PCR
Perform PCR
IMPORTANT! This kit is validated for use with the validated thermal cyclers listed in
“Instrument and software compatibility“ on page 14
1. Program the thermal cycling conditions.
IMPORTANT! If you are using the GeneAmp ™ PCR System 9700, select the Max ramping mode. If you are using the ProFlex ™ 96-well PCR System, select the
GeneAmp ™ PCR System 9700 simulation mode.
Initial incubation step
HOLD
95°C,
1 minute
Optimum cycle number [1]
Denature Anneal Extend
94°C,
3 seconds
CYCLE
59°C,
16 seconds
65°C,
29 seconds
Final extension
HOLD
60°C,
5 minutes
Final hold
HOLD
4°C, up to 24 hours
[2]
[1]
[2]
See “Optimize PCR cycle number (before first use of the kit)“ on page 16.
The infinity (∞) setting allows an unlimited hold time.
2. Load the plate into the thermal cycler, close the heated cover, then start the run.
IMPORTANT! If you are using adhesive clear film instead of caps to seal the plate wells, be sure to place a MicroAmp ™ Optical Film Compression Pad
(Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. The ProFlex ™ 96-well PCR System does not require a compression pad.
3. When the run is complete, store the amplified DNA.
If you are storing the DNA...
<2 weeks
>2 weeks
Then place at...
2°C to 8°C
–25°C to –15°C
IMPORTANT! Protect the amplified DNA from light.
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3
Perform electrophoresis
■
■
■
■
Prepare samples for electrophoresis (3500 Series and 3130
■
■
Prepare samples for electrophoresis (3500 Series and 3130
■
Set up the 3730 instruments for electrophoresis (before first use of the kit) . . . 34
■
Prepare samples for electrophoresis (3730 instruments) . . . . . . . . . . . . . . . . . . . . 36
Allelic ladder requirements for electrophoresis
To accurately genotype samples, you must run an allelic ladder with the samples.
Instrument
3500
3500xL
3130
3130 xl
3730
Number of allelic ladders to run
One injection equals
1 per 3 injections 8 samples
1 per injection 24 samples
1 per 4 injections 4 samples
1 per injection 16 samples
3 per injection 48 samples
Number of samples per allelic ladder(s)
23 samples + 1 allelic ladder
23 samples + 1 allelic ladder
15 samples + 1 allelic ladder
15 samples + 1 allelic ladder
15 samples + 1 allelic ladder
IMPORTANT! Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between runs. Follow the guidelines in the preceding table, which should account for normal variation in run speed. Perform internal validation studies to verify the required allelic ladder injection frequency, to ensure accurate genotyping of all samples in your laboratory environment.
It is critical to genotype using an allelic ladder run under the same conditions as the samples. Size values obtained for the same sample can differ between instrument platforms, because of different polymer matrices and electrophoretic conditions.
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3 Chapter 3 Perform electrophoresis
Materials required for electrophoresis
Materials required for electrophoresis
Appendix B, “Materials required but not supplied“ lists the required materials that
are not supplied with this kit.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)
Electrophoresis software setup
Genetic
Analyzer
3500
3500xL
Operating
System
Windows
Vista
™
The following table lists the data collection software and the run modules that you can use to analyze PCR products generated by this kit. For details on the procedures,
see the documents listed in Appendix E, “Documentation and support“.
Note: We conducted validation studies for the kit using the 3130xl, 3500, or 3500xL configurations.
Data
Collection
Software
3500 Data
Collection
Software v1
Additional software
HID Updater 3500
DC v2
(Cat. No. 4480670)
Plate templates, assays, run modules, and conditions (installed with the HID Updater)
Plate templates: 6dye_36_POP4 (and _xl)
Assays: GF+Norm_POP4 (and _xl) and
GF_POP4 (and _xl), which contain instrument protocol HID36_POP4 (and_xl)_J6_NT3200 with the following conditions:
• Run module: HID36_POP4
• Injection conditions: 1.2 kV/15 sec (24 sec for xl)
• Run conditions: 13 kV/1550 sec
• Dye Set J6
3500
3500xL
3500
3500xL
Windows ™ 7
Windows ™ 7
3500 Data
Collection
Software v2
3500 Data
Collection
Software v3
HID Updater 3500
DC v2
(Cat. No. 4480670)
None
Same as 3500 Data Collection Software v1 listed above
Same as 3500 Data Collection Software v1 listed above
Obtain and run the
HID Updater (v1 and v2 software only)
Perform this procedure if you are using 3500 Series Software v1 or v2.
You can run 6-dye samples on 3500 Data Collection Software. Before running on either system for the first time, run the HID Updater 3500 DC v2 (Cat. No. 4480670).
The HID Updater installs plate templates, assays, and instrument protocols that can
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Chapter 3 Perform electrophoresis
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)
3
Perform spectral calibration
be used to run VeriFiler ™ Express kit samples. For more information, refer to the release notes provided with the Updater.
Note: If you have a new instrument installed by a Thermo Fisher Scientific representative, the updater may have been run during installation.
1. Shut down the 3500/3500xL Data Collection Software.
2. Download the updater from www.thermofisher.com/us/en/home/ technical-resources/software-downloads/
3500-Series-Genetic-Analyzers-for-Human-Identification.html.
3. Open the Read me file and review the software release notes.
4. Click the updater .exe file.
5. Follow the on-screen prompts.
6. Restart the computer.
Perform a spectral calibration using the DS-36 Matrix Standard Kit (6-Dye) (J6 Dye
Set) (Cat. No. 4425042).
The following figure is an example of a passing 6-dye spectral calibration.
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3 Chapter 3 Perform electrophoresis
Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)
Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)
This procedure applies to the 3500 Series and 3130 Series instruments.
Prepare the samples for electrophoresis immediately before loading.
1. Pipet the required volumes of components into an appropriately sized polypropylene tube:
Reagent
GeneScan ™ 600 LIZ ™ Size Standard v2.0
Hi ‑Di ™ Formamide
Volume per reaction
0.4 μL
9.6 μL
Note: Include volume for additional samples to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results.
2. Vortex the tube, then briefly centrifuge.
3. Into each well of a MicroAmp ™ Optical 96-Well Reaction Plate, add:
• 10 µL of the formamide/size standard mixture
• 1 µL of PCR product or Allelic Ladder
Note: For blank wells, add 10 µL of Hi-Di ™ Formamide.
4. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom.
5. Heat the reaction plate in a thermal cycler at 95°C for 3 minutes.
6. Immediately place the plate on ice for 3 minutes.
7. Place the sample tray on the autosampler, then start the electrophoresis run.
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Chapter 3 Perform electrophoresis
Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)
3
Set up the 3130/3130 use of the kit) xl instruments for electrophoresis (before first
Electrophoresis software setup
Genetic
Analyzer
3130
3130 xl
Operating
System
Windows ™ 7
The following table lists the data collection software and the run modules that you can use to analyze PCR products generated by this kit. For details on the procedures,
see the documents listed in Appendix E, “Documentation and support“.
Note: We conducted validation studies for the VeriFiler
3500, or 3500xL configurations.
™ Express kit using the 3130xl,
Data
Collection
Software
Data
Collection
Software v4 [1]
Additional software
3130/3730 DC v4
6 ‑Dye Module v1
Run modules and conditions
• HIDFragmentAnalysis36_POP4_1 Injection conditions: 3 kV/5 sec
• Run conditions: 15 kV/1500 sec
• Dye Set J6
• HIDFragmentAnalysis36_POP4_1 Injection conditions: 3 kV/10 sec
• Run conditions: 15 kV/1500 sec
• Dye Set J6
[1] Requires activation of 6 ‑dye license.
Obtain and activate 6-dye license
1. Confirm that you are running Data Collection Software v4 (Help4About).
2. Obtain a 3130 DC v4 6-Dye Module v1 License key. Contact your local Human
Identification representative for information.
3. Ensure that all network cards in the computer are enabled.
IMPORTANT! You can run the 3130 Series Data Collection Software v4 using only the network cards that are enabled when you activate the software license.
For example, if you activate the software when your wireless network card is disabled, you will not be able to run the software when the wireless network card is enabled.
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3 Chapter 3 Perform electrophoresis
Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)
4. Select Tools4License Manager to display the Software Activation dialog box.
32
5. Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you.
6. Obtain the software license file from your email.
7. Make a copy of the software license file and keep it in a safe location.
8. Copy the software license file to the desktop of the Data Collection Software v4 computer.
9. If the Software Activation dialog box has closed, select Tools4License Manager.
10. Click Browse, then navigate to the software license file saved on your computer.
11. Click Install and Validate License.
A message is displayed when the license is installed and validated.
12. Click Close.
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Chapter 3 Perform electrophoresis
Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)
3
Perform spectral calibration
Perform a spectral calibration using the DS-36 Matrix Standard Kit (6-Dye)
(Cat. No. 4425042). Select the J6 dye set for the 3130 instrument, or the J6-RCT dye set for the 3730 instrument.
The following figure is an example of a passing 6-dye spectral calibration.
Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)
This procedure applies to the 3500 Series and 3130 Series instruments.
Prepare the samples for electrophoresis immediately before loading.
1. Pipet the required volumes of components into an appropriately sized polypropylene tube:
Reagent
GeneScan ™ 600 LIZ ™ Size Standard v2.0
Hi ‑Di ™ Formamide
Volume per reaction
0.4 μL
9.6 μL
Note: Include volume for additional samples to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results.
2. Vortex the tube, then briefly centrifuge.
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3 Chapter 3 Perform electrophoresis
Set up the 3730 instruments for electrophoresis (before first use of the kit)
3. Into each well of a MicroAmp ™ Optical 96-Well Reaction Plate, add:
• 10 µL of the formamide/size standard mixture
• 1 µL of PCR product or Allelic Ladder
Note: For blank wells, add 10 µL of Hi-Di ™ Formamide.
4. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom.
5. Heat the reaction plate in a thermal cycler at 95°C for 3 minutes.
6. Immediately place the plate on ice for 3 minutes.
7. Place the sample tray on the autosampler, then start the electrophoresis run.
Set up the 3730 instruments for electrophoresis (before first use of the kit)
Electrophoresis software setup
Genetic
Analyzer
3730
Operating
System
Windows ™ 7
The following table lists the data collection software and the run modules that you can use to analyze PCR products generated by this kit. For details on the procedures,
see the documents listed in Appendix E, “Documentation and support“.
Data
Collection
Software
Data
Collection
Software v4 [1]
Additional software
3130/3730 DC v4 6-
Dye Module v1
Run modules and conditions
• GeneMapper36_POP7_1 Injection conditions: 2 kV/10 sec
• Run conditions: 15 kV/1200 sec
• Dye Set J6
[1] Requires activation of 6 ‑dye license.
Obtain and activate the 6-dye license
1. Confirm that you are running Data Collection Software v4 (Help4About)
2. Obtain a 3730 DC v4 6-Dye Module v1 License key. Contact Thermo Fisher
Scientific for information.
3. Ensure that all network cards in the computer are enabled.
IMPORTANT! You can run the 3730 Series Data Collection Software v4 using only the network cards enabled when you activate the software license. For example, if you activate the software when your wireless network card is disabled, you will not be able to run the software when the wireless network card is enabled.
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Chapter 3 Perform electrophoresis
Set up the 3730 instruments for electrophoresis (before first use of the kit)
3
4. Select Tools4License Managerto display the Software Activation dialog box.
5. Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you.
6. Obtain the software license file from your email.
7. Make a copy of the software license file and keep in a safe location.
8. Copy the software license file to the desktop of the Data Collection Software v4 computer.
9. If the Software Activation dialog box has closed, select Tools4License Manager.
10. Click Browse, then navigate to the software license file saved on your computer.
11. Click Install and Validate License.
A message is displayed when the license is installed and validated.
12. Click Close.
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3 Chapter 3 Perform electrophoresis
Prepare samples for electrophoresis (3730 instruments)
Perform spectral calibration
Perform a spectral calibration using the DS-36 Matrix Standard Kit (6-Dye)
(Cat. No. 4425042). Select the J6 dye set for the 3130 instrument, or the J6-RCT dye set for the 3730 instrument.
The following figure is an example of a passing 6-dye spectral calibration.
Prepare samples for electrophoresis (3730 instruments)
Prepare the samples for electrophoresis immediately before loading.
1. Pipet the required volumes of components into an appropriately sized polypropylene tube:
Reagent
GeneScan ™ 600 LIZ ™ Size Standard v2.0
Hi ‑Di ™ Formamide
Volume per reaction
0.4 μL
9.6 μL
Note: Include volume for additional samples to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results.
2. Vortex the tube, then briefly centrifuge.
3. Into each well of a MicroAmp ™ Optical 96-Well Reaction Plate, add:
• 10 µL of the formamide: size standard mixture
• 1 µL of PCR product or Allelic Ladder
Note: For blank wells, add 10 µL of Hi-Di ™ Formamide.
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Chapter 3 Perform electrophoresis
Prepare samples for electrophoresis (3730 instruments)
3
4. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom.
5. Heat the reaction plate in a thermal cycler at 95°C for 3 minutes.
6. Immediately place the plate on ice for 3 minutes.
7. Place the sample tray on the autosampler, then start the electrophoresis run.
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38
4
Analyze data with GeneMapper ™
ID ‑X
Software
■
■
■
■
■
■
■
Analyze and edit sample files with GeneMapper ™ ID-X Software . . . . . . . . . . 55
■
■
For more information on using the GeneMapper ™ ID-X Software . . . . . . . . . . 56
Overview of GeneMapper
™
ID
‑X
Software
GeneMapper ™ ID‑X Software is an automated genotyping software application for forensic casework, databasing, and paternity data analysis.
GeneMapper ™ ID‑X Software v1.4 or later analyzes 4-dye, 5-dye, and 6-dye data and is required to correctly analyze data that is generated using the VeriFiler ™ Express kit.
After electrophoresis, the data collection software stores information for each sample in a .fsa or .hid file. The GeneMapper ™ ID‑X Software v1.4 or later allows you to analyze and interpret the data from the .fsa or .hid files.
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Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Allelic ladder requirements for data analysis
4
Allelic ladder requirements for data analysis
• HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies before you use multiple allelic ladder samples in an analysis.
For multiple allelic ladder samples, the GeneMapper ™ ID‑X Software calculates allelic bin offsets by using an average of all allelic ladders that use the same panel in a run folder.
• Allelic ladder samples in an individual run folder are considered to be from a single run. When the software imports multiple run folders into a project, only the ladders in their respective run folders are used for calculating allelic bin offsets and subsequent genotyping.
• Allelic ladder samples must be labeled as "Allelic Ladder" in the Sample Type column in a project. Analysis will fail if the Allelic Ladder Sample Type is not specified.
• Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples, to ensure proper allele calling.
• Alleles that are not in the Allelic Ladders do exist. Off-ladder (OL) alleles can contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the bin window of any known allelic ladder allele or virtual bin.
Note: If a sample allele peak is called as an off-ladder allele, verify the sample result according to your laboratory protocol.
File names and versions used in this section
The file names and version numbers of panel, bin, and stutter files that are shown in this section may differ from the file names that you see when you download or import files.
If you need help to determine the correct files to use, contact your local Human
Identification representative, or go to thermofisher.com/support.
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4 Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Set up the GeneMapper ™ ID ‑X Software for analysis (before first use of the kit)
Set up the GeneMapper
™
of the kit)
ID
‑X
Software for analysis (before first use
Workflow: Set up
GeneMapper
™
ID ‑X Software
Before you use GeneMapper ™ ID‑X Software to analyze data for the first time, you must do the following:
“Check panel, bin, and stutter file versions on your computer“ on page 40
▼
“Download newer versions of panel bin and stutter files if needed“ on page 40
▼
“Import panels, bins, and marker stutter“ on page 41
▼
“(Optional) Define custom table or plot settings“ on page 44
Check panel, bin, and stutter file versions on your computer
Download newer versions of panel bin and stutter files if needed
1. Start the GeneMapper ™ ID‑X Software , then log in with the appropriate user name and password.
2. Select Tools4Panel Manager.
3. Check the version of files that are currently available in the Panel Manager: a.
Select Panel Manager in the navigation pane.
b.
Expand the Panel Manager folder and any subfolders to identify the analysis file version that is already installed for your kit choice.
4. Check the version of files available for import into the Panel Manager: a.
Select Panel Manager, then select File4Import Panels to open the Import
Panels dialog box.
b.
Navigate to, then open the Panels folder, then check the version of panel, bin, and stutter files installed.
5. Check for newer versions of the files as described in the next procedure.
1. Go to thermofisher.com/us/en/home/technical-resources/software-downloads/
genemapper-id-x-software.html.
2. If the file versions listed are newer than the versions on your computer, download the file VeriFilerExpress Analysis Files.
Note: When downloading new versions of analysis files, see the associated Read
Me file for details of changes between software file versions. Perform the appropriate internal validation studies before using new file versions for analysis.
3. Unzip the file.
40 VeriFiler
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Import panels, bins, and marker stutter
Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Set up the GeneMapper ™ ID ‑X Software for analysis (before first use of the kit)
Common Callouts and Arrows
To import the latest panel, bin set, and marker stutter from the website into the
GeneMapper ID‑X Software database:
Note: If you need more advanced callouts or arrows
1. Start the GeneMapper name and password.
™ ID‑X Software, then log in with the appropriate user
4
3. Find, then open the folder containing the panels, bins, and marker stutter:
1 a.
Select Panel Manager, then select File4Import Panels to open the Import
Panels dialog box.
1 b.
Navigate to, then open the VeriFilerExpress Analysis Files folder that you unzipped in the previous procedure.
4. Select VeriFilerExpress_Panels.txt , then click Import.
Note: Importing this file creates a new folder in the navigation pane of the Panel
Manager, VeriFilerExpress_Panels. This folder contains the panel and associated markers.
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4 Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Set up the GeneMapper ™ ID ‑X Software for analysis (before first use of the kit)
5. Import the bins file: a.
Select the VeriFilerExpress_Panels folder in the navigation pane.
b.
Select File4Import Bin Set to open the Import Bin Set dialog box.
c.
Navigate to, then open the VeriFilerExpress Analysis Files folder.
d.
Select VeriFilerExpress_Bins.txt, then click Import.
Note: Importing this file associates the bin set with the panels in the
VeriFilerExpress_Panels folder.
42 VeriFiler
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Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Set up the GeneMapper ™ ID ‑X Software for analysis (before first use of the kit)
4
6. (Optional) View the imported panels and bins in the navigation pane: Doubleclick the VeriFilerExpress_Panels folder.
The panel information is displayed in the right pane and the markers are displayed below it.
7. Import the stutter file: a.
Select the VeriFilerExpress_Panels folder in the navigation panel.
b.
Select File4Import Marker Stutter to open the Import Marker Stutter dialog box.
c.
Navigate to, then open the VeriFilerExpress Analysis Files folder.
d.
Select VeriFilerExpress_Stutter.txt, then click Import.
Note: Importing this file associates the marker stutter ratio with the bin set in the VeriFilerExpress_Panels folder and overwrites any existing stutter ratios associated with the panels and bins in that folder.
8. View the imported marker stutters in the navigation pane: a.
Double-click the VeriFilerExpress_Panels folder to display the folder.
b.
Double-click the folder to display its list of markers below it.
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4 Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Set up the GeneMapper ™ ID ‑X Software for analysis (before first use of the kit) c.
Double-click a marker to display the Stutter Ratio & Distance view for the marker in the right pane.
(Optional) Define custom table or plot settings
9. Click Apply, then click OK to add the panel, bin set, and marker stutter to the
GeneMapper ™ ID‑X Software database.
IMPORTANT! If you close the Panel Manager without clicking Apply, the panels, bin sets, and marker stutter are not imported into the GeneMapper ™ ID‑X
Software database.
Default views for table and plot settings are provided with the software. For information on defining custom views, see GeneMapper ™ ID‑X Software Getting Started
Guide— Basic Features.
44 VeriFiler
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Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Create an analysis method
4
Create an analysis method
Create an analysis method
IMPORTANT! Analysis methods are version-specific, so you must create an analysis method for each version of the software. For example, an analysis method that is created in GeneMapper
Software v3.2.1.
™ ID‑X Software version 1.2 is not compatible with analysis methods that are created in earlier versions of software, or with GeneMapper ™
1. Select Tools4GeneMapper ® ID-X Manager to open the GeneMapper ID-X
Manager.
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
1 1 1
1
2. Click the Analysis Methods tab, then click New to open the Analysis Method
Editor with the General tab selected.
3. Enter the settings shown in the figures on the following pages.
Note: The Analysis Method Editor closes when you save your settings. To complete this step quickly, do not save the analysis method until you finish entering settings in all of the tabs.
4. After you enter the settings on all tabs, click Save.
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4 Chapter 4 Analyze data with GeneMapper ™
Create an analysis method
ID ‑X Software
Enter Analysis
Method settings
Enter General tab settings
1. Enter a Name and select the Security Group appropriate for your software configuration.
46
2. (Optional) Enter a Description and Instrument.
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Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Create an analysis method
4
Enter Allele tab settings
IMPORTANT! Perform appropriate internal validation studies to determine the appropriate settings to use.
1. Select the VeriFilerExpress_Bins_v1X bin set.
Figure 3 Settings used in developmental validation of the kit
2. (Optional) To apply the stutter ratios contained in the
VeriFilerExpress_Stutter.txt, select the Use marker-specific stutter ratio and
distance if available checkbox (selected by default).
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4 Chapter 4 Analyze data with GeneMapper ™
Create an analysis method
ID ‑X Software
3. If using GeneMapper ™ ID‑X Software v1.4 or later, enter values for the 4 Marker
Repeat Types.
Note: For paternity and database applications, we recommended using a 20%
Global Cut-off Value for the Tri, Tetra, and Penta loci as shown in Figure 3.
4. Enter the appropriate filter settings.
48 VeriFiler
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Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Create an analysis method
4
Enter Peak Detector tab settings
IMPORTANT! Perform appropriate internal validation studies to determine the appropriate settings to use.
Enter the appropriate values:
Figure 4 Settings used in developmental validation of the kit
Note: When using the 3730 DNA Analyzer with POP-7 ™ polymer only; a Smoothing setting of None and the Peak Window Size of 9 pts, the instances of spacing failures for the D2S441 and D1S1656 markers in some allelic ladder samples are significantly reduced. With the default Smoothing setting of Light, failures of base-pair spacing quality assessment are observed.
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4 Chapter 4 Analyze data with GeneMapper ™
Create an analysis method
ID ‑X Software
Enter Peak Quality tab settings
IMPORTANT! Perform the appropriate internal validation studies to determine the heterozygous and homozygous minimum peak height thresholds, maximum peak height threshold, and the minimum peak height ratio threshold for interpretation of data.
Enter the following values:
50 VeriFiler
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Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Create an analysis method
4
Enter SQ and GQ tab settings
IMPORTANT! The values that are shown are the software defaults and are the values we used during developmental validation. Perform appropriate internal validation studies to determine the appropriate values to use.
Enter the following values:
Note: Set the ACC GQ Weighting according to the values you determine during internal validation studies of the ACC PQV. For example, set the ACC GQ Weighting to 0.3 or higher to flag samples in which the Amelogenin result is anything other than
X, X or X, Y, or does not agree with the results for the Y indel marker.
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4 Chapter 4 Analyze data with GeneMapper ™
Create a size standard definition file if needed
ID ‑X Software
Create a size standard definition file if needed
If you cannot use the default settings that are provided, create a new size standard definition file.
About the
GS600_LIZ_ (60–
460) size standard definition file
If you use POP-7
™
polymer on a 3730 instrument
The GS600_LIZ_(60– 460) size standard definition that is provided with GeneMapper ™
ID‑X Software and used with the Local Southern size calling method contains the following peaks: 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280,
300, 314, 320, 340, 360, 380, 400, 414, 420, 440, and 460.
This size standard definition has been validated for use with this kit on the genetic
analyzers listed in “Instrument and software compatibility“ on page 14. If you need to
create your own size standard definition, see “Create a size standard definition file“ on page 53.
The 60 bp size-standard peak may occasionally be obscured by the primer peak. The issue can be addressed by either of the following steps:
• Re-inject samples in which the 60 base-pair peak is not recognized.
• Use the 80– 460 bp size-standard definition after performing appropriate validation studies (as a general rule, the 60 base-pair peak is not required for accurate fragment sizing with the 3rd Order Least Squares sizing method).
For more information, see the GeneMapper
DNA Analyzer sizing failures”.
™ ID‑X Software v1.4 New Features and
Installation Procedures User Bulletin (Pub. No. 4477684 Rev. B), “Known issues: 3730
52 VeriFiler
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Create a size standard definition file
Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Create a size standard definition file if needed
4
1. Select Tools4GeneMapper ID-X Manager to open the GeneMapper ID-X
Manager.
2. Click the Size Standards tab, then click New.
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
1 1 1
1
3. Specify settings in the Size Standard Editor: a.
Enter a name as shown in the following figure or enter a new name.
b.
In the Security Group field, select the Security Group appropriate for your software configuration.
c.
In the Size Standard Dye field, select Orange.
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4 Chapter 4 Analyze data with GeneMapper ™
Create a size standard definition file if needed
ID ‑X Software d.
In the Size Standard Table, enter the peak sizes that correspond to your size standard.
54 VeriFiler
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Chapter 4 Analyze data with GeneMapper ™
ID ‑X Software
Analyze and edit sample files with GeneMapper ™ ID ‑X Software
4
Analyze and edit sample files with GeneMapper
™
ID
‑X
Software
1. In the Project window, select Edit4Add Samples to Project, then navigate to the disk or directory that contains the sample files.
2. Apply analysis settings to the samples in the project.
Parameter
Sample Type
Analysis Method
Panel
Size Standard
Settings
Select the sample type.
Select VeriFilerExpress_AnalysisMethod (or the name of the analysis method you created).
Select VeriFiler_Express.
Use a size range of 60– 460 bp for Local Southern size calling method or a size range of 80– 460 bp for 3rd Order Least
Squares size-calling method.
[1]
[1] The VeriFiler ™ Express kit was originally validated with the GeneScan of this size standard with the VeriFiler ™ Express kit.
™ 600 LIZ ™ Size Standard v2.0 . If you use a different size standard, perform the appropriate internal validation studies to support the use
3. Click Analyze, enter a name for the project (in the Save Project dialog box), then click OK to start analysis.
• The status bar displays the progress of analysis as a completion bar.
• The table displays the row of the sample currently being analyzed in green
(or red if analysis failed for the sample).
• The Analysis Summary tab is displayed, and the Genotypes tab is available when the analysis is complete.
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4 Chapter 4 Analyze data with GeneMapper ™
Examine or edit a project
ID ‑X Software
Examine or edit a project
Display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data.
For more information on using the GeneMapper
™
ID
‑X
Software
See “Related documentation“ on page 127 for a list of available documents.
56 VeriFiler
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5
Experiments and results
■
■
■
■
■
Representative data for common sample and substrate types . . . . . . . . . . . . . . . 62
■
■
■
■
■
■
Importance of validation
Validation of a DNA typing procedure for human identification applications is an evaluation of the efficiency, reliability, and performance characteristics of the procedure. By challenging the procedure with samples that are commonly encountered in forensic and parentage laboratories, the validation process uncovers attributes and limitations that are critical for sound data interpretation (Sparkes,
Kimpton, Watson, 1996; Sparkes, Kimpton, Gilbard, 1996; Wallin, 1998).
Experiment conditions
We conducted developmental validation experiments according to the updated and revised guidelines from the Scientific Working Group on DNA Analysis Methods
(SWGDAM, December 2012). Based on these guidelines, we conducted experiments that comply with guidelines 2.0 and 3.0 and its associated subsections. This DNA methodology is not novel. (Moretti et al., 2001; Frank et al., 2001; Wallin et al., 2002; and
Holt et al., 2000).
We used conditions that produced optimum PCR product yield and that met reproducible performance standards. It is our opinion that while these experiments are not exhaustive, they are appropriate for a manufacturer of STR kits intended for forensic and/or parentage testing use.
Note: All results were analyzed on a 3500xL Genetic Analyzer in accordance with the
recommended analysis settings that are outlined in Chapter 4, “Analyze data with
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5 Chapter 5 Experiments and results
Laboratory requirements for internal validation
Laboratory requirements for internal validation
Each laboratory using this kit must perform internal validation studies. Performance of this kit is supported when used according to the following developmentally validated parameters. Modifications to the protocol should be accompanied by appropriate validation studies performed by the laboratory.
Developmental validation
Except where noted, all developmental validation studies were performed using the
GeneAmp ™ PCR System 9700 96-Well thermal cycler according to the protocol described in the Perform PCR chapter.
SWGDAM guideline 2.2.1
SWGDAM guideline 3.9.2
“Developmental validation is the acquisition of test data and determination of conditions and limitations of a new or novel DNA methodology for use on forensic, database, known or casework reference samples.” (SWGDAM, December 2012)
“The reaction conditions needed to provide the required degree of specificity and robustness should be determined. These include, but are not limited to, thermal cycling parameters, the concentration of primers, magnesium chloride, DNA polymerase, and other critical reagents.” (SWGDAM, December 2012)
58 VeriFiler
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Chapter 5 Experiments and results
Developmental validation
5
PCR components
We examined the concentration of each component of the kit and established that the concentration of each component was within the range where data indicated that the amplification met the required performance criteria for specificity, sensitivity, and reproducibility. For example, blood and buccal samples on treated-paper substrates were amplified in the presence of varying concentrations of magnesium chloride, and
the results were analyzed on a 3500xL Genetic Analyzer (Figure 5). The performance
of the multiplex is most robust within ±20% of the optimal magnesium chloride concentration.
Figure 5 Blood sample on FTA
(Y-axis scale 0 to 28,000 RFU).
™ card with the VeriFiler ™ Express kit in the presence of varying concentrations of magnesium chloride and analyzed on a 3500xL Genetic Analyzer
VeriFiler
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5 Chapter 5 Experiments and results
Developmental validation
PCR cycle number
Reactions were amplified for 24, 25, and 26 cycles on the GeneAmp ™ PCR System
9700 using a blood sample on treated paper. As expected, the amount of PCR product increased with the number of cycles. A full profile was generated for all numbers of thermal PCR cycles (24–26) and off-scale data were collected for several allele peaks at
The data demonstrates that for this workflow 25 PCR cycles optimizes signal peak height and minimizes artifactual or other undesirable peaks.
Figure 6 Representative VeriFiler ™ Express kit profiles obtained from amplification of blood sample on untreated paper using 24, 25, and 26 cycles, analyzed on a 3500xL Genetic
Analyzer (Y-axis scale 0 to 25,000 RFU).
60 VeriFiler
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Thermal cycling temperatures
Chapter 5 Experiments and results
Developmental validation
5
Thermal cycling parameters were optimized using a Design of Experiments (DOE) approach that attempts to identify the combination of temperatures and hold times that produce the best assay performance. Optimal assay performance was determined through evaluation of assay sensitivity, peak-height balance, and resistance to PCR inhibitors.
For example, annealing/extension temperatures of 58, 59, and 60°C were tested using a GeneAmp ™
PCR System 9700 (Figure 7). The PCR products were analyzed using an
3500xL Genetic Analyzer.
All of the tested annealing temperatures, 58 to 60°C produced robust profiles. The optimal combination of specificity, sensitivity, and resistance to PCR inhibition was observed at 59°C. Thermal cycler temperature is critical to assay performance; therefore, routine, regularly scheduled thermal cycler calibration is strongly recommended.
Figure 7 Electropherograms obtained from amplification of a blood sample on an FTA ™ card at annealing temperatures of 58, 59, and 60°C, analyzed on a 3500xL Genetic Analyzer
(Y-axis scale 0 to 5,000 RFU).
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5 Chapter 5 Experiments and results
Representative data for common sample and substrate types
Representative data for common sample and substrate types
For all the representative electropherograms shown (Figure 8 through Figure 12),
peak heights at the D22S1045 marker may be elevated as compared to the other markers in the TAZ ™ dye channel. This result is typical to the assay and has no impact on the ability to generate a full STR profile.
Figure 8 Electropherogram of a blood sample on an FTA ™
VeriFiler ™
(Y-axis scale 0 to 12,000 RFU).
card amplified with the
Express kit at a customer test site and analyzed on a 3500xL Genetic Analyzer
62
Figure 9 Electropherogram of a blood sample on a NUCLEIC-CARD ™ amplified with the VeriFiler ™
Genetic Analyzer (Y-axis scale 0 to 25,000 RFU).
Collection Device
Express kit at a customer test site and analyzed on a 3500xL
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Representative data for common sample and substrate types
5
Figure 10 Electropherogram of a buccal sample on an FTA ™
VeriFiler ™
(Y-axis scale 0 to 7,000 RFU).
card amplified with the
Express kit at a customer test site and analyzed on a 3500xL Genetic Analyzer
Figure 11 Electropherogram of a buccal sample on a NUCLEIC-CARD ™ amplified with the VeriFiler ™
Genetic Analyzer (Y-axis scale 0 to 7,000 RFU).
Collection Device
Express kit at a customer test site and analyzed on a 3500xL
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5 Chapter 5 Experiments and results
Accuracy, precision, and reproducibility
Figure 12 Electropherogram of a buccal sample on a cotton swab (with lysate) extracted using Prep ‑n‑Go ™ Buffer, and amplified with the VeriFiler ™ Express kit at a customer test site and analyzed on a 3500xL Genetic Analyzer (Y-axis scale 0 to 25,000 RFU).
Accuracy, precision, and reproducibility
SWGDAM guideline 3.5
Accuracy observation
“Precision and accuracy of the assay should be demonstrated: Precision characterizes the degree of mutual agreement among a series of individual measurements, values and/or results. Precision depends only on the distribution of random errors and does not relate to the true value or specified value. The measure of precision is usually expressed in terms of imprecision and computed as a standard deviation of the test results. Accuracy is the degree of conformity of a measured quantity to its actual
(true) value. Accuracy of a measuring instrument is the ability of a measuring instrument to give responses close to a true value.” (SWGDAM, December 2012)
Laser-induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology (Holt et al., 2000; and Wallin et al., 2002). However, accuracy and reproducibility of profiles have been determined from various sample types.
The following three figures show the size differences that are typically observed between sample alleles and allelic ladder alleles on the 3130xl, and 3500xL Genetic
Analyzers with POP-4
Polymer. The X-axis in the following figures represents the nominal nucleotide sizes for the VeriFiler ™
™ Polymer and the 3730xl Genetic Analyzer with POP-7 ™
Express Allelic Ladder. The dashed lines parallel to the X-axis represent the ±0.25-nt windows. The y-axis represents the deviation of each sample allele size from the corresponding Allelic Ladder allele size. All sample alleles are
64 VeriFiler
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Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
1 1 1
1
Chapter 5 Experiments and results
Accuracy, precision, and reproducibility
5 within ±0.5 nt from a corresponding allele in the Allelic Ladder, irrespective of the capillary electrophoresis platforms.
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
1 1 1
1
Figure 13 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on a 3130 xl Genetic
Analyzer. Size and ladder sizing for the VeriFiler
GeneScan ™ 600 LIZ ™ Size Standard v2.0.
™ Express kit were calculated using the
Figure 14 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on a 3500xL
Genetic Analyzer. Size and ladder sizing for the VeriFiler using the GeneScan ™ 600 LIZ ™ Size Standard v2.0.
™ Express kit were calculated
VeriFiler
™
Express PCR Amplification Kit User Guide 65
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Chapter 5 Experiments and results
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Precision and size window description
Figure 15 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on an 3730 xl
Genetic Analyzer. Size and ladder sizing for the VeriFiler using the GeneScan ™ 600 LIZ ™ Size Standard v2.0.
™ Express kit were calculated
Sizing precision enables the determination of accurate and reliable genotypes. The recommended method for genotyping is to use a ±0.5-nt “window” around the size obtained for each allele in the allelic ladder. A ±0.5-nt window allows for the detection and correct assignment of alleles. Any sample allele that sizes outside the specified window could be either:
• An “off-ladder” allele, that is, an allele of a size that is not represented in the allelic ladder.
• An allele that does correspond to an allele in the allelic ladder, but whose size is just outside a window because of measurement error.
The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times. Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument.
66 VeriFiler
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Accuracy, precision, and reproducibility
5
Precision and size window observation
Figure 16 lists typical precision results obtained from multiple runs of the VeriFiler
™
Express Allelic Ladder using the GeneScan ™ 600 LIZ ™ Size Standard v2.0. The results were obtained within a set of injections on a single capillary array. The number of repeated injections for each genetic analyzer platform is shown in the following table:
CE platform
3130 xl
3500
3500xL
3730
Capillaries
16/injection
8/injection
24/injection
48/injection
# Injections
5
12
4
4
Sizing method
3rd Order Least Square, 60–
460 bp
3rd Order Least Square, 60–
460 bp
3rd Order Least Square, 60–
460 bp
3rd Order Least Square
The mean sizes and the standard deviation for the allele sizing were calculated for all
the alleles in each run (Figure 16). The mean range and the standard deviation range
show the lowest and highest values obtained across multiple runs.
Sample alleles can occasionally size outside of the ±0.5-nt window for a respective
Allelic Ladder allele because of measurement error. The frequency of such an occurrence is lowest in detection systems with the smallest standard deviations in
sizing. The figures in “Accuracy observation“ on page 64 illustrate the tight clustering
of allele sizes obtained on the Applied Biosystems ™ genetic analyzers, where the standard deviation in sizing is typically less than 0.15 nt. The instance of a sample allele sizing outside the ±0.5-nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0.15 nt or less (Smith,
1995).
Note: The read region for the VeriFiler ™ Express kit is 75–465 nt.
For sample alleles that do not size within a ±0.5-nt window, the PCR product must be rerun to distinguish between a true off–ladder allele versus measurement error of a sample allele that corresponds to an allele in the Allelic Ladder. Repeat analysis, when necessary, provides an added level of confidence in the final allele assignment.
GeneMapper ™ ID‑X Software automatically flags sample alleles that do not size within the prescribed window around an allelic ladder allele by labeling the allele as
OL (off-ladder).
Maximum sizing precision is obtained within the same set of capillary injections.
Cross–platform sizing differences occur due to several factors including type and concentration of polymer, run temperature, and electrophoresis conditions. Variations in sizing can also occur between runs on the same instrument and between runs on different instruments of the same platform type because of these factors.
IMPORTANT! To minimize the variation in sizing between runs and to ensure
analyze the samples.
For more information on precision and genotyping, see Lazaruk et al., 1998 and
Mansfield et al., 1998.
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Accuracy, precision, and reproducibility
In the following figure, the sizes for all the alleles across multiple injections of the
VeriFiler ™ Express Allelic Ladder were calculated. Each box represents the 25th through the 75th percentile of the observed values, with the center line indicating the median. Standard error bars indicate the range of the data, with presumed outliers
(points that are different from the mean by more than twice the pooled standard
68 VeriFiler
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Chapter 5 Experiments and results
Accuracy, precision, and reproducibility
5 deviation) shown as black dots. Panel B shows the distribution of the standard deviations for all the markers combined for each CE instrument platform.
Figure 16 Precision results of multiple runs of the VeriFiler ™ Express Allelic Ladder
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5 Chapter 5 Experiments and results
Extra peaks in the electropherogram
Extra peaks in the electropherogram
Causes of extra peaks
Extra peaks:
Stutter
Peaks other than the target alleles may be detected on the electropherogram. Causes for the appearance of extra peaks include stutter products, incomplete 3´ A nucleotide addition (at the n-1 position), dye artifacts, and mixed DNA samples (see DNA
Advisory Board (DAB) Standard 8.1.2.2).
Stutter definition
Stutter is a well-characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller than the target STR allele product (minus stutter), or less frequently, one repeat larger (plus stutter) (Butler, 2005; Mulero et al., 2006). Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the minus stutter product is missing a single tetranucleotide core repeat unit relative to the main allele (Walsh et al., 1996). Although plus-stutter is normally much less significant than minus-stutter in STR loci with tetranucleotide repeats, the incidence of plus-stutter may be more significant in trinucleotide repeat-containing loci.
Contact HID Support for more information on plus stutter.
The proportion of the stutter product relative to the main allele (percent stutter) is measured by dividing the height of the stutter peak by the height of the main allele peak.
Stutter observations
The level of stutter in this kit is normal and as expected for STR chemistries that are designed to overcome inhibitors and obtain robust performance with single source reference samples. Note the following:
Peak heights were measured for amplified samples at the loci used in the kit. All data were generated on the 3500xL Genetic Analyzer. Some conclusions from these measurements and observations are:
• For each locus, the stutter percentage generally increases with allele length.
• Smaller alleles display a lower level of stutter relative to the longer alleles within each locus.
• Each allele within a locus displays a consistent stutter percentage.
• Peaks in the stutter position that are above the stutter filter percentage specified in the software are not filtered (stutter filter percentage is calculated as the mean stutter for the locus plus three standard deviations). Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated.
• The measurement of stutter percentage for allele peaks that are off-scale may be unusually high due to artificial truncation of the main allele peak.
Marker-specific stutter observed in the population study with the VeriFiler ™ Express
kit is shown in Figure 17 through Figure 23 and listed in Table 3.
Workflow
Treated paper
Untreated paper
Number of samples
49 blood samples on FTA ™ Classic Cards
425 blood samples on FTA ™ Bloodstain Cards
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Extra peaks in the electropherogram
5
Figure 17 Stutter percentages for D1S1656, D2S441, D2S1338, and D3S1358 loci
(Blue=FAM ™ dye, black=NED ™ dye, purple=SID ™ dye)
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Extra peaks in the electropherogram
Figure 18 Stutter percentages for D5S818, D7S820, D8S1179, and D10S1248 (Green=VIC ™ dye, red=TAZ ™ dye, purple=SID ™ dye)
72 VeriFiler
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Extra peaks in the electropherogram
5
Figure 19 Stutter percentages for D12S391, D13S317, and D16S539 loci (Blue=FAM ™ dye, red=TAZ ™ dye, purple=SID ™ dye)
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Extra peaks in the electropherogram
Figure 20 Stutter percentages for D18S51, D19S433, and D21S11 loci (Green=VIC ™ dye, black=NED ™ dye)
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Extra peaks in the electropherogram
5
Figure 21 Stutter percentages for D22S1045, CSF1PO, TH01, and vWA loci (Blue=FAM ™ dye, green=VIC ™ dye, black=NED ™ dye, red=TAZ ™ dye). Red and orange data points associated with D22S1045 locus indicate minus and plus stutter, respectively.
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5 Chapter 5 Experiments and results
Extra peaks in the electropherogram
Figure 22 Stutter percentages for FGA and TPOX loci (Blue=FAM
™
dye, black=NED
™
dye)
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Extra peaks in the electropherogram
5
Figure 23 Stutter percentages for D6S1043, Penta D, and Penta E loci (Red=TAZ ™ dye, purple=SID ™ dye, green=VIC ™ dye)
Stutter percentage filter settings provided with GeneMapper ™ ID ‑X Software
The settings in Table 3 were derived using the data shown earlier in this section. The
proportion of the stutter product relative to the main allele (stutter percent) is measured by dividing the height of the stutter peak by the height of the main allele peak.
IMPORTANT! The values that are shown in the table are the values that were determined during developmental validation studies using specific data sets. Always perform internal validation studies to determine the appropriate values to use for your applications.
Table 3 Marker-specific stutter filter percentages for VeriFiler ™ Express kit loci
Locus [1]
CSF1PO
D10S1248
D12S391
D13S317
D16S539
D18S51
% Stutter
10.54
12.27
14.44
10.10
10.76
14.15
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Extra peaks in the electropherogram
Extra peaks:
Addition of 3' A nucleotide
78
Locus [1]
D19S433
D1S1656
D21S11
D22S1045
D22S1045 (+3 nt)
D2S1338
D2S441
D3S1358
D5S818
D7S820
D8S1179
FGA
Penta D
Penta E
TH01
8.31
4.93
TPOX 5.20
vWA
[1] These percentages are used as stutter filters in VeriFilerExpress_Stutter.txt.
13.18
% Stutter
10.77
13.62
12.24
8.05
18.0
13.29
9.40
12.16
10.53
9.39
10.85
12.27
4.09
3′ A definition
Many DNA polymerases can catalyze the addition of a single nucleotide
(predominantly adenosine) to the 3′ ends of double-stranded PCR products (Clark,
1988; Magnuson et al., 1996). This nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence. The PCR product with the extra nucleotide is referred to as the “+A” form.
3′ A observations
The efficiency of +A addition is related to the particular sequence of the DNA at the 3´ end of the PCR product.
The VeriFiler ™ Express kit includes two main design features that promote maximum
+A addition:
• The primer sequences have been optimized to encourage +A addition.
• The PCR chemistry allows complete +A addition with a short final incubation at
60°C for 5 minutes .
This final extension step gives the DNA polymerase additional time to complete +A
addition to all double-stranded PCR products. Figure 24 shows examples of
incomplete and normal +A addition. Final extension incubation for longer than the
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Chapter 5 Experiments and results
Extra peaks in the electropherogram
5 recommended time can result in double +A addition, in which two nontemplate adenosine residues are added to the PCR product. Double +A addition can cause
"shoulders" on the right side of main allele peaks, and is therefore to be avoided.
Extra peaks:
Artifacts
Figure 24 Omitting the final extension step results in shoulders on main allele peaks due to incomplete A nucleotide addition. Examples shown are the smaller amplicons of FAM ™ ,
NED ™ , and SID ™ dye channel data from a 3500xL Genetic Analyzer using the VeriFiler ™
Express kit.
Due to improved PCR buffer chemistry, the lack of +A addition is generally less of an issue with the VeriFiler ™ Express kit than with earlier generation kits. However,
"shouldering" of allele peaks can still be observed if the amount of input DNA is greater than recommended concentration. Amplification of excess input DNA can also result in off-scale data.
Artifact definition
Artifacts and anomalies are seen in all molecular biological systems. Artifacts are typically reproducible. Anomalies are non-reproducible, intermittent occurrences that are not observed consistently in a system (for example, spikes and baseline noise).
Artifact observation
Due to improvements in PCR primer manufacturing processes, the incidence of artifacts has been greatly reduced in the VeriFiler ™ Express kit. Kit electropherograms are free of reproducible dye artifacts in the kit read region of 75– 465 nt for commonly
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Express PCR Amplification Kit User Guide 79
5 Chapter 5 Experiments and results
Extra peaks in the electropherogram
used analytical thresholds. Figure 25 shows the low baseline-level fluorescence that is
observed in a typical negative control PCR.
Figure 25 Examples of fluorescence background in data produced on a 3500xL Genetic
Analyzer (Y-axis scale 0 to 200 RFU).
Additional reproducible DNA-dependent artifacts have been characterized and
documented in Table 4. It is important to consider noise and other amplification-
related artifacts when interpreting data.
Table 4 DNA-dependent artifacts observed with the VeriFiler ™ Express kit
Artifact
TAZ60–80
SID130
SID202
SID402–411
VIC110-120
Color
Red
Purple
Purple
Purple
Green
Size
60–80 nt
130–131 nt
202– 203 nt
402– 411 nt
110–120 nt
Type
PCR artifact
PCR artifact
PCR artifact
PCR artifact
Sample-independent artifact intrinsic to the assay
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Characterization of loci
5
Characterization of loci
SWGDAM guideline 3.1
Loci in this kit
Nature of polymorphisms
Inheritance
Mapping
Genetic linkage
“The basic characteristics of a genetic marker should be determined and documented.” (SWGDAM, December 2012)
This section describes basic characteristics of the 23 autosomal STR loci, one Y indel locus, and the sex-determining marker (Amelogenin), which are amplified with the
VeriFiler ™ Express kit. Most of these loci have been extensively characterized by other laboratories.
The primers for the Amelogenin locus flank a 6-nucleotide deletion in intron 1 of the
X homolog. Amplification generates 104-nt and 110-nt products from the X and Y chromosomes, respectively. (Sizes are the actual nucleotide size according to sequencing results, including 3´ A nucleotide addition, and size may not correspond exactly to allele mobility observed on capillary electrophoresis platforms.) Except for
D22S1045, a trinucleotide STR locus, and Penta D and Penta E, pentanucleotide STR loci (Bacher and Schumm, 1998), the remaining loci are tetranucleotide short tandem repeat (STR) loci. The length differences among alleles of a particular locus are caused by differences in the number of repeat units.
We have sequenced all the alleles in the VeriFiler ™ Express kit Allelic Ladder, including microvariants. In addition, other groups in the scientific community have sequenced alleles at some of these loci (Nakahori et al., 1991; Puers et al., 1993; Möller
et al., 1994; Barber et al., 1995; Möller and Brinkmann, 1995; Barber et al., 1996; Barber and Parkin, 1996; Brinkmann et al., 1998; Momhinweg et al., 1998; Watson et al., 1998).
Among the various sources of sequence data on the loci, there is consensus on the repeat patterns and structure of the STRs.
The Centre d'Etude du Polymorphisme Humain (CEPH) has collected DNA from families of Utah Mormon, French Venezuelan, and Amish descent. These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of inheritance of various DNA loci. Each family set contains three generations, generally including four grandparents, two parents, and several offspring. Consequently, the CEPH family DNA sets are ideal for studying inheritance patterns (Begovich et al., 1992).
The VeriFiler ™ Express kit loci have been mapped, and the chromosomal locations have been published (Nakahori et al., 1991; Edwards et al., 1992; Kimpton et al., 1992;
Mills et al., 1992; Sharma and Litt, 1992; Li et al., 1993; Straub et al., 1993; Barber and
Parkin, 1996; and Lareu, et al., 1996).
Two sets of STR loci in the VeriFiler ™ Express kit are located on the same chromosomes. vWA and D12S391 are located approximately 6.3 million bp apart on the p arm of chromosome 12; D2S1338 and D2S441 are located approximately 150 million bp apart on opposite arms of chromosome 2. Linkage disequilibrium analysis was conducted on the genotype results from 1,034 individuals of three ethnic groups
(350 African American, 349 Caucasian, and 335 Hispanic). STR locus genotype results from the population study were analyzed using the Linkage Disequilibrium module of GenePop software version 4.0.10 (Raymond and Rousset, 1995; Rousset, 2008). See
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5 Chapter 5 Experiments and results
Species specificity
The relatively high probability values indicate that there is no statistically significant linkage disequilibrium found between the pairs of loci that are located on the same chromosome.
An independent analysis of data from the same collection of population samples
(Budowle, et al., 2010) also concluded that the 15 STR loci that are shared between the
NGM ™ and NGM SElect ™ kits were independent at the population level (note that the
SE33 locus was not part of this analysis). Therefore, to calculate the rarity of a profile for comparison to single-source and mixture samples, the frequencies of all loci including vWA and D12S391 could be multiplied. However, the analysis of the CEPH pedigree families demonstrated a degree of linkage between vWA and D12S391 that does not support the assumption of independence for kinship analysis.
Table 5 GenePop software LD Result (p ‑value for pairwise analysis of loci)
Locus
Chromosome map position [1]
Chromosome
Nuclear
Coordinates [1]
(million bp)
African-
American
(n = 350)
Caucasian
(n = 350)
Hispanic
(n = 293) vWA
D12S391
12p13.31
12p13.2
5.9
12.2
0.86
0.29
0.27
D2S441 2p14 68
0.11
0.32
0.19
D2S1338 2q35 218
[1] STR locus mapping data was obtained from the NCBI Map Viewer http://www.ncbi.nlm.nih.gov/projects/ mapview/map_search.cgi?taxid=9606 or the UCSC Genome Browser (http://genome.ucsc.edu/). GenePop LD analysis probability results (p values) greater than 0.05 were considered to indicate that linkage disequilibrium between the loci within the population tested was not statistically significant.
Species specificity
SWGDAM
Guideline 3.2
“The ability to detect genetic information from non-targeted species (e.g., detection of microbial DNA in a human assay) should be determined. The detection of genetic information from non-targeted species does not necessarily invalidate the use of the assay, but may help define the limits of the assay.” (SWGDAM, December 2012)
82 VeriFiler
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Nonhuman studies
Chapter 5 Experiments and results
Species specificity
5
The VeriFiler ™ Express kit provides the required specificity for detecting human alleles. Species specificity testing was performed to ensure that there is no crossreactivity with nonhuman DNA that may be present in forensic casework samples.
The following species were tested (in the specified amounts) using standard PCR and capillary electrophoresis conditions for the VeriFiler ™ Express kit kit:
• Primates: chimpanzee, gorilla, and orangutang (1.0 ng each)
• Non-primates: cow, chicken, dog, horse, mouse, pig, rabbit, rat, sheep (10.0 ng each)
• Microorganisms: Candida albicans, Enterococcus faecalis, Escherichia coli,
Fusobacterium nucleatum, Lactobacillus casei, Staphylococcus aureus, Streptococcus
mitis, Streptococcus mutans, Streptococcus salivarius, and Streptococcus viridans
(equivalent to 105 copies) (These microorganisms are commonly found in the oral cavity (Suido et al., 1986; Guthmiller et al., 2001).)
Results were evaluated for the presence of any amplified peaks that would indicate cross reactivity of the VeriFiler ™ Express kit with any of these non-human species.
“Nonhuman studies“ on page 83 shows example electropherogram results from the
species specificity tests. The chimpanzee, gorilla, and orangutang DNA samples produced partial profiles in the 75–465 nucleotide region.
Figure 26 Representative electropherograms for some species tested in a species specificity study. Data produced on a 3500xL Genetic Analyzer.
Among the non-primate species, most produced no peaks over a threshold of
175 RFU. Horse yielded reproducible VIC ™ dye peaks at 94 bp (<100 RFU) due to
Amelogenin cross-reactivity. Pig yielded reproducible TAZ ™
(<50 RFU), non-reproducible peaks.
dye peaks at 424 bp (
<200 RFU). Individual replicate PCRs of dog, mouse, and chicken yielded single, small
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5 Chapter 5 Experiments and results
Sensitivity
Sensitivity
SWGDAM guideline 3.3
Sample collection factors that can affect DNA quantity
Effect of DNA quantity on results
“The ability to obtain reliable results from a range of DNA quantities, to include the upper and lower limits of the assay, should be evaluated.” (SWGDAM, December
2012)
The VeriFiler ™ Express kit has been optimized at 25 µL PCR reaction volume to overcome the PCR inhibition expected when amplifying:
• Blood samples that are obtained directly from unpurified 1.2 mm treated paper discs
• Buccal cells that are obtained directly from unpurified 1.2 mm treated paper discs
(with the addition of Prep-n-Go ™ Buffer )
• Buccal swab sample lysate is prepared using Prep-n-Go ™ Buffer
Depending on the following conditions, DNA quantities present on the 1.2 mm disc may vary from laboratory to laboratory:
• Volume of blood that is spotted onto the treated paper
• Collecting devices that are used
• Collection methods that are applied
• Swab-to-paper transfer protocol that is used
It is essential to optimize the PCR conditions for types of blood samples and spotting
protocol. See “Optimize PCR cycle number (before first use of the kit)“ on page 16.
If too much DNA is added to the PCR reaction, the increased amount of PCR product that is generated can result in:
• Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument (“off-scale” data).
Off-scale data is a problem because:
– Quantitation (peak height and area) for off-scale peaks is not accurate. For example, an allele peak that is off-scale can cause the corresponding stutter peak to appear higher in relative intensity, thus increasing the calculated percent stutter.
– Multicomponent analysis of off-scale data is not accurate. This inaccuracy results in poor spectral separation (“pull-up”).
• Incomplete +A nucleotide addition.
To ensure minimal occurrence of offscale data when using the VeriFiler ™ Express PCR
Amplification Kit , optimize PCR cycle number according to instructions in the
Perform PCR chapter.
When the total number of allele copies added to the PCR is extremely low, unbalanced amplification of the alleles may occur because of stochastic fluctuation.
Population data
SWGDAM guideline 3.7
“The distribution of genetic markers in populations should be determined in relevant population groups.” (SWGDAM, December 2012)
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Population data
5
Population data overview
Loci in this kit
Population samples used in these studies
Concordance studies
Probability of
Identity definition
To interpret the significance of a match between genetically typed samples, you must know the population distribution of alleles at each locus in question. If the genotype of the relevant evidence sample is:
• Different from the genotype of the reference sample for a suspect, then the suspect is excluded as the donor of the biological evidence that was tested. An exclusion is independent of the frequency of the two genotypes in the population.
• The same as the genotype of the reference sample for a suspect, then the suspect is included as a possible source of the evidence sample.
The probability that another, unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant populations.
This section describes basic characteristics of the 23 autosomal STR loci, one Y indel locus, and the sex-determining marker (Amelogenin), which are amplified with the
VeriFiler ™ Express kit. Most of these loci have been extensively characterized by other laboratories.
Population data for loci contained in theVeriFiler ™ Express PCR Amplification Kit was obtained from data published by (Hill et. al., 2013). See the original data at
www.cstl.nist.gov/biotech/strbase/NISTpop.htm. Ethnicities of sample donors were:
• African-American—342 samples
• Asian—97 samples
• Caucasian—361 samples
• Hispanic—236 samples
The VeriFiler ™ Express kit contains loci for which extensive population data are available.
The primer sequences used in the VeriFiler ™
GlobalFiler ™
Express PCR Amplification Kit and
Express kit are identical except for the D6S1043, Penta E, and Penta D loci which are not shared between the two kits.
We compared allele calls between the VeriFiler the PowerPlex the PowerPlex
™
™
™ Express PCR Amplification Kit and
21 System. Genotype data from 200 blood samples on FTA ™
21 System at the D5S818 and Penta E loci. These alleles were accurately genotyped by the VeriFiler ™ Express PCR Amplification Kit.
Classic
Cards showed 99.6% concordance between the two kits. Null alleles were found with
The P
I
value is the probability that two individuals selected at random will have an identical genotype (Sensabaugh, 1982).
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5 Chapter 5 Experiments and results
Population data
Probability of identity
Table 6 shows the Autosomal STR allele frequencies at VeriFiler
™ population group.
Express kit loci by
Table 7 shows the Probability of identity (P
I individually and combined.
) values of the VeriFiler ™ Express kit loci
Table 6 Autosomal allele frequencies by population group for VeriFiler ™
Kit STR loci. (*=Alleles not detected)
Express kit
Allele
African
American
(n = 342)
Asian (n = 97)
U.S. Caucasian
(n = 361)
U.S. Hispanic
(n = 236)
*
0.2953
*
*
0.0468
0.0088
*
*
*
*
*
0.2485
*
*
*
*
0.0556
*
0.0556
*
0.0395
*
0.2500
11.3
12.0
12.1
12.3
13.0
14.0
15.0
16.0
10.1
10.2
10.3
11.0
11.1
11.2
CSF1PO
5.0
6.0
7.0
7.3
8.0
8.1
9.0
9.1
10.0
*
0.3866
*
*
0.0876
0.0155
0.0052
*
0.2165
*
*
*
*
*
*
*
0.0206
*
*
*
0.0670
*
0.2010
*
0.3601
*
*
0.0817
0.0097
*
*
*
*
*
0.3089
*
*
*
*
*
*
0.0055
*
0.0139
*
0.2202
*
0.3750
*
*
0.0593
0.0064
0.0021
*
*
*
*
0.2797
*
*
*
*
0.0127
*
0.0042
*
0.0233
*
0.2373
86 VeriFiler
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Allele
17.0
18.0
19.0
D12S391
13.0
14.0
15.0
16.0
17.0
17.1
17.3
18.0
D10S1248
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
18.1
18.2
18.3
19.0
19.1
19.3
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
*
*
0.2629
*
*
*
*
*
0.0412
0.0103
0.0825
0.1753
*
0.0052
0.2526
0.2062
0.0979
0.0309
*
*
*
*
*
0.0052
*
0.0876
0.3196
African
American
(n = 342)
Asian (n = 97)
U.S. Caucasian
(n = 361)
U.S. Hispanic
(n = 236)
0.0044
0.0044
0.2529
0.0015
*
0.0044
*
0.0015
0.0775
0.0673
0.1667
0.1477
0.0088
0.0044
0.2763
0.1974
0.0877
0.0249
0.0015
*
*
0.0029
0.0029
0.0073
0.0351
0.1301
0.2339
*
0.0208
0.1717
*
*
0.0249
*
*
0.0319
0.0222
0.1274
0.1247
*
0.0042
0.2978
0.1967
0.1330
0.0277
0.0014
0.0028
*
*
*
*
0.0014
0.0319
0.3075
*
0.0169
0.1780
*
*
0.0127
*
*
0.0445
0.0424
0.0763
0.1886
0.0021
0.0064
0.3390
0.2119
0.0996
0.0254
0.0021
0.0021
*
*
*
*
0.0042
0.0424
0.2733
87
5 Chapter 5 Experiments and results
Population data
Allele
26.0
27.0
D13S317
5.0
6.0
7.0
9.0
10.0
11.0
7.1
8.0
8.1
11.3
12.0
12.3
13.0
13.3
14.0
22.0
22.3
23.0
24.0
24.3
25.0
20.0
20.1
20.3
21.0
21.1
88
0.0336
0.0307
0.3099
*
0.4181
*
*
*
*
*
0.0278
*
0.1404
*
0.0395
0.0365
*
0.0292
0.0132
*
0.0088
*
*
African
American
(n = 342)
0.1038
0.0029
*
0.0643
*
0.0567
*
0.0258
0.0103
0.0052
0.0155
0.0103
0.0052
0.1959
*
*
0.0979
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
0.0956
*
0.0693
0.0471
*
0.0166
0.0028
0.0014
0.1108
*
*
0.1288
*
U.S. Hispanic
(n = 236)
0.0678
*
0.0572
0.0169
*
0.0064
0.0064
0.0064
0.1547
*
*
0.1123
*
0.1443
0.1031
0.2680
*
0.2062
*
*
0.2216
*
*
*
*
0.0567
*
*
0.0776
0.0471
0.3255
*
0.2687
*
*
*
*
*
0.1205
*
0.1163
*
0.0429
0.1653
0.0996
0.2182
*
0.2352
*
*
*
*
*
0.1102
*
0.1059
*
0.0614
VeriFiler
™
Express PCR Amplification Kit User Guide
Allele
D18S51
6.0
7.0
8.0
9.0
9.2
12.2
13.0
13.3
14.0
15.0
16.0
9.3
10.0
11.0
11.3
12.0
12.1
15.0
16.0
17.0
D16S539
4.0
8.0
8.3
9.0
5.0
6.0
7.0
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
*
0.1649
0.1907
*
0.1753
*
*
*
0.3557
*
*
*
*
*
0.0979
*
0.0155
*
*
*
*
*
*
*
African
American
(n = 342)
*
*
*
Asian (n = 97)
*
*
*
U.S. Caucasian
(n = 361)
0.0014
*
*
U.S. Hispanic
(n = 236)
0.0042
*
*
*
0.1170
0.3143
*
0.2047
*
*
0.0015
*
*
0.0322
*
0.1827
*
0.1228
*
0.0249
*
*
*
*
*
0.0029
*
*
0.0568
0.3144
*
0.3144
*
*
*
*
*
0.0180
*
0.1066
*
0.1634
*
0.0263
*
*
*
*
*
*
*
*
0.1504
0.2648
*
0.2775
*
*
*
*
*
0.0191
*
0.1398
*
0.1335
*
0.0127
0.0021
*
*
*
*
*
*
89
5 Chapter 5 Experiments and results
Population data
Allele
16.1
16.2
17.0
17.2
17.3
18.0
14.0
14.2
15.0
15.2
15.3
16.0
18.1
18.2
19.0
19.2
20.0
20.2
12.2
12.3
13.0
13.1
13.2
13.3
10.0
10.2
11.0
11.2
12.0
90
*
*
0.1520
*
*
0.1213
0.0716
*
0.1652
0.0015
*
0.1711
African
American
(n = 342)
0.0044
*
0.0015
*
0.0760
*
*
0.0409
*
0.0044
*
*
*
0.0994
*
0.0629
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
*
0.0014
0.1385
*
*
0.0776
0.1343
0.0014
0.1704
*
*
0.1468
*
*
0.0402
*
0.0180
*
*
*
0.1233
*
*
*
0.0083
*
0.0097
*
0.1136
*
*
0.0670
*
*
0.0309
0.2371
*
0.1804
*
*
0.1289
*
*
0.0412
*
0.0258
*
*
*
0.2165
*
*
*
*
*
*
*
0.0361
U.S. Hispanic
(n = 236)
*
*
0.1250
*
*
0.0784
0.1610
0.0021
0.1589
*
*
0.1250
*
*
0.0466
*
0.0275
*
*
*
0.1229
*
*
*
0.0021
*
0.0148
*
0.1144
VeriFiler
™
Express PCR Amplification Kit User Guide
Allele
28.0
D19S433
5.2
6.0
6.2
7.0
10.0
10.2
11.0
8.0
9.0
9.2
11.2
12.0
12.1
12.2
13.0
13.1
23.0
23.2
24.0
25.0
26.0
27.0
21.0
21.1
21.2
22.0
22.2
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
0.0102
*
0.0629
*
0.1228
*
*
*
*
*
*
*
*
0.0365
0.2456
*
0.0044
*
0.0015
*
*
*
*
African
American
(n = 342)
0.0102
*
0.0015
0.0073
*
0.0052
*
*
*
*
*
0.0052
0.0103
*
*
0.0155
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
*
*
*
*
*
*
*
0.0097
*
*
0.0069
*
U.S. Hispanic
(n = 236)
*
*
0.0021
*
*
*
*
0.0085
*
*
0.0106
*
*
0.0361
*
*
*
*
0.0258
0.2835
*
*
*
*
*
*
*
*
0.0014
*
0.0055
*
0.0706
*
*
*
*
*
*
*
*
0.0014
0.2548
*
0.0021
*
0.0148
*
0.0657
*
*
*
*
*
*
0.0021
*
0.0127
0.2225
*
91
5 Chapter 5 Experiments and results
Population data
Allele
D1S1656
8.0
9.0
18.2
19.0
19.2
10.0
11.0
12.0
13.0
13.3
14.0
14.2
14.3
15.0
15.3
16.0
16.1
15.2
16.0
16.2
17.0
17.2
18.0
13.2
14.0
14.2
14.3
15.0
92
0.1009
*
0.2573
*
0.0073
0.1579
*
*
0.0146
0.0453
0.0643
0.0292
0.1096
*
African
American
(n = 342)
0.0526
0.2105
0.0746
*
0.0804
0.0614
0.0044
0.0263
*
0.0088
*
0.0029
*
*
0.1237
0.0103
0.0309
*
0.0052
*
0.0206
0.2990
0.1031
*
0.0619
*
*
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
0.0360
0.0568
0.0152
0.0069
0.0014
*
0.0069
0.3615
0.0235
*
0.1565
0.0014
*
*
U.S. Hispanic
(n = 236)
0.0551
0.0254
0.0275
*
*
*
0.0445
0.3538
0.0381
*
0.1356
*
*
*
0.1340
*
0.0619
*
*
0.2784
*
*
*
0.0309
0.0464
*
0.2010
*
0.0665
*
0.0789
*
0.0028
0.1496
*
*
0.0028
0.0776
0.1163
0.0582
0.1357
*
0.1144
*
0.1165
*
0.0042
0.1377
*
*
0.0064
0.0275
0.0890
0.0508
0.1758
*
VeriFiler
™
Express PCR Amplification Kit User Guide
Allele
D21S11
23.2
24.0
24.2
24.3
25.0
25.2
25.3
26.0
26.2
27.0
27.1
27.2
27.3
28.0
28.1
28.2
28.3
18.3
19.0
19.3
20.0
20.3
21.0
16.3
17.0
17.1
17.3
18.0
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
*
*
0.0015
*
0.0746
*
*
*
*
*
*
*
*
0.2456
*
*
*
African
American
(n = 342)
0.1023
0.0278
*
0.0497
0.0029
0.0234
*
0.0073
*
*
*
0.0515
*
0.0052
*
*
*
0.0155
0.0722
*
0.0876
0.0155
Asian (n = 97)
U.S. Caucasian
(n = 361)
0.0499
*
0.0152
*
*
*
0.0609
0.0471
*
0.1330
0.0055
U.S. Hispanic
(n = 236)
0.0254
*
0.0042
*
*
*
0.0508
0.0424
*
0.1483
0.0064
*
*
*
*
*
*
*
*
*
*
*
*
*
0.0567
*
0.0052
*
0.0014
*
*
*
0.0222
*
*
*
*
*
*
*
*
0.1593
*
*
*
*
*
*
0.0021
0.0275
*
*
*
0.0021
*
*
*
*
0.0996
*
*
*
93
5 Chapter 5 Experiments and results
Population data
Allele
33.1
33.2
33.3
34.0
34.1
34.2
31.3
32.0
32.1
32.2
32.2
33.0
35.0
35.1
35.2
36.0
36.1
36.2
30.1
30.2
30.3
31.0
31.1
31.2
29.0
29.1
29.2
29.3
30.0
94
0.0029
0.0351
*
0.0058
*
*
*
0.0088
*
0.0614
*
0.0044
African
American
(n = 342)
0.2047
*
*
0.0015
0.1696
*
0.0175
*
0.0789
*
0.0512
0.0219
*
*
0.0088
*
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
*
0.0263
*
*
*
0.0042
*
0.0055
*
0.0900
*
0.0014
0.0014
*
*
0.0014
*
*
*
0.0291
*
0.0720
*
0.0983
0.2022
*
0.0028
*
0.2825
*
0.0464
*
*
*
*
*
0.0567
*
0.1134
*
0.0155
*
*
*
*
*
*
*
0.0052
0.0103
0.1237
*
0.0361
0.2010
*
*
*
0.3299
U.S. Hispanic
(n = 236)
0.0021
0.0339
*
0.0021
*
*
*
0.0169
*
0.1271
*
0.0042
*
*
*
*
*
*
*
0.0233
*
0.0763
*
0.0996
0.2076
*
0.0021
*
0.2733
VeriFiler
™
Express PCR Amplification Kit User Guide
Allele
18.0
19.0
D2S1338
10.0
11.0
12.0
12.0
13.0
14.0
15.0
16.0
17.0
D22S1045
7.0
8.0
9.0
10.0
11.0
37.0
37.2
38.0
38.2
39.0
13.0
14.0
15.0
16.0
17.0
18.0
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
*
*
*
*
*
0.0015
0.0556
0.1009
0.0424
0.0541
0.0029
0.0775
0.2515
0.1915
0.2091
0.0146
0.0058
*
0.0073
*
0.0409
0.1447
African
American
(n = 342)
0.0029
*
0.0015
*
0.0015
Asian (n = 97)
*
*
*
*
*
U.S. Caucasian
(n = 361)
*
*
*
*
*
U.S. Hispanic
(n = 236)
*
*
*
*
*
0.0052
*
0.0103
0.3093
0.2268
0.2216
0.0206
0.0052
*
*
*
*
0.2010
0.0125
0.0069
0.0568
0.3213
0.3823
0.0748
0.0055
*
*
*
*
*
0.1399
0.0127
0.0085
0.0275
0.4258
0.3496
0.0911
0.0064
*
*
*
*
0.0148
0.0636
0.0206
0.0567
0.1340
*
*
*
*
*
*
*
*
*
*
*
0.0014
0.0374
0.1856
0.0734
*
*
*
*
*
*
0.0297
0.1695
0.0805
95
5 Chapter 5 Experiments and results
Population data
Allele
25.0
26.0
27.0
28.0
29.0
D2S441
9.1
10.0
10.1
8.0
8.1
9.0
11.0
11.1
11.3
12.0
12.2
12.3
22.0
23.0
23.2
23.3
24.0
24.2
18.3
19.0
19.3
20.0
21.0
96
0.3626
*
0.0439
0.1652
*
0.0058
*
*
0.0029
*
0.0848
*
African
American
(n = 342)
*
0.1389
*
0.1038
0.1360
0.1374
0.1038
*
*
0.0833
*
0.0775
0.0146
0.0044
*
*
0.0515
0.1649
*
*
0.1289
*
*
0.1804
*
0.1598
0.0155
0.0464
0.0361
0.0052
*
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
0.0346
0.1053
*
*
0.1150
*
*
0.1205
*
0.1565
0.0374
0.1025
0.0305
*
*
*
U.S. Hispanic
(n = 236)
0.0572
0.1398
*
*
0.0763
*
*
0.1928
*
0.1271
0.0318
0.0784
0.0169
*
*
*
0.3505
*
0.0309
0.2165
*
0.0052
0.0052
*
*
0.0052
0.2680
*
0.3435
*
0.0609
0.0471
*
0.0042
*
*
0.0014
0.0014
0.2105
*
0.2987
*
0.0445
0.0360
*
0.0021
*
*
*
*
0.3369
*
VeriFiler
™
Express PCR Amplification Kit User Guide
Allele
15.0
15.2
16.0
16.2
17.0
17.1
10.0
11.0
12.0
13.0
13.2
14.0
D3S1358
8.0
9.0
15.0
16.0
17.0
13.0
13.3
14.0
14.1
14.3
17.2
18.0
18.2
19.0
20.0
20.1
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
*
0.2120
*
*
0.0570
*
0.0044
*
*
0.0029
*
0.0906
0.3085
0.0015
0.3187
*
*
*
*
0.0044
African
American
(n = 342)
0.0439
0.0029
0.2675
*
0.0015
0.0190
*
*
0.0258
*
0.0825
*
*
0.0103
*
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
0.0291
*
0.2410
*
*
0.0596
0.0014
*
U.S. Hispanic
(n = 236)
0.0233
*
0.2055
*
*
0.0487
0.0021
0.0021
*
0.2010
*
*
0.0670
*
0.0103
*
*
*
*
0.0258
0.3660
*
0.3299
*
*
*
*
*
*
0.2105
*
*
0.1510
*
0.0166
0.0014
*
0.0014
*
0.1066
0.2729
*
0.2382
*
*
*
0.0014
*
*
0.1843
*
*
0.1229
*
0.0042
0.0021
*
0.0064
*
0.0784
0.3220
*
0.2797
*
*
*
*
*
97
5 Chapter 5 Experiments and results
Population data
Allele
15.0
16.0
17.0
18.0
19.0
D6S1043
12.0
12.1
12.3
13.0
14.0
14.3
21.0
D5S818
6.0
7.0
8.0
9.0
10.0
10.1
11.0
11.1
11.3
10.0
11.0
12.0
7.0
8.0
9.0
98
0.2680
*
*
0.2062
*
*
*
0.0155
0.0052
0.0979
0.2268
*
0.1649
0.0155
*
*
*
*
*
*
0.0309
0.1495
0.1237
*
*
*
African
American
(n = 342)
*
Asian (n = 97)
*
U.S. Caucasian
(n = 361)
*
U.S. Hispanic
(n = 236)
*
0.2339
*
*
0.3699
*
*
*
0.0015
0.0468
0.0322
0.0731
*
0.2237
0.0161
*
0.0029
*
*
*
*
*
*
0.0015
0.0058
0.1535
0.2237
0.3560
*
*
0.3878
*
*
*
0.0028
0.0055
0.0416
0.0554
*
0.1427
0.0069
*
0.0014
*
*
*
*
*
0.0014
*
0.0166
0.2964
0.2368
0.3898
*
*
0.3390
*
*
*
0.0339
0.0085
0.0530
0.0572
*
0.1081
0.0085
*
0.0021
*
*
*
*
*
*
*
0.0042
0.1780
0.2055
VeriFiler
™
Express PCR Amplification Kit User Guide
Allele
D7S820
5.0
5.2
24.3
25.0
26.0
21.3
22.0
22.3
23.0
23.3
24.0
19.0
19.2
19.3
20.0
20.3
21.0
17.0
17.2
17.3
18.0
18.1
18.3
12.3
13.0
14.0
15.0
16.0
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
0.0015
*
*
0.0102
*
0.0058
0.1126
*
0.0015
0.0731
*
0.0175
*
0.0015
0.0015
African
American
(n = 342)
*
0.0965
0.0585
0.0541
0.0161
0.0570
*
*
0.1067
*
0.0015
Asian (n = 97)
U.S. Caucasian
(n = 361)
*
0.0014
*
*
*
*
0.0983
*
*
0.0319
*
0.0097
*
*
*
0.0609
*
*
0.0886
*
*
*
0.0859
0.0554
0.0125
0.0042
*
*
*
*
*
*
0.0928
*
*
0.0412
*
0.0052
*
*
*
0.0722
*
*
0.1598
*
*
0.0052
0.1237
0.1546
0.0361
0.0052
U.S. Hispanic
(n = 236)
0.0403
*
0.0127
*
0.0021
*
0.0763
*
0.0021
0.0318
0.0127
0.0064
*
*
*
0.0487
*
*
0.1081
0.0021
*
*
0.1017
0.1356
0.0297
0.0021
*
*
*
*
*
*
*
*
99
5 Chapter 5 Experiments and results
Population data
Allele
11.1
11.3
12.0
12.1
12.3
13.0
9.2
9.3
10.0
10.1
10.3
11.0
13.1
14.0
14.1
15.0
16.0
8.3
9.0
9.1
8.0
8.1
8.2
6.0
6.3
7.0
7.1
7.3
*
*
0.0877
*
*
0.0146
*
*
0.3363
*
*
0.2032
African
American
(n = 342)
0.0015
*
0.0117
*
*
0.2281
*
*
*
0.1155
*
*
0.0015
*
*
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
*
*
0.1593
*
*
0.0346
*
*
0.2562
*
*
0.2050
*
0.0042
*
*
*
0.1440
0.0014
*
*
0.1676
*
*
*
0.0277
*
*
*
*
0.1753
*
*
0.0258
*
*
0.2577
*
*
0.3608
*
*
*
*
*
0.1289
*
*
*
0.0464
*
*
*
0.0052
*
*
U.S. Hispanic
(n = 236)
*
*
0.1547
*
*
0.0360
*
*
0.3072
*
0.0021
0.2775
*
*
*
*
*
0.1208
*
*
*
0.0911
*
*
*
0.0106
*
*
100 VeriFiler
™
Express PCR Amplification Kit User Guide
Allele
16.0
17.0
18.0
19.0
20.0
12.3
13.0
13.3
14.0
15.0
15.3
D8S1179
4.0
5.0
6.0
7.0
8.0
9.0
10.0
10.2
11.0
12.0
FGA
12.3
13.0
14.0
15.0
15.2
16.0
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
*
*
*
*
*
*
African
American
(n = 342)
Asian (n = 97)
U.S. Caucasian
(n = 361)
U.S. Hispanic
(n = 236)
*
0.0526
0.1301
*
0.2193
*
*
*
*
*
0.0073
0.0044
0.0307
0.2939
0.1901
*
0.0643
0.0044
0.0029
*
*
*
0.1186
0.1186
*
0.2010
*
*
*
0.1237
*
*
*
*
0.2010
0.1289
*
0.0928
0.0103
0.0052
*
*
*
0.0762
0.1676
*
0.3296
*
*
*
*
*
0.0139
0.0055
0.1025
0.1662
0.1039
*
0.0332
0.0014
*
*
*
*
0.0530
0.1292
*
0.2733
*
*
*
*
*
0.0148
0.0064
0.0932
0.2627
0.1292
*
0.0318
0.0042
0.0021
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
101
5 Chapter 5 Experiments and results
Population data
Allele
22.1
22.2
22.3
23.0
23.1
23.2
20.2
20.3
21.0
21.2
21.3
22.0
23.3
24.0
24.1
24.2
24.3
25.0
18.2
19.0
19.2
19.3
20.0
20.1
16.1
16.2
17.0
17.2
18.0
102
*
0.0044
0.0015
0.1696
*
0.0015
*
*
0.1228
*
*
0.1988
African
American
(n = 342)
*
0.0015
*
0.0015
0.0015
0.0175
0.0512
0.0029
*
0.0541
*
*
0.1330
*
*
*
0.1184
Asian (n = 97)
U.S. Caucasian
(n = 361)
*
0.0125
*
0.1524
*
0.0028
*
*
0.1787
0.0055
*
0.2050
*
0.1343
*
0.0014
*
0.0789
*
0.0499
*
*
0.1233
*
*
*
*
*
0.0249
0.2062
*
*
*
*
*
*
*
0.1031
*
*
0.2423
*
0.1495
*
0.0052
*
0.0722
*
0.0567
*
*
0.0876
*
*
*
0.0103
*
0.0258
U.S. Hispanic
(n = 236)
*
0.0042
*
0.1208
*
0.0042
*
*
0.1525
*
*
0.1653
*
0.1419
*
*
*
0.1186
*
0.0805
*
*
0.0847
*
*
*
0.0021
*
0.0127
VeriFiler
™
Express PCR Amplification Kit User Guide
Allele
32
32.2
33.2
34.2
41.2
42.2
29.1
29.2
30.0
30.2
31.0
31.2
43.2
44.2
45.2
46.2
47.2
48.2
26.2
27.0
27.2
28.0
28.2
29.0
25.1
25.2
25.3
26.0
26.1
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
*
*
*
*
*
*
*
*
0.0015
0.0015
*
0.0015
*
*
*
*
*
*
African
American
(n = 342)
*
0.0015
*
0.0702
*
*
0.0234
*
0.0146
*
0.0058
Asian (n = 97)
U.S. Caucasian
(n = 361)
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
0.0042
*
*
*
*
*
*
*
0.0263
*
*
*
*
*
*
*
*
*
*
*
*
*
0.0052
*
*
*
*
*
*
0.0052
*
*
*
*
*
*
*
0.0309
*
U.S. Hispanic
(n = 236)
*
*
*
*
*
*
*
*
0.0021
*
*
*
*
*
*
*
*
*
*
0.0445
*
0.0021
*
0.0021
*
*
*
0.0614
*
103
5 Chapter 5 Experiments and results
Population data
Allele
15.0
16.0
17.0
18.0
Penta E
4.0
12.0
12.2
13.0
13.4
14.0
14.1
10.0
11.0
11.2
9.0
9.2
9.4
49.2
50.2
51.2
Penta D
2.2
7.0
7.4
8.0
3.2
5.0
6.0
104
0.1804
*
0.1031
*
0.0103
*
0.0103
*
*
*
0.3144
*
*
0.1856
0.1804
*
*
*
*
0.0103
*
*
0.0052
*
African
American
(n = 342)
*
*
*
Asian (n = 97)
*
*
*
U.S. Caucasian
(n = 361)
*
*
*
U.S. Hispanic
(n = 236)
*
*
*
0.1082
*
0.0833
0.0015
0.0249
*
0.0044
*
0.0015
*
0.1681
*
*
0.0994
0.1798
*
0.1140
0.0088
0.0439
0.0102
0.0439
*
0.1082
*
0.2327
*
0.1967
*
0.0609
*
0.0097
0.0028
0.0014
*
0.2216
*
*
0.1150
0.1260
*
0.0042
*
*
0.0042
0.0042
*
0.0208
*
0.1631
*
0.1441
*
0.0720
*
0.0106
0.0042
0.0021
*
0.2415
*
*
0.1568
0.1568
*
0.0169
0.0021
0.0064
0.0021
0.0021
*
0.0191
*
VeriFiler
™
Express PCR Amplification Kit User Guide
Allele
16.4
17.0
17.4
18.0
18.4
19.0
14.2
15.0
15.2
15.4
16.0
16.2
19.4
20.0
21.0
22.0
23.0
24.0
10.0
11.0
11.4
12.0
13.0
14.0
5.0
6.0
7.0
8.0
9.0
VeriFiler
™
Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
*
0.0439
*
0.0161
*
0.0073
*
0.0556
*
*
0.0409
*
African
American
(n = 342)
0.0950
0.0015
0.1038
0.1667
0.0512
0.0468
0.0643
*
0.1287
0.1038
0.0687
*
0.0044
*
*
*
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
*
0.0485
*
0.0332
*
0.0152
*
0.0429
*
0.0014
0.0512
*
*
0.0097
0.0028
0.0014
*
0.0014
0.0859
0.0873
*
0.1994
0.0859
0.0623
0.0762
*
0.1690
0.0139
0.0125
*
0.0825
*
0.0773
*
0.0412
*
0.1134
*
*
0.0567
*
0.0103
0.0206
0.0206
0.0309
0.0206
*
0.0619
0.1598
*
0.0928
0.0515
0.0515
0.0722
*
*
0.0052
0.0258
U.S. Hispanic
(n = 236)
*
0.0551
*
0.0339
*
0.0212
*
0.0911
*
0.0021
0.0614
*
*
0.0212
0.0064
0.0021
0.0064
*
0.0847
0.0742
*
0.1737
0.0932
0.0720
0.0360
*
0.1186
0.0254
0.0169
105
5 Chapter 5 Experiments and results
Population data
Allele
25.0
26.0
27.0
TH01
10.3
11.0
12.0
TPOX
13.0
13.3
7.0
7.1
7.3
4.0
5.0
6.0
9.0
9.3
10.0
7.3
8.0
8.3
6.0
6.3
7.0
3.0
4.0
5.0
5.3
106
African
American
(n = 342)
0.0015
*
*
Asian (n = 97)
0.0052
*
*
U.S. Caucasian
(n = 361)
*
*
*
U.S. Hispanic
(n = 236)
0.0042
*
*
*
0.1959
*
0.1594
0.0965
0.0044
*
*
0.0044
*
0.1316
*
0.4079
*
*
*
*
*
*
0.0722
*
0.4433
0.0412
0.0052
0.1701
*
0.2680
*
*
*
*
*
*
*
*
*
*
0.0956
*
0.1191
0.3449
0.0083
*
*
0.0014
*
0.2355
*
0.1939
*
0.0014
*
*
*
*
0.0911
*
0.1462
0.2182
0.0085
*
*
*
*
0.2394
*
0.2966
*
*
*
*
*
*
*
0.0892
0.0175
*
*
*
*
*
*
*
*
*
0.0014
0.0014
*
*
*
*
*
0.0085
0.0064
*
*
VeriFiler
™
Express PCR Amplification Kit User Guide
Allele vWA
15.0
15.2
16.0
17.0
18.0
18.2
10.0
11.0
12.0
13.0
14.0
19.0
20.0
21.0
22.0
23.0
11.0
12.0
13.0
14.0
15.0
16.0
8.0
9.0
10.0
10.1
10.3
Chapter 5 Experiments and results
Population data
5
0.1915
*
0.2500
0.2354
0.1491
*
*
0.0029
0.0015
0.0088
0.0804
0.0629
0.0161
0.0015
*
*
African
American
(n = 342)
0.3670
0.1959
0.0863
*
*
0.2164
0.0263
0.0015
*
*
*
0.2990
0.0464
*
*
*
*
0.5515
0.0773
0.0258
*
*
Asian (n = 97)
U.S. Caucasian
(n = 361)
0.2521
0.0416
0.0014
*
*
*
0.5249
0.1274
0.0499
*
*
U.S. Hispanic
(n = 236)
0.2542
0.1038
*
*
*
*
0.4852
0.0932
0.0487
*
*
0.0206
*
0.1392
0.3144
0.2062
*
*
*
*
*
0.1959
0.1082
0.0155
*
*
*
0.1053
*
0.2008
0.2839
0.2022
*
*
*
0.0014
0.0014
0.0928
0.1039
0.0069
0.0014
*
*
0.1441
*
0.2839
0.2458
0.1801
*
*
0.0021
*
*
0.0805
0.0508
0.0106
0.0021
*
*
VeriFiler
™
Express PCR Amplification Kit User Guide 107
5 Chapter 5 Experiments and results
Population data
108
Allele
24.0
25.0
African
American
(n = 342)
*
*
Asian (n = 97)
*
*
U.S. Caucasian
(n = 361)
*
*
U.S. Hispanic
(n = 236)
*
*
D5S818
D6S1043
D7S820
D8S1179
FGA
Penta D
Penta E
TH01
TPOX vWA
Combined
CSF1PO
D10S1248
D12S391
D13S317
D16S539
D18S51
D19S433
D1S1656
D21S11
D22S1045
D2S1338
D2S441
D3S1358
Table 7 Probability of identity (PI) values for the VeriFiler ™ Express kit STR loci
Locus
0.0424
0.0566
0.0967
0.0307
0.1027
0.1350
0.0984
0.0863
0.0444
0.0686
0.0873
0.0456
0.0676
0.0721
0.0266
0.0992
0.0405
0.0403
0.0745
0.0124
0.1405
0.2233
0.0759
4.693 × 10 −28
0.0212
0.0937
0.1067
0.1012
0.0266
0.0863
0.0650
0.0289
0.0257
0.0169
0.1017
0.0892
0.0615
2.546 × 10 −30
African
American
(n = 342)
0.0828
0.0666
0.0346
0.1339
0.0706
0.0268
0.0358
0.0310
0.0395
0.0538
Asian (n = 97)
Caucasian
(n = 361)
Hispanic
(n = 236)
0.0188
0.0497
0.1221
0.0257
0.0872
0.0768
0.1291
0.0979
0.0219
0.0767
0.0948
0.0284
0.0844
0.1437
0.0517
0.0597
0.0607
0.0356
0.0542
0.0211
0.0919
0.1783
0.0639
1.092 × 10 −28
0.0242
0.0448
0.1533
0.0285
0.1035
0.0942
0.1271
0.1010
0.0268
0.0495
0.0742
0.0266
0.0661
0.1271
0.0285
0.0808
0.0611
0.0261
0.0460
0.0149
0.0853
0.1476
0.0710
2.947 × 10 −29
VeriFiler
™
Express PCR Amplification Kit User Guide
Probability of paternity exclusion observation
Chapter 5 Experiments and results
Population data
5
The following table shows the Probability of paternity exclusion (PE) values of the
VeriFiler ™ Express kit STR loci individually and combined.
The PE value is the probability, averaged over all possible mother-child pairs, that a random alleged father will be excluded from paternity after DNA typing using the
VeriFiler ™ Express kit STR loci (Chakraborty, Stivers, and Zhong, 1996).
Table 8 Probability of paternity exclusion values for the VeriFiler ™ loci
Express kit STR
Locus
D2S1338
D2S441
D3S1358
D5S818
D6S1043
D7S820
D8S1179
FGA
Penta D
Penta E
TH01
TPOX
CSF1PO
D10S1248
D12S391
D13S317
D16S539
D18S51
D19S433
D1S1656
D21S11
D22S1045
0.6976
0.6994
0.5908
0.8347
0.4535
0.3428
0.7382
0.5259
0.4613
0.5973
0.7562
0.5323
0.5351
0.5619
0.6849
0.6068
0.5580
0.6807
0.6135
0.6916
0.6440
0.5350
0.6186
0.7457
0.7607
0.8069
0.5259
0.5557
0.7832
0.5471
0.5158
0.5293
0.7559
0.5615
African
American
(n = 342)
0.5724
0.6138
0.7214
0.4687
0.6018
0.7555
0.7167
0.7356
0.7028
0.6524
Asian (n = 97)
Caucasian
(n = 361)
Hispanic
(n = 236)
0.6302
0.7173
0.6509
0.7830
0.5476
0.3988
0.7606
0.5616
0.5842
0.4503
0.6602
0.6330
0.4708
0.5346
0.7794
0.5877
0.5436
0.7481
0.5698
0.7957
0.6661
0.4888
0.6303
0.7585
0.6782
0.8184
0.5624
0.4452
0.7474
0.5250
0.5427
0.4825
0.7475
0.5761
0.4756
0.5286
0.7559
0.6657
0.5918
0.7563
0.6172
0.7679
0.6829
0.4388
VeriFiler
™
Express PCR Amplification Kit User Guide 109
5 Chapter 5 Experiments and results
Population data vWA
Combined
0.6283
0.999999999984
527
0.5872
0.999993438259
661
0.6206
0.999989850701
339
0.6009
0.999985788729
133
110 VeriFiler
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A
Troubleshooting
Observation
Faint or no signal from both the
DNA Control 007 and the DNA test samples at all loci
Faint no signal Control and samples all loci
Possible cause
The incorrect volume of Master
Mix or Primer Set was used.
The DNA Polymerase was not activated.
The Master Mix was not vortexed thoroughly before aliquoting.
The Primer Set was exposed to too much light.
Evaporation.
Recommended action
Use the correct volume of Master Mix or
Primer Set.
Repeat the amplification with an initial hold at
95°C for 1 minute.
Vortex the Master Mix thoroughly.
Replace the Primer Set and store it protected from light.
Ensure that the plate is properly sealed with film and that a compression pad was used with the GeneAmp ™ PCR System 9700. (A compression pad should not be used with other validated thermal cyclers.)
The thermal cycler malfunctioned.
Incorrect thermal cycler conditions were used.
See the thermal cycler user manual and check the instrument calibration.
Use correct thermal cycler conditions.
A MicroAmp ™ base was used with a tray/retainer set and tubes in GeneAmp ™ PCR
System 9700.
The tubes or plate were not seated tightly in the thermal cycler during amplification.
The wrong PCR reaction tubes or plate were used.
Remove the MicroAmp
Push the tubes or plate firmly into the block after first cycle.
™ base.
Use MicroAmp ™ Reaction Tubes with Caps or the MicroAmp ™ Optical 96 ‑well Reaction Plate for the GeneAmp ™ PCR System 9700 or Veriti ™
Thermal Cycler.
Use correct genetic analyzer settings.
Insufficient PCR product was electrokinetically injected.
Degraded formamide was used. Check the storage of formamide. Do not thaw and refreeze multiple times. Try Hi ‑Di ™
Formamide.
The sample punch location was not optimal.
For blood samples on treated paper, punch in the center of the blood stain.
For buccal samples on treated paper, punch in the center of the buccal transfer or punch in the optimal location you have previously determined.
VeriFiler
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Express PCR Amplification Kit User Guide 111
A Appendix A Troubleshooting
Population data
Observation
Faint no signal Control and samples all loci
Positive signal from DNA
Control 007 but partial or no signal from DNA test samples
More than two alleles present at a locus
Possible cause
Insufficient volume of swab lysate was added to the reaction.
Proper low-TE buffer not used for treated paper substrates.
The test sample was diluted in the wrong buffer (for example, a TE buffer with an incorrect
EDTA concentration).
The sample punch location was not optimal.
Recommended action
For buccal samples collected with the Bode
Buccal DNA Collector ™ device, punch from near the tip of the collector.
Ensure the swab heads are incubated for
20 minutes in 400 µL of Prep ‑n‑Go ™ Buffer.
Prepare low-TE buffer. See “Prepare low-TE buffer“ on page 18.
Redilute DNA using low-TE buffer (with 0.1 mM
EDTA).
For blood samples on treated paper, punch in the center of the blood stain.
For buccal samples on treated paper, punch in the center of the buccal transfer or punch in the optimal location you have previously determined.
For buccal samples collected with the Bode
Buccal DNA Collector ™ device, punch from near the tip of the collector.
Ensure the swab heads are incubated for
20 minutes in 400 µL of Prep ‑n‑Go ™ Buffer.
Prepare low-TE buffer.
Insufficient lysis of the swab head occurred.
Proper low-TE buffer not used for treated paper substrates.
Exogenous DNA is present in the sample.
Stutter product (–1 repeat unit position) was amplified.
Triallelic patterns occur.
Incomplete 3´ A base addition
(n−1 nt position) occured.
Use appropriate techniques to avoid introducing foreign DNA during laboratory handling.
See Chapter 5, “Experiments and results“
Confirm the triallelic pattern per the laboratory's guidelines.
Include the final extension step of 60°C for
10 minutes in the PCR.
Incubate the amplification products at 60°C for an additional 10 minutes.
Ensure the cycle number is optimized. Use fewer PCR cycles or interpret the off-scale data according to your laboratory procedure.
The signal exceeds the dynamic range of the instrument and is causing signal "pull-up" into adjacent channels.
Poor spectral separation occurred.
The double-stranded DNA was not completely denatured.
Perform a spectral calibration.
Confirm that Filter Set J6 modules are installed and used for analysis.
Use the recommended amount of Hi ‑Di ™
Formamide and heat the sample plate at 95°C for 3 minutes.
112 VeriFiler
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Appendix A Troubleshooting
Population data
A
More than two alleles present at a locus
Observation
Poor peak height balance
Some but not all loci visible on electropherogram of DNA Test
Samples
STR profiles contain many offscale alleles
Data collected on the 3730 instrument with POP-7 polymer fails sizing
Data collected on the 3730 instrument with POP-7 ™ polymer: the D2S441 and
D1S1656 markers in some allelic ladder samples fail base-pair spacing quality assessment
™
Possible cause
Contamination was carried over from the disc punching tool.
Recommended action
Clean the disc punching tool thoroughly. If necessary, include a blank punch step in between the sample punches.
Use correct thermal cycler conditions.
Incorrect thermal cycler conditions were used.
Expected amplification pattern observed in the TAZ channel.
™ dye
Peak heights are typically elevated at
D22S1045 for buccal samples. Follow laboratory guidelines for evaluation of the generated results.
Use a 1.2 mm disc.
The punched disc you used was too large.
Insufficient lysis of the swab head occurred.
The PCR reaction volume you used is lower than the volume required for the amplification.
The PCR cycle number used was too high.
Blood samples: Too much liquid blood was spotted onto the paper substrate.
The 60-bp size-standard peak is occasionally obscured by the primer peak.
Data was analyzed using the
Light setting for Smoothing.
Ensure the swab heads are incubated for
20 minutes in 400 µL of Prep ‑n‑Go ™ Buffer.
Use the correct PCR reaction volume: 25 μL
Perform a sensitivity experiment to determine the optimal PCR cycle number based on the sample type.
Spot <100 µL of liquid blood per sample area.
Reinject samples that fail to recognize the
60 base-pair peak.
Use the 80 to 460 bp size-standard definition after performing appropriate validation studies
(as a general rule, the 60 base-pair peak is not required for accurate fragment sizing using the
3rd Order Least Squares sizing method).
For more information, see the GeneMapper
™
ID ‑X Software v1.4 New Features and
Installation Procedures User Bulletin
(Pub. No. 4477684), “Known issues: 3730 DNA
Analyzer sizing failures”.
Use the None setting for Smoothing after performing appropriate validation studies.
For more information, see the GeneMapper
™
ID ‑X Software v1.4 New Features and
Installation Procedures User Bulletin
(Pub. No. 4477684), “Known issues: 3730 DNA
Analyzer sizing failures”.
VeriFiler
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Express PCR Amplification Kit User Guide 113
B
Materials required but not supplied
Unless otherwise indicated, all materials are available through thermofisher.com.
MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.
STR kit
Item
VeriFiler ™ Express PCR Amplification Kit, 200-reaction kit
GeneScan ™ 600 LIZ ™ Size Standard v2.0, 2 × 200 µL
IMPORTANT! Do not use GeneScan
GeneScan ™ 500 LIZ ™
™ 350 ROX ™ , GeneScan ™ 500 ROX
Size Standards with this kit.
™ , or
Hi ‑Di ™ Formamide, 25 ‑mL
Source
A32014
4408399
4311320
Sample preparation required materials
Treated paper substrate
Item
Collection system: NUCLEIC-CARD ™ system or Whatman FTA ™
NUCLEIC-CARD ™ Sample Collection Device
NUCLEIC-CARD ™ matrix, 1 spot
NUCLEIC-CARD ™ COLOR matrix, 1 spot
Whatman ™ FTA ™ Classic Cards
Whatman ™ EasiCollect ™ system
Sample preparation:
Prep ‑n‑Go ™ Buffer (for use with untreated paper substrates)
Low-TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0)
Punch tool:
Harris Micro-Punch ™ tool, 1.2
‑mm
Source
4473980
4474001
4473974
MLS
MLS
4467079
Teknova T0223
MLS
114 VeriFiler
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Appendix B Materials required but not supplied
Sample preparation required materials
B
Item
BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a
1.2
‑mm punch head
BSD1000-GenePunch Automated Dried Sample Punch Instrument with a
1.2
‑mm punch head
Untreated paper substrate
Item
Collection system: Bode or paper
Buccal DNA Collector ™ Device
903 paper
Punch tool:
Harris Micro-Punch ™ tool, 1.2
‑mm
BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a
1.2
‑mm punch head
BSD1000-GenePunch Automated Dried Sample Punch Instrument with a
1.2
‑mm punch head
Swab substrate
Item
Collection system: 4N6FLOQSwabs ™ reference collection devices
4N6FLOQSwabs ™ Lollipop Swab in peelpouch
4N6FLOQSwabs ™ , Regular size tip in peelpouch
4N6FLOQSwabs ™ , Regular size tip in plain tube
4N6FLOQSwabs ™ , Regular size tip in plastic tube with Active Drying System
4N6FLOQSwabs ™ peelpouch
, Regular size tip plus 2 ‑mL Eppendorf ™ cuvette in
4N6FLOQSwabs ™ , Regular size tip plus 2 ‑mL Eppendorf ™ cuvette with evaporation duct in peelpouch
4N6FLOQSwabs ™ Dual Swab Regular size tip in plain tube
4N6FLOQSwabs ™ , Buccal shape tip in plastic tube with Active Drying System
Cotton swabs
Sample preparation
Prep ‑n‑Go ™ Buffer (for use with buccal swab substrates)
Source
Contact your local sales office.
Source
Contact Bode Cellmark Forensics
MLS
MLS
Contact your local sales office.
Source
4473997
4473979
4479433 [1]
4479439 [1]
4479431 [1]
4479440 [1]
4479432 [1]
4479436 [1]
MLS
4471406
VeriFiler
™
Express PCR Amplification Kit User Guide 115
B Appendix B Materials required but not supplied
Thermal cycler required materials
Item Source
Heated lysis protocol only: 1.5 mL tube format or 96-well deep-well plate format
1.5 mL tube format
1.5 mL tubes
Oven
MLS
VWR ™ Scientific dry heat block or equivalent
96-well deep-well plate format
PrepFiler ™ 96-Well Processing Plates
Robbins Scientific ™ Model 400 Hybridization Incubator or equivalent
Agilent ™ Benchtop Rack for 200 µL Tubes/V Bottom Plates (metal) or equivalent
4392904
MLS
Agilent Technologies 410094
IMPORTANT! Do not use a plastic plate adaptor.
[1] To order, contact your local Thermo Fisher Scientific sales representative.
Thermal cycler required materials
ProFlex
™
PCR System
Item
ProFlex ™ 96 ‑well PCR System
ProFlex ™ 2 × 96 ‑well PCR System
ProFlex ™ 3 × 32 ‑Well PCR System
GeneAmp
™
PCR System 9700
Item
GeneAmp ™ PCR System 9700, 96-Well Silver
GeneAmp ™ PCR System 9700, 96-Well Gold-Plated
Silver 96-Well Sample Block
Gold-Plated 96-Well Block
Source
4484075
4484076
4484073
Source
N8050001
4314878
N805021
4314443
116 VeriFiler
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Appendix B Materials required but not supplied
Genetic analyzer required materials
B
Genetic analyzer required materials
3500 Series Genetic Analyzer
Item
3500/3500xL Data Collection Software v3 (RUO)
3500/3500xL Data Collection Software v2 (RUO)
HID Updater 3500 Data Collection Software v2
Anode buffer container (ABC)
Cathode buffer container (CBC)
POP-4 ™ Polymer (960 samples) for 3500/3500xL Genetic Analyzers
POP-4 ™ Polymer (384 samples) for 3500/3500xL Genetic Analyzers
DS-36 Matrix Standard Kit (Dye Set J6)
Conditioning reagent
8-Capillary array, 36 cm for 3500 Genetic Analyzers
24-Capillary array, 36 cm for 3500xL Genetic Analyzers
96-well retainer & base set (Standard) 3500/3500xL Genetic Analyzers
8-Tube retainer & base set (Standard) for 3500/3500xL Genetic Analyzers
8-Strip Septa for 3500/3500xL Genetic Analyzers
96-Well Septa for 3500/3500xL Genetic Analyzers
Septa Cathode Buffer Container, 3500 series
[1]
[2]
Contact your Thermo Fisher Scientific HID representative.
Contact your Thermo Fisher Scientific HID representative.
3130 Series Genetic Analyzer
Item
3130 Data Collection Software v4
3130 xl Data Collection Software‑v4
3130/3730 Data Collection Software ‑v4 6‑Dye Module v1
96 ‑Well Plate Septa
Reservoir Septa
3100/3130 xl Genetic Analyzer Capillary Array, 36‑cm
POP-4 ™ Polymer for 3130/3130 xl Genetic Analyzers
Running Buffer, 10✕
VeriFiler
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Express PCR Amplification Kit User Guide
Source
A26287 [1]
4475183 [2]
4480670
4393927
4408256
4393710
4393715
4425042
4393718
4404683
4404687
4410228
4410231
4410701
4412614
4410715
Source
4475105
4475126
4480670
4315933
4315932
4315931
4352755
402824
117
B Appendix B Materials required but not supplied
Analysis software required materials
Item
DS ‑36 Matrix Standard Kit (Dye Set J6)
MicroAmp ™ Optical 96-Well Reaction Plate
3730 Series Genetic Analyzer
Item
3730/3730 xl Data Collection Software‑v4
3130/3730 Data Collection Software v4 6 ‑Dye Module v1
96 ‑Well Plate Septa
Reservoir Septa
3730 DNA Analyzer 48-Capillary Array, 36-cm
3730 xl DNA Analyzer 48-Capillary Array, 36-cm
POP-7 ™ Polymer for 3730/3730 xl DNA Analyzers
Running Buffer, 10✕
DS ‑36 Matrix Standard Kit (Dye Set J6)
MicroAmp ™ Optical 96-Well Reaction Plate
250 ‑μL Glass Syringe (array-fill syringe)
5.0
‑mL Glass Syringe (polymer-reserve syringe)
Analysis software required materials
GeneMapper
™
ID ‑X Software
Item
GeneMapper ™ ID ‑X Software v1.5 Full Installation
GeneMapper ™ ID ‑X Software v1.5 Client Installation
GeneMapper ™ ID ‑X Software v1.4 Full Installation
GeneMapper ™ ID ‑X Software v1.4 Client Installation
118 VeriFiler
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Source
4425042
N8010560
Source
4475154
4480670
4315933
4315932
4331247
4331244
4335611
4335613
4425042
N8010560
4304470
628 ‑3731
Source
A27884
A27886
4479707
4479711
Miscellaneous required materials
Plates and tubes
Item
MicroAmp ™ 96-Well Tray
MicroAmp ™ Reaction Tube with Cap, 0.2 mL
MicroAmp ™ 8-Tube Strip, 0.2 mL
MicroAmp ™ Optical 8-Cap Strips
MicroAmp ™ 96-Well Tray/Retainer Set
MicroAmp ™ 96-Well Base
MicroAmp ™ Clear Adhesive Film
MicroAmp ™ Optical Adhesive Film
MicroAmp ™ Optical 96-Well Reaction Plate
Laboratory supplies
Item
Various procedures
Aerosol resistant pipette tips
Microcentrifuge tubes
Pipettors
Tape, labeling
Tube, 50-mL Falcon ™
Tube decapper, autoclavable
Deionized water, PCR grade
Vortex
[1] Major laboratory supplier
Appendix B Materials required but not supplied
Miscellaneous required materials
B
Source
MLS [1]
MLS
MLS
MLS
MLS
MLS
MLS
MLS
Source
N8010541
N8010540
N8010580
N8010535
403081
N8010531
4306311
4311971
N8010560
VeriFiler
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C
Common Callouts and Arrows
Plate layouts
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
■
■
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
Example PCR plate layout
1 1 1
1
The following layout is recommended for use with the sensitivity experiment in the
Perform PCR chapter. Create 3 identical plates for amplification at 4 different cycle numbers.
120 VeriFiler
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Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
Example electrophoresis plate layout
1 1 1
1
Appendix C Plate layouts
Example electrophoresis plate layout
The following layout is recommended for use with the sensitivity experiment in the
Perform PCR chapter.
C
VeriFiler
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Express PCR Amplification Kit User Guide 121
D
PCR work areas
■
■
■
Work area setup and lab design
Many resources are available for the appropriate design of a PCR laboratory. If you are using this kit for:
• Forensic DNA testing, see "Forensic Laboratories: Handbook for Facility
Planning, Design, Construction, and Moving", National Institute of Justice, 1998
• Parentage DNA testing, see the "Guidance for Standards for Parentage
Relationship Testing Laboratories", American Association of Blood Banks, 7th edition, 2004
The sensitivity of this kit (and other PCR-based tests) enables amplification of minute quantities of DNA, necessitating precautions to avoid contamination of samples yet to be amplified (Kwok and Higuchi, 1989).
Process samples carefully to prevent contamination by human DNA. Wear gloves at all times and change them frequently. Close sample tubes when not in use. Limit aerosol dispersal by handling sample tubes and reagents carefully.
Note: We do not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology.
PCR setup work area materials
IMPORTANT! Do not remove these items from the PCR Setup Work Area.
• Calculator
• Gloves, disposable
• Marker pen, permanent
• Microcentrifuge
• Microcentrifuge tubes, 1.5-mL, or 2.0-mL, or other appropriate nuclease-free tube
(for master mix preparation)
• Microcentrifuge tube rack
• Pipette tips, sterile, disposable hydrophobic filter-plugged
• Pipettors
122 VeriFiler
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Appendix D PCR work areas
Amplified DNA work area
D
• Tube decapper, autoclavable
• Vortex
Amplified DNA work area
IMPORTANT! Place the thermal cyclers in the Amplified DNA Work Area.
Use only the validated thermal cyclers listed in “Instrument and software compatibility“ on page 14.
VeriFiler
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Express PCR Amplification Kit User Guide 123
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document.
· Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicable Safety Data
Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see the “Documentation and
Support” section in this document.
124 VeriFiler
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Safety
Chemical safety
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions:
· Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and
Support” section in this document.
· Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing).
· Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood).
· Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS.
· Handle chemical wastes in a fume hood.
· Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)
· After emptying a waste container, seal it with the cap provided.
· Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory.
· Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations.
· IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply.
VeriFiler
™
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Safety
Biological hazard safety
Biological hazard safety
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment.
· U.S. Department of Health and Human Services, Biosafety in Microbiological
and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)
21-1112, Revised December 2009; found at: www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf
· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at: www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
126 VeriFiler
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E
Documentation and support
■
■
■
Related documentation
Document title
STR kits
VeriFiler ™ Express Kit – PCR and CE Quick Reference
VeriFiler ™ Express Kit – PCR Setup – Swab Substrate Quick Reference
VeriFiler ™ Express Kit – PCR Setup – Treated Paper Substrate Quick Reference
VeriFiler ™ Express Kit – PCR Setup – Untreated Paper Substrate Quick Reference
Thermal cyclers
ProFlex ™ PCR System User Guide
GeneAmp ™ PCR System 9700 Base Module User Manual
3500 Series Genetic Analyzer
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v1 User Guide
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v2 User Guide
HID Updater 3500 Data Collection Software v2.0 User Bulletin
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software 3 User Guide
3500 Series Data Collection Software v3 User Bulletin: New Features and HID Validation
Summary
3500 Series Data Collection Software v3.1 User Bulletin: New Features and HID Validation
Summary
3130 Series Genetic Analyzer
3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide
3130/3130xl Genetic Analyzers Using Data Collection Software v3.0 User Bulletin
3130/3130xl Genetic Analyzers Getting Started Guide
Pub. No.
100043592
100043591
100043589
100043590
MAN0007697
4303481
4401661
4476988
NA
100025036
MAN0010812
MAN0014110
4352716
4363787
4352715
VeriFiler
™
Express PCR Amplification Kit User Guide 127
E Appendix E Documentation and support
Related documentation
Document title
3130/3130xl Genetic Analyzers Quick Reference Card
3130/3130xl Genetic Analyzers AB Navigator Software Administrator Guide
3730 Series Genetic Analyzer
3730/3730xl Genetic Analyzer Getting Started Guide
GeneMapper ™ ID ‑X Software all versions
GeneMapper ™ ID ‑X Software Bin Overlap User Bulletin
GeneMapper ™ ID ‑X Software v1.0
GeneMapper ™ ID ‑X Software v1.0 Getting Started Guide— Basic Features
GeneMapper ™ ID ‑X Software v1.0 Quick Reference— Basic Features
GeneMapper ™ ID ‑X Software v1.0 Installation Guide
GeneMapper ™ ID ‑X Software v1.0 Administrator Guide
GeneMapper ™ ID ‑X Software v1.0 Reference Guide
GeneMapper ™ ID ‑X Software v1.1
GeneMapper ™ ID ‑X Software v1.1 Getting Started Guide— Mixture Analysis Tool
GeneMapper ™ ID ‑X Software v1.2
GeneMapper ™
Bulletin
ID ‑X Software v1.2 Verification Experiments and Installation Procedures User
GeneMapper ™ ID ‑X Software v1.2 Quick Reference— Mixture Analysis Tool
GeneMapper ™ ID ‑X Software v1.2 Reference Guide
GeneMapper ™ ID ‑X Software v1.3
GeneMapper ™
Bulletin
ID ‑X Software v1.3 Verification Experiments and Installation Procedures User
GeneMapper ™ ID ‑X Software v1.4
GeneMapper ™ ID ‑X Software v1.4 New Features and Installation Procedures User Bulletin
GeneMapper ™ ID ‑X Software v1.5
GeneMapper ™ ID ‑X Software v1.5 New Features and Verification User Bulletin
GeneMapper ™ ID ‑X Software v1.5 Getting Started Guide— Basic Features
GeneMapper ™ ID ‑X Software v1.5 Quick Reference— Basic Features
GeneMapper ™ ID ‑X Software v1.5 Getting Started Guide— Mixture Analysis Tool
GeneMapper ™ ID ‑X Software v1.5 Quick Reference— Mixture Analysis Tool
GeneMapper ™ ID ‑X Software v1.5 Installation Guide
128
4477684
100031708
100031701
100031702
100031704
100031705
100031706
VeriFiler
™
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Pub. No.
4362825
4359472
4359476
100029546
4375574
4375670
4476603
4376327
4375671
4396773
4462639
4426482
4426481
4470483
Appendix E Documentation and support
Customer and technical support
E
Document title
GeneMapper ™ ID ‑X Software v1.5 Administrator Guide
GeneMapper ™ ID ‑X Software v1.5 Reference Guide
Pub. No.
100031703
100031707
Customer and technical support
For support:
• In North America—Send an email to [email protected], or call 888-821-4443 option 1.
• Outside North America—Contact your local support office.
• For latest services and support information for all locations, go to
thermofisher.com/support.
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life
Technologies' website at www.thermofisher.com/us/en/home/global/
terms-and-conditions.html. If you have any questions, please contact Life
Technologies at www.thermofisher.com/support.
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130
References
Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994.
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Index
6-dye
spectral calibration 29, 33, 36
A
accuracy and reproducibility 64
allelic ladder, requirements for electrophoresis 27
allelic ladder, volume per reaction 36
B
C
characterization of loci, validation 81
D
DS-36 matrix standard 29, 33, 36 dye set for 6-dye samples 29, 33, 36
E electrophoresis
data collection software 28, 31
references 28, 31 run module 28, 31
setup of the 3130 and 3130xl instruments 31
setup of the 3500 and 3500xL instruments 28
G
GeneScan 600 LIZ Size Standard v2.0 52
GeneScan size standard, about 11
H
I
import panels, bins, and marker stutter 41
instrument and software compatibility 14
L
LIZ size standard
M
P panels
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Index
PCR
R
run module for electrophoresis, 3500 and 3500xL in-
run module, electrophoresis, 3130 and 3130xl 31
S
spectral calibration 29, 33, 36
swab
sample preparation guidelines 23
T
thermal cyclers
treated paper
sample preparation guidelines 18
U
V
W
136 VeriFiler
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Key features
- 6-dye STR multiplex assay
- Amplifies 25 loci
- Optimized for paternity and single-source samples
- Direct amplification from blood and buccal samples on treated or untreated paper substrates and buccal samples on swabs
- Fast amplification time (approx 45 min)
- Improved primer synthesis and purification
- Clean electrophoretic backgrounds
- Validated for use with 3500, 3130, and 3730 series Genetic Analyzers
- Compatible with GeneMapper ID-X software
