cms_041201

AmpFℓSTR™ Identifiler™ PCR Amplification Kit
USER GUIDE
for use with:

200 reaction kit
Catalog Number 4322288

Publication Number 4323291
Revision K
For Forensic or Paternity Use Only.

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, THERMO FISHER SCIENTIFIC INC. 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. 4323291

Revision Date Description
K 24 August 2018 Updated branding and trademarks, no technical changes.
J August 2012 Add validation experiments and results for primer manufacturing process
improvements and buffer and enzyme kit component changes.
H May 2012 •
™
Remove Mac OS procedures.
• Add 3100, 3100-Avant, 3130, 3130xl, 3500, 3500xL Genetic Analyzer
information.
Add GeneMapper ID Software and GeneMapper ID‑X Software
™ ™
•
information.
G March 2012 Change to limited licensing information.

F April 2011 Change to limited licensing information.
E September 2010 Change to limited licensing information.
D August 2006 Change to limited licensing information.
C May 2005 Change to limited licensing information.
™
B May 2001 Update GeneAmp PCR System 9700 part numbers.
A May 2001 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. TaqMan is a registered
trademark of Roche Molecular Systems, Inc., used under permission and license. AmpliTaq Gold is a trademark of Roche Molecular Systems, Inc.
Windows and Windows Vista are trademarks of Microsoft Corporation. EasiCollect is a trademark of Whatman Limited. FTA is a trademark of
Whatman International Limited. Whatman is a trademark of GE Healthcare Companies. Minitab is a trademark of Minitab, Inc. Mac OS is atrademark
of Apple, Inc.
©2018 Thermo Fisher Scientific Inc. All rights reserved.
Contents
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
■ CHAPTER 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
About the primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Loci amplified by the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Allelic ladder profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Control DNA 9947A profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Instrument and software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Data Collection and GeneMapper™ ID or ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Instrument and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
About multicomponent analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
How multicomponent analysis works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Standards for samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
■ CHAPTER 2 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Required user-supplied reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
DNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Importance of quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Methods of quantifying DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Amplification using bloodstained FTA™ cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 3

Contents
■ CHAPTER 3 Perform Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Allelic ladder requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Section 3.1 3100/3100-Avant and 3130/3130xl instruments . . . . . . . . . . . . . . . . . . . . . . . 27

Set up the 3100/3100-Avant or 3130/3130xl instrument for electrophoresis . . . . . . . . . . . . . . . . . . . 27
Reagents and parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Electrophoresis software setup and reference documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instrument . . . . . . . 28
Section 3.2 3500/3500xL Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Set up the 3500/3500xL instrument for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Prepare samples for electrophoresis on the 3500/3500xL instrument . . . . . . . . . . . . . . . . . . . . . . . 29
Section 3.3 310 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Set up the 310 instrument for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Prepare samples for electrophoresis on the 310 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
■ CHAPTER 4 Analyze Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Section 4.1 GeneMapper™ ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Overview of GeneMapper™ ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Set up GeneMapper™ ID Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
File names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Before using the software for the first time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Import panels and bins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
General tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Allele tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Peak Detector tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Peak Quality tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Quality Flags tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Create size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Analyze and edit sample files with GeneMapper™ ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

Contents
Section 4.2 GeneMapper™ ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Overview of GeneMapper™ ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Set up GeneMapper™ ID-X Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Panel, bin, and stutter file version . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Before using the software for the first time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Check panel, bin, and stutter file version . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Import panels, bins, and marker stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
General tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Allele tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Peak Detector tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Peak Quality tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
SQ & GQ tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Analyze and edit sample files with GeneMapper™ ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
■ CHAPTER 5 Experiments and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Section 5.1 Developmental Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
DAB 8.1.1 Developmental Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
PCR components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Thermal cycler parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
AmpliTaq Gold™ DNA Polymerase activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
DAB 8.1.2 Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Precision and size windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Extra Peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Causes of extra peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Stutter products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Addition of 3´ A nucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
DAB 8.1.2.1 Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Nature of the polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Contents
Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
DAB 8.1.2.2 Species Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
DAB 8.1.2.2 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Effect of DNA quantity on results and importance of quantitation . . . . . . . . . . . . . . . . . . . . . . . 85
Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
DAB 8.1.2.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Lack of amplification of some loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Differential and preferential amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Effect of inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Degraded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Multiplex amplifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Mixture studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
DAB 8.1.2.2 Mixture Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Mixed specimen studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Data interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Minimum sample requirement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
DAB 8.1.2.3 Population Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
DAB 8.1.2.3.1 Population Distribution Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Population samples used in these studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Allele frequencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Analyzing the four databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Low-frequency alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Mutation rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Estimating germline mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Additional mutation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Probability of identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Probability of paternity exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Section 5.2 Performance Verification After Primer Manufacturing Process

Improvements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Section 5.3 Performance Validation After Buffer and Enzyme Component

Replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Reproducibility study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Intracolor balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Stutter percentages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Sensitivity study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Mean referenced peak height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
DNA concentration and peak height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Contents
Allelic dropout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Genotype concordance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Inhibition study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Mean referenced peak height, minimum referenced peak height, and intracolor balance 114
Allelic dropout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
■ APPENDIX B Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Equipment and materials not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
■ APPENDIX C PCR Work Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
PCR setup work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
■ APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Specific chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Obtain SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Obtain support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Limited Product Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Contents

About This Guide
IMPORTANT! Before using this product, read and understand the information the
“Safety” appendix in this document.
Purpose
The Applied Biosystems AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide
provides information about the Applied Biosystems instruments, chemistries, and
software associated with the AmpFlSTR™ Identifiler™ PCR Amplification Kit.

About This Guide
Purpose

1 Overview
■ Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
■ Workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
■ Instrument and software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
■ Materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Product overview
Purpose The AmpFlSTR™ Identifiler™ PCR Amplification Kit is a short tandem repeat
(STR) multiplex assay that amplifies 15 tetranucleotide repeat loci and the
Amelogenin gender-determining marker in a single PCR amplification:
• All thirteen of the required loci for the Combined DNA Index System (CODIS)
(Budowle et al., 1998).
• Two additional loci, D2S1338 and D19S433.
Product The Identifiler™ Kit contains all the necessary reagents for the amplification of human
description genomic DNA.
The reagents are designed for use with the following Applied Biosystems instruments:
• 3100/3100-Avant Genetic Analyzer
• Applied Biosystems 3130/3130xl Genetic Analyzer
• Applied Biosystems 3500/3500xL Genetic Analyzer
• 310 Genetic Analyzer
• GeneAmp™ PCR System 9700 with the Silver 96-Well Block
• GeneAmp™ PCR System 9700 with the Gold-plated Silver 96-Well Block
• Veriti™ 96-Well Thermal Cycler
About the primers The Identifiler™ Kit employs the same primer sequences for all loci common to other
AmpFlSTR™ kits (except the MiniFiler™ kit). A degenerate unlabeled primer for the
D8S1179 locus was added to the AmpFlSTR™ Identifiler™ Primer Set in order to
address a mutation observed in a population of Chamorros and Filipinos from Guam
(Budowle et al.,1998b and Budowle et al., 2000). The addition of the degenerate primer
allows for the amplification of those alleles in samples containing this mutation
without altering the overall performance of the Identifiler™ Kit. The original
validation data in this guide (Section 5.1 on page 66) were generated prior to the
addition of the degenerate primer. Data showing equivalence with the degenerate
primer has been published.

Chapter 1 Overview
1 Product overview
Non-nucleotide linkers are used in primer synthesis for the following loci: CSF1PO,
D13S317, D16S539, D2S1338, and TPOX. For these primers, non-nucleotide linkers are
placed between the primers and the fluorescent dye during oligonucleotide synthesis
(Butler, 2005, Grossman et al., 1994, and Baron et al., 1996). Non-nucleotide linkers
enable reproducible positioning of the alleles to facilitate interlocus spacing. The
combination of a five-dye fluorescent system and the inclusion of non-nucleotide
linkers allows for simultaneous amplification and efficient separation of the 15 STR
loci and Amelogenin during automated DNA fragment analysis.
Loci amplified by The following table shows the loci amplified, their chromosomal locations, and the
the kit corresponding fluorescent marker dyes. The AmpFlSTR™ Identifiler™ Allelic Ladder
is used to genotype the analyzed samples. The alleles contained in the allelic ladder
and the genotype of the AmpFlSTR™ Control DNA 9947A are also listed in the table.
Table 1 Identifiler™ Kit loci and alleles
Chromosome Alleles included in AmpFlSTR™ Dye Control DNA

Locus designation
location Identifiler™ Allelic Ladder label 9947A
D8S1179 8 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 6-FAM™ 13†
D21S11 21q11.2-q21 24, 24.2, 25, 26, 27, 28, 28.2, 29, 29.2, 30, 30‡
30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35,
35.2, 36, 37, 38
D7S820 7q11.21-22 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 10, 11
CSF1PO 5q33.3-34 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 10, 12
D3S1358 3p 12, 13, 14, 15, 16, 17, 18, 19 VIC™ 14, 15
TH01 11p15.5 4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3 8, 9.3
D13S317 13q22-31 8, 9, 10, 11, 12, 13, 14, 15 11§
D16S539 16q24-qter 5, 8, 9, 10, 11, 12,13, 14, 15 11, 12
D2S1338 2q35-37.1 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 19, 23
27, 28
D19S433 19q12-13.1 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15, NED™ 14, 15
15.2, 16, 16.2, 17, 17.2
vWA 12p12-pter 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 17, 18
23, 24
TPOX 2p23-2per 6, 7, 8, 9, 10, 11, 12, 13 8††
D18S51 18q21.3 7, 9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2, 15, 15, 19
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27
Amelogenin X: p22.1-22.3 X, Y PET™ X
Y: p11.2
D5S818 5q21-31 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 11‡‡
FGA 4q28 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26.2, 27, 23, 24
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
† For CODIS purposes, profile reported as 13, 13.
‡ For CODIS purposes, profile reported as 30, 30.
§ For CODIS purposes, profile reported as 11, 11.
††For CODIS purposes, profile reported as 8, 8.
‡‡For CODIS purposes, profile reported as 11, 11.

Chapter 1 Overview
Product overview 1
Allelic ladder Figure 1 shows the allelic ladder for the Identifiler™ Kit. See “Allelic ladder
profile requirements” on page 25 for information on ensuring accurate genotyping.
Figure 1 GeneMapper™ ID-X Software plot of the AmpFlSTR™ Identifiler™ Allelic Ladder

Chapter 1 Overview
1 Product overview
Control DNA 9947A Figure 2 shows amplification of Control DNA 9947A using the Identifiler™ Kit.
profile
Figure 2 1 ng of Control DNA 9947A amplified with the Identifiler™ Kit and analyzed on the Applied Biosystems 3130xl
Genetic Analyzer

Chapter 1 Overview
Workflow overview 1
Workflow overview
Perform
Extract
DNA
PCR
AutoMate Express™ System + PrepFiler™ Express Kit

Quantify
DNA
Quantifiler™ Duo DNA Quantification Kit

reactions
Prepare
AmpFlSTR™ Identifiler™ PCR Amplification Kit

Perform
PCR
GeneAmp™ PCR System 9700 Cycler Veriti™ 96-Well Thermal Cycler
Perform
electro-
phoresis
3100/3100-Avant 3130/3130xl 3500/3500xL 310 Genetic

Genetic Analyzer Genetic Analyzer Genetic Analyzer Analyzer
Analyze
data
GeneMapper™ ID-X or GeneMapper™ ID Software

Chapter 1 Overview
1 Instrument and software overview
Instrument and software overview

This section provides information about the Data Collection Software
versions required to run the AmpFlSTR™ Identifiler™ PCR Amplification Kit
on specific instruments.
Data Collection The Data Collection Software provides instructions to firmware running on the
and GeneMapper™ instrument and displays instrument status and raw data in real time. As the
instrument measures sample fluorescence with its detection system, the Data
ID or ID-X
Collection Software collects the data and stores it. The Data Collection Software stores
Software information about each sample in a sample file (.fsa), which is then analyzed by the
GeneMapper™ ID or ID-X Software.
Instrument and Table 2 Software specific to each instrument

software
Operating Data Collection
compatibility Instrument
system Software
Analysis software
3500/3500xL • Windows™ XP 3500 Series GeneMapper™ ID-X

• Windows Data Collection Software v1.2 or higher
Vista™ Software v1.0
3130/3130xl Windows™ XP 3.0 • GeneMapper™ ID

Software v3.2.1
3100/3100-Avant Windows™ NT 1.1 (3100)
and
1.0 (3100-Avant) • GeneMapper™ ID-X
Software v1.0.1 or higher
Windows 2000 2.0
310 Windows XP 3.1
• Windows™ NT 3.0
• Windows 2000
Note: We conducted validation studies for the AmpFlSTR™ Identifiler™ PCR

Amplification Kit using the 310 Genetic Analyzer running Mac OS™. This
configuration is now obsolete.
About Applied Biosystems fluorescent multi-color dye technology allows the analysis of
multicomponent multiple loci, including loci that have alleles with overlapping size ranges. Alleles for
overlapping loci are distinguished by labeling locus-specific primers with different
analysis colored dyes.
Multicomponent analysis is the process that separates the five different fluorescent dye
colors into distinct spectral components. The four dyes used in the Identifiler™ Kit to
label samples are 6-FAM™, VIC™, NED™, and PET™ dyes. The fifth dye, LIZ™, is
used to label the GeneScan™ 500 LIZ™ Size Standard or the GeneScan™ 600 LIZ™
Size Standard v2.0.

Chapter 1 Overview
Instrument and software overview 1
How Each of these fluorescent dyes emits its maximum fluorescence at a different
multicomponent wavelength. During data collection on the Applied Biosystems instruments, the
fluorescence signals are separated by diffraction grating according to their
analysis works
wavelengths and projected onto a charge-coupled device (CCD) camera in a
predictably spaced pattern. The 6-FAM™ dye emits at the shortest wavelength and it
is displayed as blue, followed by the VIC™ dye (green), NED™ dye (yellow), PET™
dye (red), and LIZ™ dye (orange).
Although each of these dyes emits its maximum fluorescence at a different
wavelength, there is some overlap in the emission spectra between the dyes (Figure 3).
The goal of multicomponent analysis is to correct for spectral overlap.
Figure 3 Emission spectra of the five dyes used in the AmpFlSTR™ Identifiler™ Kit
Dyes
6-FAM VIC NED PET LIZ
100
Normalized Emission
80
60
40
20
0
500 550 600 650 700
Wavelength (nm)

Chapter 1 Overview
1 Materials and equipment
Materials and equipment

Kit contents and The AmpFlSTR™ Identifiler™ PCR Amplification Kit (Part No. 4322288)
storage contains materials sufficient to perform 200 amplifications at 25 µL/
amplification.
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. Keep freeze-thaw cycles to a minimum.
Component Description 200✕ Volume Storage

AmpFlSTR™ PCR Contains MgCl2, deoxynucleotide 2 tubes, 1.1 mL –15 to –25°C on receipt,
Reaction Mix triphosphates, and bovine serum albumin in each 2 to 8°C after initial use
buffer with 0.05% sodium azide.
AmpFlSTR™ Identifiler™ Contains fluorescently labeled primers and 1 tube, 1.1 mL
Primer Set non-labeled primers.
AmpFlSTR™ Identifiler™ Contains amplified alleles. 1 tube, 0.05 mL
Allelic Ladder
See Table 1 on page 12 for a list of alleles
included in the allelic ladder.
AmpliTaq Gold™ Contains enzyme, with an activity of 5 U/µL. 2 tubes, –15 to –25°C
DNA Polymerase 0.05 mL/tube
AmpFlSTR™ Contains 0.10 ng/µL human female 9947A 1 tube, 0.3 mL 2 to 8 °C
Control DNA 9947A DNA in 0.05% sodium azide and buffer†.
See Table 1 on page 12 for profile.
† The AmpFlSTR™ Control DNA 9947A is included at a concentration appropriate to its intended use as an amplification control (i.e., to provide
confirmation of the capability of the kit reagents to generate a profile of expected genotype). The AmpFlSTR™ Control DNA 9947A is not
designed to be used as a DNA quantitation control, and you may see variation from the labelled concentration when quantitating aliquots of
the AmpFlSTR™ Control DNA 9947A.
Standards for For the Identifiler™ Kit, the panel of standards needed for PCR amplification,
samples PCR product sizing, and genotyping are:
• AmpFlSTR™ Control DNA 9947A – A positive control for evaluating the
efficiency of the amplification step and STR genotyping using the AmpFlSTR™
Identifiler™ Allelic Ladder.
• GeneScan™ 500 LIZ™ Size Standard or GeneScan™ 600 LIZ™ Size Standard
v2.0 – Used for obtaining sizing results. These standards, which have been
evaluated as internal size standards, yield precise sizing results for Identifiler™
Kit PCR products. Order the GeneScan™ 500 LIZ™ Size Standard
(Part No. 4322682) or the GeneScan™ 600 LIZ™ Size Standard v2.0
(Part No. 4408399) separately.
• AmpFlSTR™ Identifiler™ Allelic Ladder – Allelic ladder developed by Life
Technologies for accurate characterization of the alleles amplified by the
Identifiler™ Kit. The AmpFlSTR™ Identifiler™ Allelic Ladder contains most of
the alleles reported for the 15 autosomal loci. Refer to Table 1 on page 12 for a
list of the alleles included in the AmpFlSTR™ Identifiler™ Allelic Ladder.

2 Perform PCR
■ Required user-supplied reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

■ DNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
■ Prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
■ Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
■ Amplification using bloodstained FTA™ cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Required user-supplied reagents

In addition to the Identifiler™ Kit reagents, the use of low-TE buffer (10 mM Tris,
0.1 mM EDTA, pH 8.0) is recommended. You can prepare the buffer as described in
the procedure below or order it from Teknova (Cat No. T0223).
To prepare low-TE buffer:
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 and autoclave the solutions.

3. Store at room temperature.
DNA quantification
Importance of Quantifying the amount of DNA in a sample before amplification allows you to
quantification determine whether or not sufficient DNA is present to permit amplification and to
calculate the optimum amount of DNA to add to the reaction. The optimum amount of
DNA for the Identifiler™ Kit is 1.0 ng in a maximum input volume of 10 µL for 28
PCR cycles.

Chapter 2 Perform PCR
2 DNA quantification
If too much DNA is added to the PCR reaction, then 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 are problematic 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, and it results in
poor spectral separation (“pull-up”).
• Incomplete A-nucleotide addition.
When the total number of allele copies added to the PCR is extremely low, allelic
dropout can occur resulting in a partial profile.
Methods of Life Technologies provides several kits for quantifying DNA in samples. See the
quantifying DNA references cited in the following table for details about these kits.
Product Description
Quantifiler™ Human DNA Properties:
Quantification Kit
The Quantifiler™ Human and Quantifiler™ Y Human Male Kits are highly specific for
(Part No. 4343895)
human DNA, and they individually detect total human or male DNA, respectively. The
and kits detect single-stranded and degraded DNA.
Quantifiler™ Y Human Male How they work:
DNA Quantification Kit The Quantifiler™ DNA Quantification Kits consist of target-specific and internal control
(Part No. 4343906) 5' nuclease assays.
For more information, see The Quantifiler™ Human and Quantifiler™ Y Human Male Kits contain different
Quantifiler™ Human DNA target-specific assays (human DNA or human male DNA, respectively) that each
Quantification Kits User’s consist of two locus-specific PCR primers and one TaqMan™ MGB probe labeled with
Manual (Pub. No. 4344790) FAM™ dye for detecting the amplified sequence. The kits each contain a separate
internal PCR control (IPC) assay, which consists of an IPC template DNA (a synthetic
sequence not found in nature), two primers for amplifying the IPC template, and one
TaqMan™ MGB probe labeled with VIC™ dye for detecting the amplified IPC.
Quantifiler™ Duo DNA Properties:
Quantification Kit
The Quantifiler™ Duo Kit is highly specific for human DNA. This kit combines the
(Part No. 4387746)
detection of both total human and male DNA in one PCR reaction.The kit detects single-
For more information, see stranded and degraded DNA.
Quantifiler™ Duo DNA How it works:
Quantification Kit User's Manual
(Pub. No. 4391294) The Quantifiler™ Duo DNA Quantification Kit consists of target-specific and internal
control 5' nuclease assays.
The Quantifiler™ Duo kit combines two human-specific assays in one PCR reaction (for
total human DNA and human male DNA). The two human DNA specific assays each
consist of two PCR primers and a TaqMan™ probe. The TaqMan™ probes for the human
DNA and human male DNA assays are labeled with VIC™ and FAM™ dyes, respectively.
In addition, the kit contains an internal PCR control (IPC) assay similar in principle to
that used in the other Quantifiler kits, but labeled with NED™ dye.

Prepare the amplification kit reactions 2
Prepare the amplification kit reactions

1. Calculate the volume of each component needed to prepare the reactions, using
the table below.
DNA sample Volume per reaction

AmpFlSTR™ PCR Reaction Mix 10.5 µL
AmpliTaq Gold™ DNA Polymerase 0.5 µL
AmpFlSTR™ Identifiler™ Primer Set 5.5 µL
Note: The volumes indicated above include a slight overfill to account for the loss
that occurs during reagent transfers.
2. Prepare reagents. Thaw the PCR Reaction Mix and the Identifiler™ Primer Set,
then vortex all reagent tubes, including the enzyme, for 3 seconds and centrifuge
briefly before opening the tubes.
IMPORTANT! Thawing is required only during first use of the Primer Set and PCR
Reaction Mix. After first use, these reagents are stored at 2 to 8°C and do not
require subsequent thawing. Do not refreeze these reagents.
3. Prepare the master mix: Pipette the required volumes of components into an
appropriately sized polypropylene tube.
4. Vortex the master mix for 3 seconds, then centrifuge briefly.

5. Dispense 15 µL of the reaction mix into each reaction well of a MicroAmp™
Optical 96-Well Reaction Plate or each MicroAmp™ tube.
6. Prepare the DNA samples:
DNA sample To prepare...

Negative control Add 10 µL of low-TE buffer (10mM Tris, 0.1mM EDTA, pH 8.0).
Test sample Dilute a portion of the test DNA sample with low-TE buffer so
that 1.0 ng of total DNA is in a final volume of 10 µL. Add 10 µL
of the diluted sample to the reaction mix.
Positive control Add 10 µL of 9947A control DNA (0.1 ng/µL).
The final reaction volume (sample or control plus master mix) is 25 µL.
7. Seal the plate with MicroAmp™ Clear Adhesive Film or MicroAmp™ Optical
Adhesive Film, or cap the tubes.
8. Centrifuge the tubes at 3000 rpm for about 20 seconds in a tabletop centrifuge
(with plate holders if using 96-well plates).
9. Amplify the samples in a GeneAmp™ PCR System 9700 with the silver or
gold-plated silver 96-well blocks or a Veriti™ 96-Well Thermal Cycler.
Note: The Identifiler™ Kit is not validated for use with the GeneAmp PCR System
9700 with the aluminium 96-well block. Use of this thermal cycling platform may
adversely affect performance of the Identifiler™ Kit.

2 Perform PCR
Perform PCR
1. Program the thermal cycling conditions:
• When using the GeneAmp PCR System 9700 with either 96-well silver or
gold-plated silver block, select the 9600 Emulation Mode.
• When using the Veriti™ 96-Well Thermal Cycler, refer to the following
document for instructions on how to configure the Veriti instrument to run
in the 9600 Emulation Mode: User Bulletin: Veriti™ 96-Well Thermal Cycler
AmpFlSTR™ Kit Validation (Pub. No. 4440754).
Initial Denature Anneal Extend Final Final hold

incubation step extension
HOLD CYCLE (28) HOLD HOLD
95°C 94°C 59°C 72°C 60°C 4–25°C
11 min 1 min 1min 1min 60 min ∞
2. Load the plate into the thermal cycler and close the heated cover.
IMPORTANT! If using the 9700 thermal cycler with silver or gold-plated silver
block and adhesive clear film instead of caps to seal the plate wells, be sure to
place a MicroAmp™ compression pad (Part No. 4312639) on top of the plate to
prevent evaporation during thermal cycling. The Veriti™ Thermal Cycler does
not require a compression pad.
3. Start the run.

4. On completion of the run, store the amplified DNA and protect from light.
If you are storing the DNA... Then place at...

< 2 weeks 2 to 8°C
> 2 weeks –15 to –25°C
IMPORTANT! Store the amplified products so that they are protected from light.
Amplification using bloodstained FTA™ cards

FTA™ cards can be useful for collecting, storing, and processing biological samples. A
small punch disc of the card containing the sample can be placed directly into an
amplification tube, purified, and amplified, without transferring the disc. Our studies
indicate that a 1.2-mm bloodstained disc contains approximately 5–20 ng DNA. An
appropriate cycle number for this high quantity of DNA is 25 cycles as determined by
our validation studies. However, it is recommended that each laboratory determine
the optimum cycle number based on internal validation studies.
In the example shown in Figure 4, a 1.2-mm disc of a bloodstained FTA card was
purified using three washes with FTA Purification Reagent and two washes with
1✕ low-TE buffer. The purified punch disc was then amplified in the MicroAmp™
tube for 25 cycles.

Amplification using bloodstained FTA™ cards 2
Figure 4 AmpFlSTR™ Identifiler™ Kit PCR Amplification Kit results from a 1.2-mm FTA bloodstain disc (25-
cycle amplification), analyzed on the Applied Biosystems 3130xl Genetic Analyzer

2 Amplification using bloodstained FTA™ cards

3 Perform Electrophoresis
■ Allelic ladder requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

■ Section 3.1 3100/3100-Avant and 3130/3130xl instruments . . . . . . . . . . . . . . . . . . 27
Set up the 3100/3100-Avant or 3130/3130xl instrument for electrophoresis . . . . 27
Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl
instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
■ Section 3.2 3500/3500xL Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Set up the 3500/3500xL instrument for electrophoresis . . . . . . . . . . . . . . . . . . . . . 29
Prepare samples for electrophoresis on the 3500/3500xL instrument. . . . . . . . . . 29
■ Section 3.3 310 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Set up the 310 instrument for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Prepare samples for electrophoresis on the 310 instrument . . . . . . . . . . . . . . . . . 31
Allelic ladder requirements

To accurately genotype samples, you must run an allelic ladder sample along with the
unknown samples.
One
Number of allelic Number of samples per
Instrument injection
ladders to run allelic ladder(s)
equals
3100-Avant or 3130 1 per 4 injections 4 samples 15 samples + 1 allelic ladder
3100 or 3130xl 1 per injection 16 samples 15 samples + 1 allelic ladder
3500 1 per 3 injections 8 samples 23 samples + 1 allelic ladder
3500xL 1 per injection 24 samples 23 samples + 1 allelic ladder
310 1 per 10 injections 1 sample 9 samples + 1 allelic ladder
IMPORTANT! Variation in laboratory temperature can cause changes in fragment

migration speed and sizing variation between both single- and multiple-capillary runs
(with larger size variations seen between samples injected in multiple-capillary runs).
We recommend the above frequency of allelic ladder injections, which should account
for normal variation in run speed. However, during internal validation studies, verify
the required allelic ladder injection frequency to ensure accurate genotyping of all
samples in your laboratory environment.

Chapter 3 Perform Electrophoresis
3 Allelic ladder requirements
It is critical to genotype using an allelic ladder run under the same conditions as the
samples because size values obtained for the same sample can differ between
instrument platforms because of different polymer matrices and electrophoretic
conditions.

Section 3.1 3100/3100-Avant and 3130/3130xl instruments
Set up the 3100/3100-Avant or 3130/3130xl instrument for electrophoresis 3
Section 3.1 3100/3100-Avant and 3130/3130xl

instruments
3100/3100-Avant and 3130/3130xl Instruments

Set up the 3100/3100-Avant or 3130/3130xl instrument for
electrophoresis
Reagents and parts “Ordering Information” on page 121 lists the required materials not supplied with the
AmpFlSTR™ Identifiler™ PCR Amplification Kit.
Electrophoresis The following table lists Data Collection Software and the run modules that can be
software setup and used to analyze Identifiler™ Kit PCR products. For details on the procedures, refer
to the documents listed in the table.
reference
documents
Data
Genetic Operating
Collection Run modules and conditions References
Analyzer System
Software
Applied 3.0 Windows™ • HIDFragmentAnalysis36_POP4_1 Applied Biosystems 3130/3130xl
Biosystems XP Injection conditions: Genetic Analyzers Using Data
3130/3130xl Collection Software v3.0, Protocols for
– 3130 = 3 kV/5 sec
Processing AmpFlSTR™ PCR
– 3130xl = 3 kV/10 sec Amplification Kit PCR Products User
• Dye Set G5 Bulletin (Pub. No. 4363787)
3100 2.0 Windows™ • HIDFragmentAnalysis36_POP4_1 3100/3100-Avant Genetic Analyzers Using
2000 Injection condition: 3kV/10 sec Data Collection Software v2.0, Protocols
• Dye Set G5 for Processing AmpFlSTR™ PCR
Amplification Kit PCR Products User
Bulletin (Pub. No. 4350218)
1.1 Windows™ • GeneScan36vb_DyeSetG5Module 3100/3100-Avant Genetic Analyzers
NT Injection condition: 3kV/10 sec Protocols for Processing AmpFlSTR™
• GS600v2.0Analysis.gsp PCR Amplification Kit PCR Products User
Bulletin (Pub. No. 4332345)
3100-Avant 1.0 Windows™ • GeneScan36Avb_DyeSetG5Module 3100/3100-Avant Genetic Analyzers
NT Injection condition: 3 kV/5sec Protocols for Processing AmpFlSTR™
PCR Amplification Kit PCR Products
• GS600v2.0Analysis.gsp
User Bulletin (Pub. No. 4332345)

3 Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instrument
Prepare samples for electrophoresis on the 3100/3100-Avant or

3130/3130xl instrument
Prepare the samples for electrophoresis immediately before loading.
1. Calculate the volume of Hi-Di™ Formamide and size standard needed to prepare
the samples:
Volume per Volume per

Reagent Reagent
reaction reaction
GeneScan™ 500 0.3 µL OR GeneScan™ 600 0.5 µL

LIZ™ Size Standard LIZ™ Size
Standard v2.0
Hi-Di™ Formamide 8.7 µL Hi-Di™ Formamide 8.5 µL
Note: Include additional samples in your calculations 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. Pipette the required volumes of components into an appropriately sized

polypropylene tube.
3. Vortex the tube, then centrifuge briefly.

4. Into each well of a MicroAmp™ Optical 96-Well Reaction Plate, add:
• 9 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.
5. Seal the reaction plate with appropriate septa, then centrifuge the plate to ensure
that the contents of each well are collected at the bottom.
6. Heat the reaction plate in a thermal cycler for 3 minutes at 95°C.

7. Immediately place the plate on ice for 3 minutes.
8. Prepare the plate assembly, then place on the autosampler.
9. Ensure that a plate record is completed and link the plate record to the plate.
10. Start the electrophoresis run.

Set up the 3500/3500xL instrument for electrophoresis 3
Section 3.2 3500/3500xL Series instruments
Set up the 3500/3500xL instrument for electrophoresis

3500/3500 xL Instruments
reference
documents
Data
Genetic Operating
Analyzer System
Software
Applied 3500 Data Windows™ • HID36_POP4 Applied Biosystems 3500/3500xL
Biosystems Collection XP Injection conditions: 1.2kV/15 sec Genetic Analyzer User Guide
3500 Software (Pub. No. 4401661)
or • Dye Set G5
v1.0
Applied Windows • HID36_POP4 Applied Biosystems 3500 and 3500xL
Biosystems Vista ™ Genetic Analyzers Quick Reference
Injection conditions: 1.2kV/24 sec
3500xL Card (Pub. No. 4401662)
• Dye Set G5
Prepare samples for electrophoresis on the 3500/3500xL

instrument
1. Calculate the volume of Hi-Di™ Formamide and GeneScan™ 600 LIZ™ Size
Standard v2.0 needed to prepare the samples:
Reagent Volume per reaction
GeneScan™ 600 LIZ™ Size Standard v2.0 0.5 µL

Hi-Di™ Formamide 8.5 µL


results and experiments.

3 Prepare samples for electrophoresis on the 3500/3500xL instrument

polypropylene tube.

4. Into each well of a MicroAmp™ Optical 96-Well Reaction Plate, or each
MicroAmp™ optical strip tube, add:
• 1 µL of PCR product or allelic ladder
5. Seal the reaction plate or strip tubes with the appropriate septa, then centrifuge to
ensure that the contents of each well are collected at the bottom.
6. Heat the reaction plate or strip tubes in a thermal cycler for 3 minutes at 95°C.
7. Immediately put the plate or strip tubes on ice for 3 minutes.
8. Prepare the plate assembly, then place on the autosampler.
9. Ensure that a plate record is completed and link the plate record to the plate.

Set up the 310 instrument for electrophoresis 3
Section 3.3 310 Instrument
Set up the 310 instrument for electrophoresis

310 Instruments
reference
documents
Data
Operating
System
Software
3.1† Windows XP • GS STR POP4 (1mL) G5 v2.md5 310 Genetic Analyzer User’s Manual (Windows)
or or Injection condition: (Pub. No. 4317588)
3.0† Windows™ 15 kV/5 sec 310 Protocols for Processing AmpFlSTR™ PCR
NT and Amplification Kit Products with Microsoft Windows
Windows NT Operating System: User Bulletin
2000 (Pub. No. 4341742)
† We conducted concordance studies for the Identifiler™ Kit using this configuration.
Prepare samples for electrophoresis on the 310 instrument

1. Calculate the volume of Hi-Di™ Formamide and size standard needed to prepare
the samples:
Reagent Volume per reaction

GeneScan™ 500 LIZ™ Size Standard or 0.75 µL
GeneScan™ 600 LIZ™ Size Standard
Hi-Di™ Formamide
v2.0 24.5 µL


3 Prepare samples for electrophoresis on the 310 instrument

results and experiments.

polypropylene tube.

4. Into each 0.2 mL sample tube, add:
• 1.5 µL of PCR product or allelic ladder
5. Seal the tubes with the appropriate septa, then briefly centrifuge to ensure that
the contents of each tube are mixed and collected at the bottom.
6. Heat the tubes in a thermal cycler for 3 minutes at 95°C.

7. Immediately place the tubes on ice for 3 minutes.
8. Place the sample tray on the autosampler.
9. Ensure that an injection list is prepared.

4 Analyze Data
■ Section 4.1 GeneMapper™ ID Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Overview of GeneMapper™ ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Set up GeneMapper™ ID Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . 34
Analyze and edit sample files with GeneMapper™ ID Software. . . . . . . . . . . . . . 46
Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
For more information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
■ Section 4.2 GeneMapper™ ID-X Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Overview of GeneMapper™ ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Set up GeneMapper™ ID-X Software for data analysis . . . . . . . . . . . . . . . . . . . . . . 49
Analyze and edit sample files with GeneMapper™ ID-X Software. . . . . . . . . . . . 61
Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Section 4.1 GeneMapper™ ID Software
Overview of GeneMapper™ ID Software

GeneMapper™ ID Software is an automated genotyping software for forensic
casework, databasing, and paternity data analysis.
After electrophoresis, the data collection software stores information for each
sample in a .fsa file. Using GeneMapper™ ID Software v3.2.1 software, you can then
analyze and interpret the data from the .fsa files.
Instruments Refer to “Instrument and software overview” on page 16 for a list of compatible
instruments.

Chapter 4 Analyze Data
4 Set up GeneMapper™ ID Software for data
analysis
Before you start When using GeneMapper™ ID Software v3.2.1 to perform human identification
(HID) analysis with AmpFlSTR™ kits, be aware that:
• HID analysis requires at least one allelic ladder sample per run folder. Your
laboratory can use multiple ladder samples in an analysis, provided individual
laboratories conduct the appropriate validation studies.
For multiple ladder samples, the GeneMapper™ ID Software calculates allelic bin
offsets by using an average of all ladders that use the same panel within 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 ladder(s)
within 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. Failure to apply this setting for ladder samples results in
failed analysis.
• 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 AmpFlSTR™ Allelic Ladders do exist. Off-ladder (OL)
alleles may contain full and/or partial repeat units. An off-ladder allele is an allele
that occurs outside the ±0.5-nt bin window of any known allelic ladder allele or
virtual bin.
Note: If a sample allele peak is called as an off-ladder allele, the sample result
needs to be verified according to the laboratory’s protocol.
Set up GeneMapper™ ID Software for data analysis

File names The file names shown in this section may differ from the file names you see when you
download or import files. If you need help determining the correct files to use, contact
your local Life Technologies Human Identification representative, or go to
www.lifetechnologies.com/supportSoftware, Patches &
UpdatesGeneMapper™ ID Software.
Before using the Before you can analyze sample (.fsa) files using GeneMapper™ ID Software v3.2.1 for
software for the the first time, you need to:
first time • Import panels and bins into the Panel Manager, as explained in “Import panels
and bins” on page 35.
• Create an analysis method, as explained in , “Create an analysis method” on page
39.
• Create a size standard, as explained in “Create size standard” on page 44.
• Define custom views of analysis tables.
Refer to Chapter 1 of the GeneMapper™ ID Software Versions 3.1 and 3.2 Human
Identification Analysis Tutorial (Pub. No. 4335523) for more information.
• Define custom views of plots.
Refer to Chapter 1 of the GeneMapper™ ID Software Versions 3.1 and 3.2 Human
Identification Analysis Tutorial (Pub. No. 4335523) for more information.

Set up GeneMapper™ ID Software for data analysis 4
Import panels and To import the Identifiler™ panel and bin set into the GeneMapper™ ID Software
bins v3.2.1 database:
1. Start the GeneMapper™ ID Software, then log in with the appropriate user name
and password.
IMPORTANT! If you need logon instructions, refer to page 2-7 of the

GeneMapper™ ID Software Version 3.1 Human Identification Analysis User Guide
(Pub. No. 4338775).
GeneMapper™ ID Software
2. Select ToolsPanel Manager.
3. Find, then open the folder containing the panels and bins:
a. Select Panel Manager in the navigation pane.
Highlight this.
b. Select FileImport Panels to open the Import Panels dialog box.

c. Navigate to, then open the x:\Applied Biosystems\GeneMapper\Panels
folder, where x is the drive on which the GeneMapper™ ID Software is
installed.
4. Select AmpFLSTR_Panels_v2.txt, then click Import.
Note: Importing this file creates a new folder in the navigation pane of the Panel
Manager, AmpFLSTR_Panels_v2. This folder contains the panels and associated
markers.
5. Import AmpFLSTR_Bins_v2.txt:
a. Select the AmpFLSTR_Panels_v2 folder in the navigation pane.

analysis
b. Select FileImport Bin Set to open the Import Bin Set dialog box.
c. Navigate to, then open the x:\Applied Biosystems\GeneMapper\Panels
folder.
d. Select AmpFLSTR_Bins_v2.txt, then click Import.
Note: Importing this file associates the bin set with the panels in the
AmpFLSTR_Panels_v2 folder.
6. View the imported panels in the navigation pane:

a. Double-click the AmpFLSTR_Panels_v2 folder.

b. Double-click the Identifiler_v2 folder to display the panel information in the

right pane and the markers below it.
7. View the markers and display the Bin view in the navigation pane:
a. Select the AmpFLSTR_Panels_v2 folder to display its list of kits in the right
pane.
b. Double-click the Identifiler_v2 folder to display its list of markers below it.

analysis
c. Select D8S1179 to display the Bin view for the marker in the right pane.
8. Click Apply, then OK to add the AmpFlSTR™ panel and bin set to the
GeneMapper™ ID Software database.
IMPORTANT! If you close the Panel Manager without clicking OK, the panels
and bins are not imported into the GeneMapper™ ID Software database.

Create an analysis The HID Advanced analysis method for the Identifiler™ Kit uses the
method AmpFLSTR_Bins_v2 file described in step 5 on page 35.
Use the following procedure to create a HID analysis method for the Identifiler™ Kit.
1. Select ToolsGeneMapper Manager to open the GeneMapper Manager.
2. Select the Analysis Methods tab, then click New to open the New Analysis
Method dialog box.
3. Select HID and click OK to open the Analysis Method Editor with the General
Tab selected.
4. Enter the settings in each tab of the Analysis Method Editor as shown in the
figures below unless the instructions state otherwise.
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.
5. After you enter settings in all tabs, click Save.

analysis
General tab
settings
In the Name field, either type the name as shown, or enter a name of your choosing.
The Description and Instrument fields are optional.

Allele tab settings
• In the Bin Set field, select the AmpFLSTR_Bins_v2 bin set imported previously
and configure the stutter distance parameters as shown.
• GeneMapper™ ID Software v3.2.1 allows you to specify four types of marker
repeat motifs: tri, tetra, penta, and hexa. You can enter parameter values for each
type of repeat in the appropriate column.
• Specify the stutter ratio:
– To apply the stutter ratios listed in the Allele tab for single-source data,
deselect the “Use marker-specific stutter ratio if available” check box
(selected by default). Perform appropriate internal validation studies to
determine the appropriate filter setting to use.
Note: Applying global stutter ratios may reduce the editing required for
single-source sample data.
– To apply the stutter ratios contained in the AmpFLSTR_Panels_v2.txt file,
select the “Use marker-specific stutter ratio if available” check box (selected
by default). Perform appropriate internal validation studies to determine the
appropriate filter setting to use.

analysis
Peak Detector tab

settings
Perform
internal
validation
studies to
determine
settings
IMPORTANT! Perform the appropriate internal validation studies to determine

the peak amplitude thresholds for interpretation of Identifiler™ Kit data.
Fields include:
• Peak amplitude thresholds – The software uses these parameters to specify the
minimum peak height, in order to limit the number of detected peaks. Although
GeneMapper™ ID Software displays peaks that fall below the specified amplitude
in electropherograms, the software does not label or determine the genotype of
these peaks.
• Size calling method – The Identifiler™ Kit has been validated using the Local
Southern sizing method. Before using other sizing methods, perform internal
validation studies.

Peak Quality tab

settings
Perform
internal
validation
studies to
determine
settings
IMPORTANT! Perform the appropriate internal validation studies to determine the
minimum heterozygous and homozygous minimum peak height thresholds and
the minimum peak height ratio threshold for interpretation of Identifiler™ Kit data.

analysis
Quality Flags tab

settings
IMPORTANT! The values shown are the software defaults and are the values we used
during developmental validation. Perform the appropriate internal validation studies to
determine the appropriate values for interpretation of Identifiler™ Kit data.
Create size The size standards for the Identifiler™ Kit use the following size standard peaks
standard in their definitions:
GeneScan™ 500 LIZ™ Size Standard GeneScan™ 600 LIZ™ Size Standard v2.0
75, 100, 139, 150, 160, 200, 300, 340, 350, 400, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220,
and 450 240, 250, 260, 280, 300, 314, 320, 340, 360,
380, 400, 414, 420, 440 and 460
Note: The 250-nt peak in the GeneScan™ 500 LIZ™ Size Standard is not included in
the size standard definition. This peak can be used as an indicator of precision within
a run.
Use the following procedure to create the appropriate size standard:
1. Select ToolsGeneMapper Manager to open the GeneMapper Manager.

2. Select the Size Standards tab, click New, select the Basic or Advanced radio
button, then click OK.
3. Enter a name (for example, CE_G5_Identifiler_GS500 as shown below). In the Size
Standard Dye field, select Orange. In the Size Standard Table, enter the sizes
specified in on page 44. The example below is for the GeneScan™ 500 LIZ™ Size
Standard.

4 Analyze and edit sample files with GeneMapper™ ID Software
Analyze and edit sample files with GeneMapper™ ID Software

1. In the Project window, select FileAdd Samples to Project, then navigate to the
disk or directory containing the sample files.
2. Apply analysis settings to the samples in the project.
Parameter Settings
Sample Type Select the sample type.
Analysis Method Identifiler_AnalysisMethod_v1 (or the name of the analysis
method you created)
Panel Identifiler_v2
Size Standard CE_G5_Identifiler_GS500† (or the name of the size standard you
created)
† The Identifiler™ Kit was originally validated using the GeneScan™ 500 LIZ™ Size Standard. If you use the
GeneScan™ 600 LIZ™ Size Standard v2.0 as an alternative, perform the appropriate internal validation
studies to support the use of this size standard with the Identifiler™ Kit.
Note: For more information about how the Size Caller works, refer to the
GeneScan™ Analysis Software for the Windows™ NT Operating System Overview of
the Analysis Parameters and Size Caller User Bulletin (Pub. No. 4335617).
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 extending to the right with the percentage
indicated
– With text messages on the left
• The table displays the row of the sample currently being analyzed in green
(or red if analysis failed for the sample).
• The Genotypes tab becomes available after analysis (see the figure on the
next page).

Examine and edit a project 4
Project window after analysis
For more information about any of these tasks, refer to the GeneMapper™ ID
Software Version 3.1 Human Identification Analysis User Guide (Pub. No. 4338775).
Examine and edit a project

You can display electropherogram plots from the Samples and Genotypes tabs of the
Project window to examine the data. These procedures start with the Samples tab of
the Project window (assuming the analysis is complete).
For more information

For details about GeneMapper™ ID Software features, allele filters, peak
detection algorithms, and project editing, refer to:
• GeneMapper™ ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial
(Pub. No. 4335523)
• GeneMapper™ ID Software Version 3.1 Human Identification Analysis User Guide (Pub.
No. 4338775)
• Installation Procedures and New Features for GeneMapper™ ID Software Software
Version v3.2 User Bulletin (Pub. No. 4352543)

Chapter 4 GeneMapper™ ID-X Software
4 Overview of GeneMapper™ ID-X Software
Section 4.2 GeneMapper™ ID-X Software
Overview of GeneMapper™ ID-X Software

GeneMapper™ ID-X Software is an automated genotyping software for forensic
casework, databasing, and paternity data analysis.
After electrophoresis, the data collection software stores information for each sample
in a .fsa file or a .hid file. Using GeneMapper™ ID-X Software, you can then analyze
and interpret the data from .fsa files (GeneMapper™ ID-X Software v1.0.1 or higher)
or .hid files (GeneMapper™ ID-X Software v1.2 or higher).
Instruments Refer to “Instrument and software overview” on page 16 for a list of compatible
instruments.
Before you start When using GeneMapper™ ID-X Software v1.0.1 or higher to perform human
identification (HID) analysis with AmpFlSTR™ kits, be aware that:
• HID analysis requires at least one allelic ladder sample per run folder. Your
laboratory can use multiple ladder samples in an analysis, provided individual
laboratories conduct the appropriate validation studies.
For multiple ladder samples, the GeneMapper™ ID-X Software calculates allelic
bin offsets by using an average of all ladders that use the same panel within 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 ladder(s)
within 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. Failure to apply this setting for ladder samples results in
failed analysis.
• 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 AmpFlSTR™ Allelic Ladders do exist. Off-ladder (OL)
alleles may contain full and/or partial repeat units. An off-ladder allele is an allele
that occurs outside the ±0.5-nt bin window of any known allelic ladder allele or
virtual bin.
Note: If a sample allele peak is called as an off-ladder allele, the sample result
needs to be verified according to the laboratory’s protocol.

Set up GeneMapper™ ID-X Software for data analysis 4
Set up GeneMapper™ ID-X Software for data analysis

Panel, bin, and The file names shown in this section may differ from the file names you see when you
stutter file version download or import files. If you need help determining the correct files to use, contact
your local Life Technologies Human Identification representative, or go to
www.lifetechnologies.com/supportSoftware, Patches &
UpdatesGeneMapper™ ID-X Software.
GeneMapper™ ID-X Software

The instructions and examples in this section refer to the latest version of panel, bin,
and stutter file available at the time of publication.
Before using the Before you use GeneMapper™ ID-X Software (v1.0.1 or higher for .fsa files, v1.2 or
software for the higher for .hid files) to analyze data for the first time, you must do the following:
first time 1. Check the version of panel, bin, and stutter files installed with the GeneMapper™
ID-X Software as explained in “Check panel, bin, and stutter file version” below.
2. Check www.lifetechnologies.com/supportSoftware, Patches &

UpdatesGeneMapper™ ID-X Software to determine if newer files are
available.
3. If updated files are available, download and import the files into the
GeneMapper™ ID-X Software, as explained in “Import panels, bins, and marker
stutter” on page 50.
Note: When downloading new versions of analysis files, refer to the associated
Read Me file for details of changes between software file versions. If you have
validated previous file versions for data analysis, conduct the appropriate
internal verification studies before using new file versions for operational
analysis.
4. Create an analysis method, as explained in “Create an analysis method” on page

55.
5. Define custom views of analysis tables.
Refer to Chapter 1 of the GeneMapper™ ID-X Software Version 1.0 Getting Started
Guide (Pub. No. 4375574) for more information.
6. Define custom views of plots.

Refer to Chapter 1 of the GeneMapper™ ID-X Software Version 1.0 Getting Started
Guide (Pub. No. 4375574) for more information.
For more For quick set up instructions, refer to the GeneMapper™ ID-X Software Version 1.0
information Getting Started Guide (Pub. No. 4375574).
For details about GeneMapper™ ID-X Software features, refer to:

• GeneMapper™ ID-X Software Version 1.0 Getting Started Guide (Pub. No. 4375574)
• GeneMapper™ ID-X Software Version 1.0 Quick Reference Guide (Pub. No. 4375670)
• GeneMapper™ ID-X Software Version 1.0 Reference Guide (Pub. No. 4375671)

4 Set up GeneMapper™ ID-X Software for data
analysis
Check panel, bin, 1. Start the GeneMapper™ ID-X Software, then log in with the appropriate user
and stutter file name and password.
version
IMPORTANT! For logon instructions, refer to the GeneMapper™ ID-X
Software Version 1.0 Getting Started Guide (Pub. No. 4375574).

3. Check the version of files imported into the Panel Manager:
b. Expand the Panel Manager folder and any sub-

folders to identify the analysis file version 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 FileImport Panels to open the Import
Panels dialog box.
b. Navigate to, then open the Panels folder and check the version of panel, bin,
and stutter files installed.
5. If newer versions are available on the website, download and import as described
below.
Import panels, To import the Identifiler™ Kit panel, bin set, and marker stutter from our web site
bins, and marker into the GeneMapper™ ID-X Software database:
stutter 1. Download and open the file containing panels, bins, and marker stutter:
a. Go to www.lifetechnologies.com/supportSoftware, Patches &
UpdatesGeneMapper™ ID-X Software. Download the file AmpFLSTR
Analysis Files GMIDX.
b. Unzip the file.
2. Start the GeneMapper™ ID-X Software, then log in with the appropriate user
name and password.
IMPORTANT! For logon instructions, refer to the GeneMapper™ ID-X

Software Version 1.0 Getting Started Guide (Pub. No. 4375574).

4. Find, then open the folder containing the panels, bins, and marker stutter:
b. Select FileImport Panels to open the Import

Panels dialog box.
c. Navigate to, then open the AmpFLSTR Analysis
Files GMIDX folder that you unzipped in step 1
on page 50.

5. Select AmpFLSTR_Panels_v2X (or the version you installed), then click Import.
Note: Importing this file creates a new folder in the navigation pane of the Panel
Manager “Identifiler_v1.1X”. This folder contains the panel and associated
markers.

6. Import AmpFLSTR_Bins_V2X.txt:
a. Select the AmpFLSTR_Panels_v2X folder in the navigation pane.
b. Select File Import Bin Set to open the Import Bin Set dialog box.
c. Navigate to, then open the AmpFLSTR Analysis Files GMIDX folder.

analysis
d. Select AmpFLSTR_Bins_V2X.txt, then click Import.

Note: Importing this file associates the bin set with the panels in the
AmpFLSTR_Panels_v2X folder.
7. View the imported panels in the navigation pane:

a. Double-click the AmpFLSTR_Panels_v2X folder.
b. Double-click the Identifiler_v1.1X folder to display the panel information in
the right pane.

8. Select and expand Identifiler_v1.1X in the navigation pane, then select D8S1179
to display the Bin view for the marker in the right pane.

9. Import AmpFLSTR_Stutter_v2X:
a. Select the AmpFLSTR_Panels_v2X folder in the navigation panel.
b. Select FileImport Marker Stutter to open the Import Marker Stutter dialog
box.
c. Navigate to, then open the AmpFLSTR Analysis Files GMIDX folder.

analysis
d. Select AmpFLSTR_Stutter_v2X, then click Import.

Note: Importing this file associates the marker stutter ratio with the bin set
in the AmpFLSTR_v2X folder.
10. View the imported marker stutters in the navigation pane:

a. Double-click the AmpFLSTR_Panels_v2X folder to display its list of kits in
the right pane.
b. Double-click the Identifiler_v1.1X folder to display its list of markers below
it.
c. Double-click D16S539 to display the Stutter Ratio & Distance view for the
marker in the right pane.
11. Click Apply, then OK to add the Identifiler™ Kit 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 will not be imported into the GeneMapper™
ID-X Software database.

Create an analysis Use the following procedure to create an analysis method for the Identifiler™ Kit.
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 created
for GeneMapper™ ID-X version 1.2 is not compatible with earlier versions of
GeneMapper™ ID-X Software or with GeneMapper™ ID Software version 3.2.1.

1. Select ToolsGeneMapper™ ID-X Manager to open the
GeneMapper™ ID-X Manager.
2. Select the Analysis Methods tab, then click New to open the Analysis Method
Editor with the General tab selected.
3. The figures below show the settings for each tab of the Analysis Method Editor.
Configure the Analysis Method Editor tab settings as shown in the figures below,
unless the instructions state otherwise.
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 settings in all tabs, click Save.

analysis
General tab
settings
In the Name field, either type the name as shown or enter a name of your choosing. In
the Security Group field, select the Security Group appropriate to your software
configuration from the dropdown list. The Description and Instrument fields are
optional.

Allele tab settings

• In the Bin Set field, select the AmpFLSTR_Bins_v2X bin set and configure the
stutter distance parameters as shown.
• GeneMapper™ ID-X Software v1.0.1 or higher allows you to specify 4 types of
marker repeat motifs: tri, tetra, penta and hexa. You can enter parameter values
for each type of repeat in the appropriate column.
• Specify the stutter ratio:
– To apply the stutter ratios listed in the Allele tab for single-source data,
deselect the “Use marker-specific stutter ratio if available” check box
(selected by default). Perform appropriate internal validation studies to
determine the appropriate filter setting to use.
Note: Applying global stutter ratios may reduce the editing required for
single-source sample data.
– To apply the stutter ratios contained in the AmpFLSTR_Panels_v2.txt file,
select the “Use marker-specific stutter ratio if available” check box (selected
by default). Perform appropriate internal validation studies to determine the
appropriate filter setting to use.

analysis
Peak Detector tab

settings
Perform
internal
validation
studies to
determine
settings

appropriate peak amplitude thresholds for interpretation of Identifiler™ Kit data.
Fields include:
• Peak amplitude thresholds – The software uses these parameters to specify the
minimum peak height, in order to limit the number of detected peaks. Although
GeneMapper™ ID-X Software displays peaks that fall below the specified
amplitude in electropherograms, the software does not label or determine the
genotype of these peaks.
• Size calling method – The Identifiler™ Kit has been validated using the Local
Southern sizing method. Select alternative sizing methods only after you perform
the appropriate internal validation studies.
• Normalization – A Normalization checkbox is available on this tab in
GeneMapper™ ID-X Software v1.2 for use in conjunction with data run on the
Applied Biosystems 3500 Series Genetic Analyzers. Users of this version of
software should perform laboratory evaluations to determine whether to use the
Normalization feature for analysis of Identifiler™ Kit data.

Peak Quality tab

settings
Perform
internal

validation
studies to
determine
settings

minimum heterozygous and homozygous minimum peak height thresholds,
maximum peak height threshold and the minimum peak height ratio threshold
for interpretation of Identifiler™ Kit data.

analysis
SQ & GQ tab
settings
IMPORTANT! The values 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.

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 FileAdd Samples to Project, then navigate to the
disk or directory containing the sample files.
2. Apply analysis settings to the samples in the project.

Parameter Settings
Sample Type Select the sample type.
Analysis Method Identifiler_AnalysisMethod_v2X (or the name of the analysis
method you created)
Panel Identifiler_v1.1X
Size Standard CE_G5_GS500(75-450)
For more information about how the Size Caller works, or about size standards,
refer to the GeneMapper™ ID-X Software v1.2 Reference Guide (Pub. No. 4426481A).
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
extending to the right with the percentage indicated.
• 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 becomes
available upon completion of the analysis.

4 Examine and edit a project
Analysis summary window after analysis
Examine and edit a project

You can display electropherogram plots from the Samples and Genotypes tabs of the
Project window to examine the data. These procedures start with the Analysis
Summary tab of the Project window (assuming the analysis is complete).
For more information

For more information, refer to:
• GeneMapper™ ID-X Software Version 1.0 Getting Started Guide (Pub. No. 4375574)
• GeneMapper™ ID-X Software Version 1.1(Mixture Analysis) Getting Started Guide
(Pub. No. 4396773)

For more information 4
• GeneScan™ Analysis Software for the Windows NT™ Operating System Overview of
the Analysis Parameters and Size Caller User Bulletin (Pub. No. 4335617).

4 For more information

5 Experiments and Results
■ Section 5.1 Developmental Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Extra Peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Mixture studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Data interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Mutation rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Probability of identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Probability of paternity exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
■ Section 5.2 Performance Verification After Primer Manufacturing Process
Improvements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
■ Section 5.3 Performance Validation After Buffer and Enzyme Component
Replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Reproducibility study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Sensitivity study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Inhibition study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Chapter 5 Experiments and Results
5 Overview
Section 5.1 Developmental Validation
Overview
This chapter provides results of the developmental validation experiments we
performed using the AmpFlSTR™ Identifiler™ PCR Amplification Kit.
The data contained in this section was generated during the original developmental
validation of the Identifiler™ Kit before its release in 2001. Since that time, we have
made a series of improvements to the Identifiler™ Kit. For information on changes
to the Identifiler™ Kit since 2001 and associated data, see:
• “Performance Verification After Primer Manufacturing Process Improvements”
on page 107
• “Performance Validation After Buffer and Enzyme Component Replacement” on
page 108
Importance of Validation of a DNA typing procedure for human identification applications is an

validation evaluation of the procedure’s efficiency, reliability, and performance characteristics. By
challenging the procedure with samples commonly encountered in forensic and
parentage laboratories, the validation process uncovers attributes and limitations
which are critical for sound data interpretation in casework (Sparkes, Kimpton,
Watson et al., 1996; Sparkes, Kimpton, Gilbard et al., 1996; Wallin et al., 1998).
Experiment We performed experiments to evaluate the performance of the Identifiler™ Kit

conditions according to the DNA Advisory Board (DAB) Quality Assurance Standards, effective
October 1, 1998 (DNA Advisory Board, 1998). The DAB standards describe the quality
assurance requirements that a laboratory should follow to ensure the quality and
integrity of the data and competency of the laboratory.
These DAB standards describe the quality assurance requirements that a laboratory
should follow to ensure the quality and integrity of the data and competency of the
laboratory. DAB defines a laboratory as a facility in which forensic DNA testing is
performed.
Based on these standards, we conducted experiments which comply with Standards
8.1.1 and 8.1.2 and its associated subsections. Whereas this DNA methodology is not
novel, Standard 8.1.2 and its related subsections have been addressed (Holt et al., 2001
and Wallin et al., 2001). This chapter will discuss many of the experiments we
performed and examples of the results we obtained. We used conditions that produced
maximum PCR product yield and a window in which reproducible performance
characteristics were met. These experiments, while not exhaustive, are appropriate
for a manufacturer, in our opinion. Each laboratory using the Identifiler™ Kit should
perform appropriate validation studies.

Developmental validation 5
Developmental validation
DAB 8.1.1 “Developmental validation that is conducted shall be appropriately documented.” (DNA
Developmental Advisory Board, 1998).
Validation Critical reagent concentrations and reaction conditions (such as thermal cycling
parameters, AmpliTaq Gold™ DNA polymerase activation, cycle number) to produce
reliable, locus-specific amplification and appropriate sensitivity have been
Developmental Validation
determined.
PCR components The concentration of each component of the Identifiler™ Kit was examined. The PCR
components are Tris-HCl (pH 8.3), KCl, dNTPs, primers, AmpliTaq Gold™ DNA
Polymerase, MgCl2, bovine serum albumin, and sodium azide. The concentration for a
particular component was established to be in the window that meets the reproducible
performance characteristics of specificity and sensitivity (Figure 5).
Figure 5 A 1 ng amplification of genomic DNA varying the MgCl2 concentration, analyzed on the
310 Genetic Analyzer
– 20%
– 8%
Optimal
+ 8%
+ 20%
Thermal cycler Thermal cycling parameters were established for amplification of the Identifiler™ Kit
parameters in the GeneAmp™ PCR Systems 9600 and 9700. Thermal cycling times and
temperatures of GeneAmp PCR systems were verified. Annealing and denaturation
temperature windows were tested around each stipend to verify that a ±1.5°C window
produced a specific PCR product with the desired sensitivity of at least 1 ng of
AmpFlSTR™ Control DNA 9947A.
The effects of denaturation and annealing temperatures on the amplification of
Identifiler™ Kit loci were examined using AmpFlSTR™ Control DNA 9947A and two
DNA samples.

5 Developmental validation
The denaturation temperatures tested were 92.5, 94, and 95.5°C, all for 1-minute hold
times on the GeneAmp PCR System 9700. The annealing temperatures tested were 55,
57, 59, 61, and 63°C (Figure 6), also for 1-minute hold times in the GeneAmp PCR
System 9700. The PCR products were analyzed using the 310 Genetic Analyzer.
Neither preferential nor differential amplification was observed in the denaturation
temperature experiments. Of the tested annealing temperatures, 55, 57, 59, and 61°C
produced robust profiles. At 63°C, the yield of the majority of loci was significantly
reduced. This should pose no problem with routine thermal cycler calibration and
when following the recommended amplification protocol. Preferential amplification
was not observed at any of the tested annealing temperatures.
Figure 6 An amplification of 1 ng of genomic DNA, amplified while varying the annealing
temperature, analyzed on the 310 Genetic Analyzer
55°C
57°C
59°C
standard
protocol
61°C
63°C
AmpliTaq Gold™ Identifiler™ Kit reactions were amplified for 27, 28, 29, 30, and 31 cycles on the
DNA Polymerase GeneAmp™ PCR System 9700 using 1.0 ng of three DNA samples. As expected, PCR
product increased with the number of cycles. A full profile was generated at 27 cycles;
activation
off-scale data were collected for several allele peaks at 31 cycles.
While none of the cycle numbers tested produced nonspecific peaks, 28 cycles was
found to give optimal sensitivity when the amplified products were examined on 310
Genetic Analyzers. Additionally, the cycle number was set to avoid detection of low
quantities of DNA (20 pg or less). At 28 cycles, 1.0 ng of AmpFlSTR™ Control
DNA 9947A amplifies reliably and specifically following the conditions outlined in
this guide.

Accuracy, precision, and reproducibility 5
Accuracy, precision, and reproducibility

DAB 8.1.2 Accuracy “Novel forensic DNA methodologies shall undergo developmental validation to ensure the
accuracy, precision and reproducibility of the procedure.” (DAB, 1998).
Laser-induced fluorescence detection systems of length polymorphism at short
tandem repeat loci is not a novel methodology (Holt et al., 2001 and Wallin et al., 2001).
However, accuracy and reproducibility of Identifiler™ Kit profiles have been
determined from various sample types.
Figure 7 illustrates the size differences that are typically observed between sample
alleles and allelic ladder alleles on the 310 Genetic Analyzer with POP-4™ polymer.
The x-axis in Figure 7 represents the nominal base pair sizes for the AmpFlSTR™
Identifiler™ Allelic Ladder, and the dashed lines parallel to the x-axis represent the
±0.5-bp windows. The y-axis is the deviation of each sample allele size from the
corresponding allelic ladder allele size. The data include a total of 2269 alleles from 70
population database samples. All sample alleles are within 0.5 bp of a corresponding
allele in an allelic ladder.
Figure 7 Size deviation of 70 samples and two allelic ladders from one injection of allelic ladder
on a single 310 Genetic Analyzer run
Precision and size Sizing precision allows for determining accurate and reliable genotypes. Sizing
windows precision was measured on the 310 Genetic Analyzer. The recommended method for
genotyping is to use a ±0.5-bp “window” around the size obtained for each allele in the
AmpFlSTR™ Identifiler™ Allelic Ladder. A ±0.5-bp window allows for the detection
and correct assignment of alleles. An allele that sizes only one base pair different from
an allele in the allelic ladder will not be incorrectly typed and will be identified as off-
ladder. Any sample allele that sizes outside a window could be either of the following:
• An “off-ladder” allele, for example, an allele of a size that is not represented in the
AmpFlSTR™ Identifiler™ Allelic Ladder
• An allele that does correspond to an allelic ladder allele, but whose size is just
outside a window because of measurement error

5 Accuracy, precision, and reproducibility
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 in one capillary run. Table 3 on page 70 indicates typical precision results
obtained from the seven injections of the AmpFlSTR™ Identifiler™ Allelic Ladder
analyzed on the 310 Genetic Analyzer (47-cm capillary and POP-4™ polymer). The
internal size standard used was GeneScan™ 500 LIZ™ Size Standard. These results
were obtained within a set of injections on a single capillary.
As indicated above, sample alleles may occasionally size outside of the ±0.5-bp
window for a respective allelic ladder allele because of measurement error. The
frequency of such an occurrence is lowest in detection systems having the smallest
standard deviations in sizing. Figure 7 on page 69 illustrates the tight clustering of
allele sizes obtained on the 310 Genetic Analyzer, where the standard deviation in
sizing is typically less than 0.15 bp. The instance of a sample allele sizing outside of the
±0.5-bp window because of measurement error is relatively rare when the standard
deviation in sizing is approximately 0.15 bp or less (Smith, 1995).
For sample alleles that do not size within a ±0.5-bp window, the PCR product must be
rerun to distinguish between a true off-ladder allele vs. measurement error of a sample
allele that corresponds with an allele in the allelic ladder. Repeat analysis, when
necessary, provides an added level of confidence to the final allele assignment.
GeneMapper™ ID Software and GeneMapper™ ID-X Software automatically flags
sample alleles that do not size within the prescribed window around an allelic ladder
allele.
It is important to note that while the precision within a set of capillary injections is very
good, the determined allele sizes vary between platforms. Cross-platform sizing
differences arise from a number of parameters, including type and concentration of
polymer mixture, run temperature, and electrophoresis conditions. Variations in sizing
can also be found between runs on the same instrument and between runs on different
instruments because of these parameters. We strongly recommend that the allele sizes
obtained be compared to the sizes obtained for known alleles in the AmpFlSTR™
Identifiler™ Allelic Ladder from the same run and then converted to genotypes. For
more information on precision and genotyping, see Lazaruk et al., 1998 and Mansfield
et al.,1998.
1187 population database DNA samples have been typed using the Identifiler™ Kit
(see“About the primers” on page 11). These samples have been previously genotyped
with concordant results of the same loci, using other AmpFlSTR™ kits.
Table 3 Example of precision results of seven injections of the AmpFlSTR™ Identifiler™ Allelic
Ladder
Allele Mean Standard Deviation

Amelogenin
X 107.02 0.04
Y 112.61 0.02
CSF1PO
6 304.69 0.08
7 309.01 0.10


8 313.30 0.10
9 317.55 0.11
10 321.97 0.12
11 325.86 0.11
12 329.97 0.13
13 334.00 0.10
14 338.04 0.11
15 341.84 0.08
D2S1338
15 307.30 0.11
16 311.65 0.11
17 315.91 0.12
18 320.16 0.12
19 324.34 0.12
20 328.44 0.08
21 332.58 0.11
22 336.62 0.09
23 340.57 0.11
24 344.18 0.07
25 347.78 0.07
26 351.39 0.07
27 355.08 0.07
28 358.77 0.05
D3S1358
12 111.96 0.06
13 116.04 0.04
14 119.99 0.04
15 123.89 0.02
16 128.06 0.05
17 132.24 0.05
18 136.30 0.06
19 140.43 0.03
D5S818
7 134.14 0.05
8 138.21 0.04
9 142.56 0.04


10 147.02 0.06
11 151.31 0.01
12 155.63 0.05
13 159.81 0.06
14 164.04 0.07
15 167.95 0.05
16 172.09 0.05
D7S820
6 255.15 0.08
7 259.21 0.07
8 263.24 0.07
9 267.26 0.09
10 271.32 0.08
11 275.35 0.06
12 279.42 0.07
13 283.42 0.06
14 287.48 0.10
15 291.58 0.06
D8S1179
8 123.29 0.07
9 127.32 0.05
10 131.41 0.05
11 135.49 0.04
12 139.73 0.04
13 144.25 0.03
14 148.71 0.06
15 153.16 0.07
16 157.51 0.07
17 161.72 0.05
18 165.84 0.07
19 169.92 0.05
D13S317
8 216.87 0.05
9 220.83 0.05
10 224.77 0.07
11 228.88 0.07


12 232.81 0.05
13 236.68 0.07
14 240.69 0.06
15 244.68 0.09
D16S539
5 252.37 0.08
8 264.30 0.07
9 268.32 0.08
10 272.32 0.06
11 276.37 0.07
12 280.37 0.09
13 284.34 0.07
14 288.44 0.09
15 292.51 0.07
D18S51
7 262.07 0.08
9 270.22 0.06
10 274.34 0.09
10.2 276.36 0.06
11 278.41 0.08
12 282.49 0.05
13 286.57 0.06
13.2 288.63 0.05
14 290.77 0.04
14.2 292.78 0.05
15 294.91 0.07
16 299.07 0.06
17 303.50 0.07
18 307.94 0.09
19 312.40 0.11
20 316.71 0.09
21 320.99 0.14
22 325.24 0.11
23 329.40 0.11
24 333.54 0.15
25 337.67 0.11


26 341.56 0.09
27 345.24 0.08
D19S433
9 101.99 0.05
10 105.88 0.05
11 109.78 0.04
12 113.64 0.02
12.2 115.61 0.02
13 117.56 0.03
13.2 119.55 0.02
14 121.46 0.03
14.2 123.47 0.02
15 125.45 0.05
15.2 127.43 0.05
16 129.44 0.05
16.2 131.46 0.05
17 133.42 0.03
17.2 135.44 0.06
D21S11
24 184.86 0.04
24.2 186.82 0.02
25 188.77 0.03
26 192.69 0.05
27 196.56 0.04
28 200.41 0.05
28.2 202.36 0.05
29 204.32 0.03
29.2 206.31 0.02
30 208.29 0.07
30.2 210.24 0.05
31 212.23 0.05
31.2 214.14 0.06
32 216.14 0.04
32.2 218.10 0.04
33 220.14 0.05
33.2 222.07 0.04


34 224.10 0.07
34.2 226.02 0.06
35 228.07 0.06
35.2 230.01 0.07
36 232.04 0.07
37 236.00 0.03
38 239.94 0.08
FGA
17 214.81 0.07
18 218.80 0.06
19 222.79 0.07
20 226.81 0.06
21 230.76 0.08
22 234.78 0.07
23 238.81 0.05
24 242.83 0.07
25 246.88 0.06
26 250.96 0.06
26.2 253.00 0.09
27 254.97 0.08
28 259.02 0.10
29 263.12 0.08
30 267.26 0.09
30.2 269.07 0.10
31.2 273.17 0.09
32.2 277.24 0.08
33.2 281.33 0.09
42.2 319.83 0.14
43.2 324.04 0.14
44.2 328.26 0.13
45.2 332.42 0.16
46.2 336.43 0.14
47.2 340.42 0.14
48.2 344.15 0.10
50.2 351.45 0.05
51.2 355.13 0.05


TH01
4 163.29 0.04
5 167.36 0.03
6 171.40 0.05
7 175.40 0.03
8 179.38 0.04
9 183.36 0.05
9.3 186.93 0.02
10 187.29 0.04
11 191.23 0.03
13.3 201.94 0.05
TPOX
6 222.07 0.04
7 226.02 0.06
8 229.91 0.03
9 233.86 0.06
10 237.88 0.07
11 241.83 0.06
12 245.77 0.07
13 249.78 0.08
vWA
11 154.59 0.08
12 158.87 0.07
13 163.00 0.05
14 167.27 0.05
15 171.15 0.05
16 175.15 0.04
17 179.15 0.04
18 183.08 0.04
19 187.00 0.04
20 190.93 0.05
21 194.80 0.05
22 198.62 0.06
23 202.44 0.05
24 206.69 0.08

Extra Peaks in the electropherogram 5
Extra Peaks in the electropherogram

Causes of extra To further demonstrate reproducibility, 1187 population database DNA samples have
peaks been typed using the Identifiler™ Kit. These samples have been previously
genotyped with concordant results of the same loci using other AmpFlSTR™ kits.
Peaks other than the target alleles may be detected on the electropherogram displays.
Several causes for the appearance of extra peaks, including the stutter product (at the
n–4 position), incomplete 3´ A nucleotide addition (at the n–1 position), artifacts, and
mixed DNA samples (see “DAB 8.1.2.2 Mixture Studies” on page 91).
Stutter products The PCR amplification of tetranucleotide STR loci typically produces a minor product
peak four bases shorter (n–4) than the corresponding main allele peak. This is referred
to as the stutter peak or product. Sequence analysis of stutter products at
tetranucleotide STR loci has revealed that the stutter product is missing a single
tetranucleotide core repeat unit relative to the main allele (Walsh et al.,1996).
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. Such measurements have been made for amplified samples at the loci used in the
Identifiler™ Kit. All data were generated on the 310 Genetic Analyzer.
Some of the general conclusions from these measurements and observations are as
follows:
• For each Identifiler™ Kit locus, the percent stutter generally increases with allele
length, as shown in Figure 8 through Figure 12 on the following pages. Smaller
alleles display a lower level of stutter relative to the longer alleles within each
locus. This is reflected in Figure 8 through Figure 11, where minimal data points
are plotted for some smaller alleles, as stutter could not be detected for many of
these samples.
• For the alleles within a particular locus, the percent stutter is generally greater for
the longer allele in a heterozygous sample (this is related to the first point above).
• Each allele within a locus displays percent stutter that is reproducible.
• The highest percent stutter observed for each allele is as follows: CSF1PO, 9.2%;
D2S1338, 11.1%; D3S1358, 10.7%; D5S818, 6.8%; D7S820, 8.2%; D8S1179, 8.2%;
D13S317, 8.0%; D16S539, 10.4%; D18S51, 17.0%; D19S433, 13.3%; D21S11, 9.4%;
FGA, 14.7%; TH01, 5.1%; TPOX, 4.8% and vWA, 12.6%.
• The highest observed percent stutter for each locus is included as the filter in the
GeneMapper™ ID Software and the GeneMapper™ ID-X Software. Peaks in the
stutter position that are above the highest observed percent stutter will not be
filtered. Peaks in the stutter position that have not been filtered and remain
labeled can be further evaluated. For evaluation of mixed samples, see “Mixture
studies” on page 91.

5 Extra Peaks in the electropherogram
• The percent stutter does not change significantly with the recommended quantity
of input DNA, for on-scale data. The measurement of percent stutter may be
unusually high for main peaks that are off-scale.
• The percent stutter for allele 15 in D3S1358 (Figure 9) is artificially increased due
to a reproducible artifact (Figure 4-8) observed in the green dye lanes at this
position. When analyzing samples which contain a D3S1358 allele 15, we
recommend careful examination due to the contribution that this identified
artifact may add to the observed peak height or area. The highest percent stutter
for D3S1358 is not inconclusive of allele 15.
Figure 8 Stutter percentages for the D8S1179, D21S11, D7S820, and CSF1PO loci
16
15
14
13
12
11
10
9
Stutter
7
Percent
0
8 9 10 11 12 13 14 15 16 17 25 26 27 28 29 30 31 32 33 34 35 36 8 9 10 11 12 13 14 8 9 10 11 12 13 14
D8S1179 D21S11 D7S820 CSF1PO
Allele

Figure 9 Stutter percentages for the D3S1358, TH01, D13S317, D16S539, and D2S1338 loci. See
the comment on page 78 regarding stutter at allele 15 of D3S1358
30
25
20
Percent Stutter
15
10
0
12 13 14 15 16 17 18 19 6 7 8 9 10 11 8 9 10 11 12 13 14 8 9 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25 26
D3S1358 TH01 D13S317 D16S539 D2S1338
Allele
Figure 10 Stutter percentages for the D19S433, vWA, TPOX, and D18S51 loci
18
17
16
15
14
13
12
11
10
Percent Stutter
0
9 10 11 12 13 14 15 16 17 11 12 13 14 15 16 17 18 19 20 21 6 7 8 9 10 11 12 11 12 13 14 15 16 17 18 19 20 21 22 23
D19S433 vWA TPOX D18S51
Allele

5 Extra Peaks in the electropherogram
Figure 11 Stutter percentages for the D5S818 and FGA loci

16
14
12
10
Percent Stutter
0
7 8 9 10 11 12 13 14 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
D5S818 FGA
Allele
Figure 12 Sample 1 in panel A and panel B has a profile of 15, 16 for D3S1358. The amount
of stutter can not be accurately measured due to the VIC™ dye artifact. Note the degree of
magnification (y-axis) used in panels B and C to illustrate the artifact. Data was produced on
the 310 Genetic Analyzer.
Sample 1
A
Sample 1
B
Negative
Control
C

Addition of 3´ A AmpliTaq Gold™ enzyme, like many other DNA polymerases, can catalyze the
nucleotide addition of a single nucleotide (predominately adenosine) to the 3´ ends of
double-stranded PCR products (Clark, 1988; Magnuson et al.,1996). This non-template
addition results in a PCR product that is one base pair longer than the actual target
sequence, and the PCR product with the extra nucleotide is referred to as the “+A”
form (Figure 13).
The efficiency of “A addition” is related to the particular sequence of the DNA at the 3
´ end of the PCR product. The Identifiler™ Kit includes two main design features
that promote maximum A addition:
• The primer sequences have been optimized to encourage A addition.
• The final extension step is 60°C for 60 minutes.
This final extension step gives the AmpliTaq Gold™ DNA Polymerase extra time to
complete A addition to all double-stranded PCR product. STR systems that have not
been optimized for maximum A addition may have “split peaks”, where each allele is
represented by two peaks one base pair apart.
Figure 13 Split peaks resulting from incomplete A nucleotide addition due to omission of the
60-minute extension step
The AmpliTaq Gold™ DNA Polymerase generally requires extra time to complete the
A nucleotide addition at the 3´ end of the PCR products.
Lack of full A nucleotide addition may be observed in Identifiler™ Kit results when the
amount of input DNA is greater than recommended protocols. This is because more
time is needed for AmpliTaq Gold™ DNA Polymerase to add the A nucleotide to all
molecules as more PCR product is generated. Amplification of too much input DNA
will also result in off-scale data.
Artifacts Artifacts, or anomalies, have been seen in data produced on the 310 Genetic Analyzer
when using the Identifiler™ Kit. The shape of these artifacts is not consistent with the
shape of labeled DNA fragments as seen on the 310 Genetic Analyzer. Artifacts may or
not be reproducible.

5 Characterization of loci
Artifacts can be intermittent and are not always reproducible. In our experience, non-
reproducible artifacts can be correlated to sources other than the kit (that is, spikes). An
intermittent artifact is not observed in the same position upon re-injection.
Figure 14 demonstrates reproducible artifacts while using the Identifiler™ Kit. Consider
these artifacts when interpreting data.
Figure 14 Reproducible anomalies in the blue, green, yellow, and red dye electropherograms
when using the Identifiler™ Kit. Genotyping may result in the detection of these artifacts as off-
ladder alleles, or “OL Alleles”. Note the degree of magnification (y-axis) used in this figure to
illustrate these artifacts. Data produced on the 310 Genetic Analyzer.
Characterization of loci
DAB 8.1.2.1 “Documentation exists and is available which defines and characterizes the locus.” (DAB,
Documentation 1998).
Overview This section describes basic characteristics of the 16 loci that are amplified with the
Identifiler™ Kit. These loci have been previously characterized.
Nature of the The primers for the Amelogenin locus flank a six-base pair deletion within intron 1 of
polymorphisms the X homologue. Amplification results in 107-bp and 113-bp products from the X and
Y chromosomes, respectively. (Sizes are the actual base pair size according to
sequencing results, including 3' A nucleotide addition.) The remaining Identifiler™
Kit loci are all tetranucleotide short tandem repeat (STR) loci. The length differences
among alleles of a particular locus result from differences in the number of 4–bp repeat
units.

Species specificity 5
We have subjected to DNA sequencing some alleles in the AmpFlSTR™ Identifiler™

Allelic Ladder containing partial repeat units in population database and nonhuman
primate DNA samples (Lazaruk, et al., 2001). In addition, other groups in the forensic
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 Identifiler™
Kit loci, there is consensus on the repeat patterns and structure of the STRs.
Inheritance The AmpFlSTR™ loci have been validated by family studies to demonstrate their
mode(s) of inheritance.
The Centre d’Etude du Polymorphisme Humain (CEPH) has collected DNA from
39 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).
Four CEPH family DNA sets were examined. One and a half nanograms of DNA
from each sample were amplified using the AmpFlSTR™ SGM Plus™ kit, followed by
analysis using an 377 DNA Sequencer. The families examined included #1331
(11 offspring), #13291 (9 offspring), #13292 (9 offspring), and #13294 (8 offspring),
representing 37 meiotic divisions. The results confirmed that the loci are inherited
according to Mendelian rules, as has been reported in the literature (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).
Mapping The Identifiler™ Kit loci Amelogenin, CSF1PO, D2S1338, D3S1358, D5S818, D7S820,
D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX and vWA
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).
Species specificity
DAB 8.1.2.2 “Species specificity, sensitivity, stability and mixture studies are conducted.” (DAB, 1998).
Species Specificity The Identifiler™ Kit provides the required degree of specificity such that it is specific
to primates. Other species do not amplify for the loci tested, with the exception of the
Amelogenin locus.
Nonhuman Studies
Nonhuman DNA may be present in forensic casework samples. The Identifiler™ Kit
provides the required degree of specificity such that it is specific to primates for the
species tested (with the exception of the Amelogenin locus) (Figure 15).

5 Species specificity
Figure 15 Representative electropherograms of a primate, non-primates, a microorganism, and

a negative control are shown. All samples were analyzed on an 310 Genetic Analyzer. The
peaks shown in orange are the GeneScan™ 500 LIZ™ Size Standard.
Chimp
Horse
Pig
Dog
E. coli
Negative Control
The following experiments were conducted to investigate interpretation of

Identifiler™ Kit results from nonhuman DNA sources.
The extracted DNA samples were amplified in Identifiler™ Kit reactions and
analyzed using the 310 Genetic Analyzer.
• Primates – Gorilla, chimpanzee, orangutan, and macaque (1.0 ng each).
• Non-primates – Mouse, dog, pig, cat, horse, chicken and cow (2.5 ng each).
• Bacteria and yeast – Brochothrix, Escherichia, Neisseria, Pseudomonas, Bacillus,
Staphylococcus (approximately 5 ng each), and Saccharomyces (1 ng).
The primate DNA samples all amplified, producing fragments within the 100–400 base
pair region (Lazaruk, et al., 2001; Wallin et al.,1998).
The microorganisms, chicken, cow, cat and mouse did not yield detectable product.
Horse, dog, and pig produced a 103-bp fragment near the Amelogenin locus in PET™
dye.

Sensitivity 5
Sensitivity
Sensitivity
Effect of DNA The amount of input DNA added to the Identifiler™ Kit should be between 0.5 and
quantity on results 1.25 ng (Figure 16 on page 86). The DNA sample should be quantitated prior to
and importance of amplification using a system such as the Quantifiler™ Human DNA Quantitation
Kit (Part No. 4343895). The final DNA concentration should be in the range of
quantitation 0.05–0.125 ng/μL so that 0.5–1.25 ng of DNA will be added to the PCR reaction in a
volume of 10 μL. If the sample contains degraded DNA, amplification of additional
DNA may be beneficial.
If too much DNA is added to the PCR reaction, then the increased amount of PCR
product that is generated can result in the following:
• Fluorescence intensity that exceeds the linear dynamic range for detection by the
instrument (“off-scale” data).
Off-scale data is a problem for two reasons:
– 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, which results in
poor spectral separation (“pull-up”).
• Incomplete A nucleotide addition.
The sample can be re-amplified using less DNA.
When the total number of allele copies added to the PCR is extremely low, unbalanced
amplification of the two alleles of a heterozygous individual may occur (Walsh
et al.,1992; Wallin et al.,1998) due to stochastic fluctuation in the ratio of the two
different alleles (Sensabaugh et al.,1991). The PCR cycle number and amplification
conditions have been specified to produce low peak heights for a sample containing
20 pg human genomic DNA. Low peak heights should be interpreted with caution.
Individual laboratories may find it useful to determine an appropriate minimum peak
height threshold based on their own results and instruments using low amounts of
input DNA.

5 Stability
Figure 16 Effect of amplifying various amounts of DNA ranging from 16 pg to 1 ng. Note that the y-axis scale is magnified
for the lower amounts of DNA. Data analyzed using the 310 Genetic Analyzer
Stability
Stability
Lack of As with any multi-locus system, the possibility exists that not every locus will amplify.
amplification of This is most often observed when the DNA sample contains PCR inhibitors or when
the DNA sample has been severely degraded. Because each locus is an independent
some loci
marker whose results are not based upon information provided by the other markers,
results generally can still be obtained from the loci that do amplify.

Stability 5
Differential and Differential amplification can be defined as the difference in the degree of
preferential amplification of each locus within a co-amplified system, such that one or more loci
may amplify to a greater extent compared to the other loci. Preferential amplification is
amplification
used in this guide to describe differences in the amplification efficiency of two alleles
at a single locus.
Preferential amplification of alleles in systems that distinguish alleles based on length
polymorphisms is most likely to be observed when the alleles differ significantly in
base pair size. Since most Identifiler™ Kit loci have small size ranges, the potential
for preferential amplification of alleles is low.
Effect of inhibitors Heme compounds have been identified as PCR inhibitors in DNA samples extracted
from bloodstains (DeFranchis et al.,1988; Akane et al., 1994). It is believed that the
inhibitor is co-extracted and co-purified with the DNA and subsequently interferes
with PCR by inhibiting polymerase activity.
Bovine serum albumin (BSA) can prevent or minimize the inhibition of PCR, most
likely by binding to the inhibitor (Comey et al., 1994). Since the presence of BSA can
improve the amplification of DNA from blood-containing samples, BSA has been
included in the AmpFlSTR™ PCR Reaction Mix at 4 μg per 25-μL amplification. BSA
has also been identified as an aid in overcoming inhibition from samples containing
dyes, such as in denim (Comey et al., 1994).
To examine the effects of hematin on the amplification results obtained by the
Identifiler™ Kit, DNA samples were amplified using the Identifiler™ Kit reagents
(including the BSA-containing PCR reaction mix) in the presence of varying
concentrations of purified hematin. The concentrations of hematin used were 0 μM,
10 μM, 12 μM, 14 μM, 16 μM, 18 μM, and 20 μM. When the amount of hematin was
increased to a concentration that started to inhibit the PCR, CSF1PO and D2S1338 were
the first loci to exhibit decreased amplification, followed by D16S539 and D18S51.
Differential amplification was observed in the presence of increasing amounts of
hematin. Moreover, as the concentration of hematin was increased, the overall yield of
products was reduced particularly for the larger loci.

5 Stability
Figure 17 DNA amplified with the Identifiler™ Kit in the presence of varying concentrations of hematin: 0, 10 μM, 12 μM, 14
μM, 16 μM, 18 μM, and 20 μM, analyzed on the 310 Genetic Analyzer
Degraded DNA As the average size of degraded DNA approaches the size of the target sequence, the
amount of PCR product generated is reduced. This is due to the reduced number of
intact templates in the size range necessary for amplification.
Degraded DNA was prepared to examine the potential for differential amplification of
loci. High molecular weight DNA was incubated with the enzyme DNase I for varying
amounts of time. The DNA was examined by agarose gel analysis to determine the
average size of the DNA fragments at each time point.
Four nanograms of degraded DNA (or 1 ng undegraded DNA) was amplified using
the Identifiler™ Kit (all 16 primer pairs together). As the DNA became increasingly
degraded the loci became undetectable according to size. Preferential amplification
was not observed. The loci failed to robustly amplify in the order of decreasing size as
the extent of degradation progressed: CSF1PO and D2S1338 were the first loci to
exhibit decreased amplification, followed by D16S539 and D18S51 and so forth. A
similar result at each time point was obtained whether the DNA samples were
amplified for each locus alone or co-amplified with the Identifiler™ Kit (Figure 18 on
page 89).

Stability 5
Figure 18 Multiplex amplifications of a DNA sample in the absence of DNase I and the sample incubated for 30 sec, 1 min,
4 min, and 8 min with DNase I, analyzed using the 310 Genetic Analyzer
Multiplex DNA samples were amplified in 16 separate reactions containing primers for only one
amplifications Identifiler™ Kit locus (singleplex) and a reaction containing all primers for the
Identifiler™ Kit loci (multiplex). DNA used as PCR template consisted of a sample that
had been degraded for 1 min with DNase I.
Amplified samples were analyzed using the 310 Genetic Analyzer. Similar results were
obtained (genotype and peak height) whether the DNA samples were amplified for
each locus alone or co-amplified in the Identifiler™ Kit reaction(Figure 19 on page
90).
When degraded DNA is suspected to have compromised amplification of one or more
loci, the molecular weight of the DNA can be assessed by agarose gel analysis. If the
DNA is degraded to an average of 400 base pairs in size or less, adding more DNA
template to the Identifiler™ Kit amplification reaction may help produce a typeable
signal for the loci. Adding more DNA to the amplification may provide more of the
necessary size template for amplification.

5 Stability
Figure 19 Multiplex and singleplex amplifications of a DNA sample incubated for 1 min with
DNase I, analyzed on the 310 Genetic Analyzer

Mixture studies 5
Mixture studies
Mixture Studies Evidence samples may contain DNA from more than one individual. The possibility of
multiple contributors should be considered when interpreting the results. We
recommend that individual laboratories assign a minimum peak height threshold
based on validation experiments performed in each laboratory to avoid typing when
stochastic effects are likely to interfere with accurate interpretation of mixtures.
Mixed specimen Evidence samples that contain body fluids and/or tissues originating from more than
studies one individual are an integral component of forensic casework. Therefore it is essential
to ensure that the DNA typing system is able to detect DNA mixtures. In the case of
STRs, stutter peaks may be informative in the interpretation of mixed samples.
Furthermore, alleles amplified with the Identifiler™ Kit have similar peak height
values for a heterozygous genotype within a locus. This balance can be used as an aid
in detecting and interpreting mixtures.
Detection of mixed samples

Each of the following can aid in determining whether a sample is a mixture:
• The presence of more than two alleles at a locus.
• The presence of a peak at a stutter position that is significantly greater in
percentage than what is typically observed in a single-source sample.
• Significantly imbalanced alleles for a heterozygous genotype.
The peak height ratio is defined as the height of the lower peak (in RFU) divided
by the height of the higher peak (in RFU), expressed as a percentage. Mean,
median, and minimum and maximum peak height ratios observed for alleles in
the Identifiler™ Kit loci in unmixed population database samples are listed in
Table 4.
Table 4 Peak height ratios
Number of
Allele Mean† Median† Minimum† Maximum†
Observations (n)
CSF1PO 84 86 88 63.6 99.8
D2S1338 93 84 86 42.8 99.7
D3S1358 91 88 90 64.3 99.7
D5S818 82 89 91 64.9 99.7
D7S820 96 89 90 66.2 99.5
D8S1179 89 90 93 57.5 99.8
D13S317 96 87 87 63.3 100.0
D16S539 92 88 91 61.5 99.9
D18S51 99 82 83 56.3 99.9
D19S433 98 88 92 48.8 100.0
D21S11 92 88 89 66.4 99.6
FGA 94 85 87 60.9 99.5

5 Mixture studies
Number of
Allele Mean† Median† Minimum† Maximum†
Observations (n)
TH01 99 86 88 48.8 99.9
TPOX 87 87 92 55.9 99.8
vWA 101 86 88 62.8 99.1
† Peak height ratios were determined for those heterozygous samples with peak heights > 200 RFU.
For all 15 loci, the mean peak height ratios indicate that the two alleles of a
heterozygous individual are generally very well balanced.
If an unusually low peak height ratio is observed for one locus and there are no other
indications that the sample is a mixture, the sample may be reamplified and
reanalyzed to determine if the imbalance is reproducible. Possible causes of imbalance
at a locus are degraded DNA, presence of inhibitors, extremely low amounts of input
DNA, or the presence of an allele containing a rare sequence that does not amplify as
efficiently as the other allele.
Resolution of genotypes in mixed samples

A sample containing DNA from two sources can be comprised (at a single locus) of
any of the seven genotype combinations listed below.
• Heterozygote + heterozygote, no overlapping alleles (four peaks)
• Heterozygote + heterozygote, one overlapping allele (three peaks)
• Heterozygote + heterozygote, two overlapping alleles (two peaks)
• Heterozygote + homozygote, no overlapping alleles (three peaks)
• Heterozygote + homozygote, overlapping allele (two peaks)
• Homozygote + homozygote, no overlapping alleles (two peaks)
• Homozygote + homozygote, overlapping allele (one peak)
Specific genotype combinations and input DNA ratios of the samples contained in a
mixture determine whether it is possible to resolve the genotypes of the major and
minor component(s) at a single locus.
The ability to obtain and compare quantitative values for the different allele peak
heights on Applied Biosystems instruments provides additional valuable data to aid in
resolving mixed genotypes. This quantitative value is much less subjective than
comparing relative intensities of bands on a stained gel.
Ultimately, the likelihood that any sample is a mixture must be determined by the
analyst in the context of each particular case, including the information provided from
known reference sample(s).
Limit of detection of the minor component

Mixtures of two DNA samples were examined at various ratios (1:1 to 1:10). The total
amount of genomic input DNA mixed at each ratio was 1 ng.
The samples were amplified in a GeneAmp™ PCR System 9700 with a silver or gold-
plated silver block and were electrophoresed and detected using a 310 Genetic
Analyzer.

Mixture studies 5
The results of the mixed DNA samples are shown in Figure 20, where sample A and
sample B were mixed according to the ratios provided.
The profiles of the samples in Figure 20 are listed in Table 5.
Table 5 Mixture profiles
Profile
Allele Sample A Sample B
Amelogenin X X, Y
CSF1PO 10, 12 11,12
D2S1338 17, 25 20, 23
D3S1358 15, 18 15,16
D5S818 11, 13 11
D7S820 9, 10 7,12
D8S1179 13 12,13
D13S317 11 11
D16S539 11, 12 9, 10
D18S51 17, 19 12, 15
D19S433 13 14,15
D21S11 30, 30.2 28, 31
FGA 23.2, 24 24, 26
TH01 7, 9 7, 9.3
TPOX 8, 9 8
vWA 17, 19 14,16
For these 1-ng total DNA mixture studies, the limit of detection is when the minor
component is present at approximately one-tenth of the concentration of the major
component and a threshold of 50 RFU. The limit of detection for the minor component
is influenced by the combination of genotypes in the mixture.

5 Data interpretation
Figure 20 Results of the two DNA samples mixed together at defined ratios and amplified with the Identifiler™ Kit.
Sample A and Sample B are a female and male sample, respectively. The ratios of Sample A to Sample B (A:B ratios)
shown are 10:1, 3:1, 1:1, 1:3, and 1:10, respectively. The alleles attributable to the minor component, even when the major
component shares an allele, are highlighted in panels 2, 3, 5, and 6. All alleles are highlighted in panel 4.
Panel 1 Sample A
2 10:1
3 3:1
4 1:1
5 1:3
6 1:10
7 Sample B
Data interpretation
Minimum sample The Identifiler™ Kit has been optimized to reliably amplify and type
requirement approximately 0.5–1.25 ng of sample DNA.
The PCR cycle number and amplification conditions have been specified to produce
low peak heights for a sample containing 20 pg human genomic DNA. Thus, the
overall sensitivity of the assay has been adjusted to avoid or minimize stochastic
effects. We have successfully typed samples containing less than 0.5 ng DNA.
Note: Individual laboratories may find it useful to determine an appropriate
minimum peak height threshold based on their own results/instruments using low
amounts of input DNA.
Population data
DAB 8.1.2.3 “Population distribution data are documented and available.” (DAB, 1998).
Population Data
DAB 8.1.2.3.1 “The population distribution data would include the allele and genotype distributions for the
Population locus or loci obtained from relevant populations. Where appropriate, databases should be tested
for independence expectations.” (DAB, 1998).
Distribution Data

Population data 5
Overview To interpret the significance of a match between genetically typed samples, it is

necessary to 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
suspect’s reference sample, then the suspect is “excluded” as the donor of the
biological evidence tested. An exclusion is independent of the frequency of the two
genotypes in the population.
If the suspect and evidence samples have the same genotype, 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 population(s).
Population The Identifiler™ Kit, prior to the addition of the D8S1179 degenerate primer, was
samples used in used to generate the population data provided in this section. Samples were collected
from individuals throughout the United States with no geographical preference.
these studies
Number
Population of Samples provided by
samples
African-American 357 Kentucky State Police and the Federal Bureau of
Investigation
U.S. Caucasian 349
U.S. Hispanic 290 Minnesota Bureau of Criminal Apprehension/Memorial
Blood Center of Minneapolis
Native American 191
Allele frequencies Table 6 shows the Identifiler™ Kit allele frequencies in four populations, listed
as percentages.
Table 6 Identifiler™ Kit allele frequencies
African-American U.S. Caucasian U.S. Hispanic Native American

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
CSF1PO
6 † † † †
7 4.62 0.14† 0.34† †
8 7.56 0.29† 0.17† 0.52†

9 3.78 1.72 0.86† 8.38
10 27.87 24.21 23.10 30.89
11 20.59 31.81 28.28 21.99
11.3 0.14† † † †
12 29.13 32.81 39.66 32.72

13 5.32 7.31 6.38 4.71
14 0.98 1.43 0.86† 0.79†
15 † 0.29† 0.34† †
D2S1338
15 0.14† † † †
16 5.32 4.73 2.41 2.62

5 Population data

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
17 10.78 17.34 21.21 9.95
18 5.60 6.30 4.14 7.07
19 14.15 13.75 22.76 29.58
20 6.02 14.61 13.79 9.69
21 14.01 2.58 2.59 2.36
22 13.17 4.01 7.41 15.18
23 10.78 11.46 11.38 11.78
24 9.80 11.75 8.45 7.85
25 8.12 10.60 5.17 3.14
26 1.96 2.72 0.69† 0.79†
27 0.14† 0.14† † †
28 † † † †

Population data 5

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
D3S1358
<11 0.42† 0.14† † †
11 † † † 0.26†
12 0.56† † 0.17† †
13 0.70† 0.29† 0.17† †
14 12.04 15.76 7.41 6.81

15 30.53 25.36 39.14 40.84
15.2 0.14† † † †
16 28.57 22.78 26.72 32.98

17 19.47 18.19 16.03 9.95
18 6.72 16.48 8.97 8.38
19 0.84 1.00 1.03 0.79†
20 † † 0.34† †
D5S818
7 0.14† † 6.72 15.71
8 5.46 † 0.69† †
9 1.68 4.15 5.17 6.02

10 6.72 5.44 5.17 4.19
11 25.49 39.26 39.14 41.10
12 36.41 35.24 29.31 23.30
13 21.57 15.47 12.59 9.42
14 2.38 0.14† 0.69† 0.26†
15 † 0.29† 0.18† †
16 † † 0.17† †
17 0.14† † 0.17† †

5 Population data

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
D7S820
6 † 0.14† 0.17† †
7 0.42† 1.29 1.72 0.52†

8 18.77 16.48 11.72 13.09
9 13.73 17.62 6.21 8.12
10 34.45 27.22 27.41 21.99
11 19.89 18.05 28.79 28.80
12 10.78 14.76 20.17 24.08
13 1.54 3.72 3.45 3.40
14 0.42† 0.72 0.34† †
15 † † † †
D8S1179
8 0.42† 2.29 0.34† 0.52†
9 0.42† 1.15 0.34† 0.26†
10 2.38 9.74 8.45 4.71
11 3.92 6.02 5.86 3.40
12 13.31 14.04 12.07 11.52
13 23.25 32.52 32.93 37.43
14 30.11 21.35 26.21 30.63
15 20.17 9.89 10.86 9.42
16 4.62 2.72 2.41 1.57
17 1.12 0.29† 0.52† 0.52†
18 0.28† † † †
19 † † † †
D13S317
8 3.08 12.18 9.66 4.97
9 2.52 7.74 21.72 17.80
10 3.78 4.44 9.14 13.61
11 24.51 29.80 23.10 24.35
12 46.22 30.80 20.86 23.04
13 15.41 11.17 10.17 7.85
14 4.34 3.72 5.34 8.12
15 0.14† 0.14† † 0.26†

Population data 5

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
D16S539
5 † † † †
8 3.22 1.72 1.72 0.79†

9 19.05 10.46 9.31 12.30
10 10.92 5.59 15.69 15.45
11 31.51 31.95 30.17 30.89
12 18.77 30.23 29.48 27.75
13 14.85 16.76 11.55 10.73
14 1.54 3.01 2.07 2.09
15 0.14† 0.29† † †
D18S51
7 † † † †
9 0.14† † † †
10 0.28† 0.86 0.52† 0.79†

10.2 0.14† † † †
11 0.28† 1.15 1.21 †
12 7.00 13.90 10.34 14.92

13 4.34 12.18 14.48 9.16
13.2 0.42† † † †
14 6.86 16.76 15.52 26.96

14.2 0.28† † † †
15 19.47 13.61 16.55 12.04

16 16.53 13.61 11.72 10.73
17 18.21 12.32 14.14 14.66
18 11.90 7.74 6.72 2.62
19 6.02 4.44 4.14 3.93
20 4.90 1.72 2.24 1.83
21 2.10 1.00 1.03 1.31
22 0.70† 0.43† 0.52† 0.79†
23 0.42† 0.14† 0.52† 0.26†
24 † 0.14† 0.17† †
25 † † 0.17† †
26 † † † †
27 † † † †

5 Population data

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
D19S433
9 † 0.14† 0.17† †
10 1.54 † † †
11 7.14 0.72 0.52† 0.52†

11.2 0.14† † 0.17† †
12 10.78 7.74 6.21 3.14

12.2 6.30 0.57† 1.90 †
13 29.83 28.94 16.03 17.80

13.2 5.74 1.72 8.62 15.45
14 21.01 34.10 31.72 24.87
14.2 4.20 0.86 5.00 3.66
15 4.76 15.76 13.45 13.35
15.2 3.36 2.72 8.79 10.73
16 2.38 4.15 4.31 3.93
16.2 2.38 1.72 2.93 1.83
17 † 0.29† 0.17† 0.79†
17.2 0.28† 0.29† † 2.88
18.2 0.14† 0.29† † 1.05†

Population data 5

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
D21S11
24 † † † †
24.2 0.14† 0.43† 0.17† †
24.3 0.28† † † †
25 † † † †
25.2 † 0.14† 0.17† †
26 0.14† 0.14† 0.17† †
27 5.04 4.58 1.21 0.52†

28 22.97 16.76 9.14 6.28
28.2 † † † †
29 19.33 20.49 21.21 16.75

29.2 0.14† † 0.52† 0.26†
29.3 0.14† † † †
30 17.23 25.21 29.31 34.29

30.2 1.40 3.30 2.93 1.83
31 7.98 7.16 6.72 5.76
31.2 7.98 9.46 8.62 18.85
32 1.12 1.43 1.55 0.79†
32.2 5.88 7.16 12.93 9.69
33 0.56† † † 0.52†
33.2 3.78 3.30 4.14 3.66
34 1.26 † † †
34.1 0.14† † † †
34.2 0.14† 0.29† 0.86† 0.79†

35 2.94 † 0.34† †
35.1 0.14† † † †
35.2 † 0.14† † †
36 0.84 † † †
37 0.28† † † †
38 0.14† † † †

5 Population data

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
FGA
16 † 0.14† † †
16.1 0.14† † † †
17 † 0.29† 0.17† †
17.2 0.14† † † †
18 0.70† 2.72 0.52† 1.31

18.2 1.40 † † †
19 6.72 6.16 7.07 10.21

19.2 0.28† † † †
20 7.00 13.90 7.41 12.30

20.2 † 0.14† † †
21 12.89 16.91 14.66 12.83

21.2 † 0.29† 0.17† †
22 21.57 16.91 17.24 10.47

22.2 0.28† 1.29 0.34† 0.26†
22.3 0.14† † † †
23 14.99 15.19 11.90 15.97

23.2 0.14† † 0.86† 0.26†
24 17.51 13.75 15.34 15.71
24.2 † 0.14† 0.17† †
25 7.98 8.60 14.14 14.14

26 3.50 2.72 6.90 4.45
26.2 † † † 0.52
27 1.82 0.72 2.41 0.79†
28 1.40 0.14† 0.69† 0.52†
29 0.56† † † †
30 † † † †
30.2 0.14† † † †
31.2 † † † †
32.2 † † † †
33.2 † † † †
34.2 0.14† † † †
42.2 † † † †
43.2 † † † †
44.2 0.28† † † †
45.2 † † † 0.26†
46.2 0.14† † † †

Population data 5

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
FGA (continued)
47.2 † † † †
48.2 0.14† † † †
50.2 † † † †
51.2 † † † †
TH01
4 † † † †
5 0.28† 0.43† 0.17† †
6 11.06 20.49 22.76 20.68

7 42.86 21.78 33.62 43.98
8 20.73 11.46 8.45 5.24
8.3 † 0.14† † †
9 12.32 16.19 14.14 6.28

9.3 11.62 29.08 20.34 23.56
10 0.98 0.43† 0.52† 0.26†
11 † † † †
13.3 0.14† † † †
TPOX
6 6.72 0.14† 0.34† †
7 2.24 † 0.34† 0.26†

8 36.13 53.30 49.66 37.96
9 21.15 11.60 7.24 4.19
10 9.24 4.30 4.66 3.40
11 21.43 25.93 27.24 39.27
12 3.08 4.73 10.52 14.92
13 † † † †

5 Population data

Allele
(n = 357) (n = 349) (n = 290) (n = 191)
vWA
11 0.28† † 0.17† †
12 † † † 0.26†
13 1.26 0.43† † 0.26†
14 7.14 8.31 6.90 4.45
15 20.03 11.32 10.00 7.07
16 26.75 23.35 34.31 32.98
17 20.59 24.50 21.55 33.51
18 14.71 22.49 18.45 15.45
19 6.72 8.31 7.07 4.71
20 1.96 1.15 1.38 1.05†
21 0.28† † 0.17† 0.26†
22 0.28† † † †
23 † † † †
24 † 0.14† † †
† A minimum allele frequency (0.7% for the African-American database, 0.7% for the U.S. Caucasian database, 0.9% for the U.S. Hispanic database,
and 1.3% for the Native American database) is suggested by the National Research Council in forensic calculations.
Analyzing the four Analysis across the four databases of 2274 total chromosomes per locus revealed the
databases following number of different alleles: 10 CSF1PO alleles, 13 D2S1338 alleles, at least
12 D3S1358 alleles, 11 D5S818 alleles, 9 D7S820 alleles, 11 D8S1179 alleles, 8 D13S317
alleles, 8 D16S539 alleles, 20 D18S51 alleles, 17 D19S433 alleles, 26 D21S11 alleles,
31 FGA alleles, 9 TH01 alleles, 7 TPOX alleles, and 13 vWA alleles.
In addition to the alleles that were observed and recorded in the our databases, other
known alleles have been published or reported to us by other laboratories (see
STRBase, www.cstl.nist.gov/div831/strbase).
Low-frequency Some alleles of the Identifiler™ Kit loci occur at a low frequency. For these alleles, a
alleles minimum frequency (5/ 2n, where n = the number of individuals in the database) was
assigned for the Identifiler™ Kit African-American, U.S. Caucasian, U.S. Hispanic,
and Native American databases, as suggested in the 1996 report of the Committee on
DNA Forensic Science (National Research Council, 1996). These databases are
summarized in Table 6 on page 95 through page 104. The minimum reportable
genotype frequency at each locus is as follows: 1.19 x 10–4 for the African-American
database; 1.19 x 10–4 for the U.S. Caucasian database; 1.70 x 10–4 for the U.S. Hispanic
database; and 2.97 x 10–4 for the Native American database [p2 + p(1–p) θ, where θ =
0.01]. Hence, the minimum combined multilocus genotype frequency at 15 loci is as
follows: 1.36 x 10–59 for the African-American database; 1.36 x 10–59 for the U.S.
Caucasian database; 2.86 x 10–57 for the U.S. Hispanic database; and 1.23 x 10–53 for
the Native American database.

Mutation rate 5
Mutation rate
Estimating Estimation of spontaneous or induced germline mutation at genetic loci may be
germline achieved through comparison of the genotypes of offspring to those of their parents.
From such comparisons the number of observed mutations are counted directly.
mutations
In previous studies, genotypes of ten STR loci amplified by the AmpFlSTR™ SGM™
Plus PCR Amplification Kit were determined for a total of 146 parent-offspring allelic
transfers (meioses) at the Forensic Science Service, Birmingham, England. One
length-based STR mutation was observed at the D18S11 locus; mutation was not
detected at any of the other nine STR loci. The D18S11 mutation was represented by an
increase of one 4-bp repeat unit, a 17 allele was inherited as an 18 (single-step
mutation). The maternal/paternal source of this mutation could not be distinguished.
Additional Additional studies (Edwards et al.,1991; Edwards et al.,1992; Weber and Wong, 1993;
mutation studies Hammond et al.,1994; Brinkmann et al.,1995; Chakraborty et al.,1996; Chakraborty
et al.,1997; Brinkmann et al.,1998; Momhinweg et al.,1998; Szibor et al.,1998) of direct
mutation rate counts produced:
• Larger sample sizes for some of the Identifiler™ Kit loci.
• Methods for modifications of these mutation rates (to infer mutation rates
indirectly for those loci where these rates are not large enough to be measured
directly and/or to account for those events undetectable as Mendelian errors).
Probability of identity
Table 7 shows the Probability of Identity (PI) values of the Identifiler™ Kit
loci individually and combined.
Table 7 Probability of Identity values for the Identifiler™ Kit STR loci
Locus African-American U.S. Caucasian U.S. Hispanic Native American

CSF1PO 0.079 0.132 0.141 0.123
D2S1338 0.023 0.027 0.038 0.043
D3S1358 0.097 0.076 0.112 0.158
D5S818 0.104 0.147 0.115 0.110
D7S820 0.085 0.063 0.083 0.081
D8S1179 0.074 0.064 0.089 0.104
D13S317 0.132 0.079 0.056 0.056
D16S539 0.077 0.097 0.090 0.082
D18S51 0.033 0.031 0.031 0.046
D19S433 0.042 0.087 0.049 0.044
D21S11 0.037 0.044 0.047 0.074
FGA 0.034 0.035 0.032 0.031
TH01 0.109 0.079 0.097 0.134
TPOX 0.089 0.188 0.168 0.159

5 Probability of paternity exclusion

vWA 0.066 0.066 0.080 0.103
Combined 1.31 x 10–18 5.01 x 10–18 7.65 x 10–18 3.62 x 10–17
The PI value is the probability that two individuals selected at random will have an
identical Identifiler™ Kit genotype (Sensabaugh, 1982). The PI values for the
populations described in this section are then approximately 1/7.64 x 1017
(African-American), 1/2.00 x 1017 (U.S. Caucasian), 1/1.31 x 1017 (U.S. Hispanic),
and 1/2.76 x 1016 (Native American).
Probability of paternity exclusion

Table 8 shows the Probability of Paternity Exclusion (PE) values of the Identifiler™
Kit STR loci individually and combined.
Table 8 Probability of Paternity Exclusion for the Identifiler™ Kit STR loci

CSF1PO 0.545 0.496 0.450 0.409
D2S1338 0.748 0.725 0.671 0.399
D3S1358 0.591 0.630 0.495 0.510
D5S818 0.506 0.440 0.525 0.601
D7S820 0.591 0.582 0.574 0.492
D8S1179 0.580 0.680 0.599 0.601
D13S317 0.383 0.487 0.638 0.370
D16S539 0.649 0.566 0.567 0.428
D18S51 0.760 0.731 0.767 0.329
D19S433 0.601 0.531 0.678 0.360
D21S11 0.737 0.708 0.586 0.399
FGA 0.760 0.766 0.739 0.309
TH01 0.492 0.566 0.618 0.646
TPOX 0.521 0.329 0.392 0.687
vWA 0.709 0.625 0.555 0.528
Combined 0.9999996 0.9999992 0.9999990 0.9999527

Chapter 5 Performance Verification After Primer Manufacturing Process Improvements
5
Section 5.2 Performance Verification After Primer
Performance Validation: Primer Manufacting Process Improvements

Manufacturing Process Improvements
As part of our continual efforts to improve the quality of our products, several
improvements and updates have been made to the manufacturing process of the
Identifiler™ Kit (Part No.4322288) since its introduction in 2001.
Effective from kit lot number 0310018, modifications were made to the manufacturing
process of the Identifiler™ Kit to reduce the occurrence of artifacts in the PET™ dye and
VIC™ dye channels that may interfere with the interpretation of casework samples.
We amplified negative control samples using lot number 0301011 and lot number
0310018 and generated data using the 310 Genetic Analyzer with the Windows™ NT OS
using the G5 module. Results show that the VIC™ and PET™ labeled artifacts are
greatly reduced in the after the manufacturing process improvements (Figure 21).
Figure 21 Comparison of the observed VIC™ dye- and PET™ dye-labeled artifacts for negative control amplifications
with Identifiler™ Kit lot numbers before and after kit lot number 0310018. The artifacts have been highlighted for
illustrative purposes.
VIC™ dye labelled artifact at ~120 bp PET™ dye labeled artifacts between
Amelogenin and D5S818 loci
Lot No. 0301011 Lot No. 0301011

(representative of lots (representative of lots
0103001– 0308017) 0103001– 0308017)
Before
process
improvements
Lot No. 0310018 Lot No. 0310018

(representative of lots (representative of lots
After 0310018 and later) 0310018 and later)
process
improvements
For more information, refer to AmpFlSTR™ Identifiler™ PCR Amplification Kit

Human Identification Application Note (Pub. no. 040302).

Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement
5 Overview
Section 5.3 Performance Validation After Buffer and

Enzyme Component Replacement
Overview
As part of an ongoing program to exercise greater control over raw materials used in
the AmpFlSTR™ PCR Amplification Kits, manufacturing of the AmpliTaq Gold™
enzyme and 10✕ PCR Buffer II (Tris-KCl buffer) components is transitioning from
Roche Molecular Systems to Life Technologies. Manufacturing of both components by
Life Technologies will be conducted according to the same specifications used
previously by Roche. The in-house components are established raw materials in our
next generation kits (for example, the NGM™, NGM SElect™ and Identifiler™ Plus
Kits).
Experiments
We performed studies to compare the performance of the Identifiler™ Kit containing
the in-house components (updated kit) with the performance of the original kit,
focusing on studies most relevant to forensic DNA testing (see SWGDAM Guidelines
effective January 1, 2011). These studies, while not exhaustive, are in our opinion
appropriate for a manufacturer.
Our studies compared the performance of two Roche-manufactured enzyme and
buffer lots (Control mixes) with three new lots of buffer and two new lots of enzyme
manufactured by Life Technologies (Test mixes). Studies were performed using Test
mixes containing both the enzyme and buffer manufactured by Life Technologies.
Test
Control A mix Control B mix Test A mix Test B mix Test C mix
Material
Buffer Control Buffer Control Buffer Test Buffer Test Buffer Test Buffer
Lot 1 Lot 2 Lot 1 Lot 2 Lot 3
Enzyme Control Control Test Enzyme Control Test Enzyme

Enzyme Enzyme Lot 1 Enzyme Lot 2
Lot 1 Lot 2 Lot 2
Each of the five mixes listed above were used to conduct reproducibility, sensitivity,
and inhibition studies. All amplifications were performed using a GeneAmp™ PCR
System 9700 with either silver or gold-plated silver block using the recommended
amplification conditions and cycle number for the Identifiler™ Kit. All data was run
on an Applied Biosystems 3130xl Genetic Analyzer running Data Collection Software
v3.0 and analyzed using GeneMapper™ ID-X Software. Subsequent data analysis was
performed using Minitab™ Statistical Software. To minimize the effect of injection-to-
injection variation on result interpretation, peaks heights for all studies were
normalized using an in-house, multicolor reference standard.

Section 5.3 Performance Validation After Buffer and Enzyme Component Replacement
Reproducibility study 5
Reproducibility study
Performance Validation: Buffer and Enzyme Replacement

For the reproducibility study, 12 replicates of control DNA 007 at 1 ng input and three
negative control replicates were amplified. The results were evaluated for intracolor
balance, stutter percentage, and the presence, signal intensity, and location of artifacts.
Intracolor balance No significant difference (<10% increase or decrease) in the level of intracolor balance
was observed between the Test and Control mixes (Figure 22).
Figure 22 Reproducibility study: intracolor balance

5 Reproducibility study
Stutter Stutter percentage results for each marker were comparable across all Test and Control
percentages mixes (Figure 23).
Figure 23 Reproducibility study: mean stutter percentage
Artifacts Known artifacts observed showed the same morphology, signal intensity, and location
in all Test and Control mixes and did not exceed 50 RFU (Figure 24). No new artifacts
were observed in the Test mixes.
Figure 24 Reproducibility study: known artifacts (Y-scale 100 RFU)
VIC™ dye labeled artifact at ~70 bp NED™ dye labeled artifact at ~88 bp
Control A
Control B
Test A
Test B
Test C

Sensitivity study 5
Sensitivity study

For the sensitivity study, dilution series of three genomic DNA samples were
amplified: 1 ng (three replicates each), 0.5 ng, 0.25 ng, and 0.125 ng (four replicates
each). The results were evaluated for mean referenced peak height, degree of linearity
between input DNA concentration and peak height, level of allelic dropout at 125 pg,
and genotype concordance.
Mean referenced Mean referenced peak height observations were consistent between all Test and
peak height Control mixes (Figure 25) demonstrating equivalent performance (Figure 26).
Figure 25 Sensitivity study: mean referenced peak heights three genomic DNA samples

5 Sensitivity study
Figure 26 Sensitivity study: representative electropherograms for Sample 2 amplified using

125 pg input DNA (Y-scale 500 RFU)
Control A
Control B
Test A
Test B
Test C
DNA concentration The calculated slope and R2 values for each of the plotted curves were equivalent,
and peak height showing comparable relationships between peak height and DNA input amount for
the Test and Control mixes (Figure 27). In general, the Test mixes showed a slight
increase in peak height compared to the Control mixes.
Figure 27 Sensitivity study: linear regression plot of combined mean referenced peak height for
three genomic DNA samples

Sensitivity study 5
Allelic dropout Allelic dropout was observed only for amplifications of 125 pg where dropout of a
single allele was observed for Test A Sample 1 (Figure 28) and Control B Sample 3
(Figure 29). These results can be explained by stochastic variation and sampling from

dilute DNA solutions. Allelic dropout results can therefore be considered equivalent
between Test and Control mixes.
Figure 28 Sensitivity study: electropherogram of 125 pg Sample 1 amplified with Test A mix.
One allele at the FGA locus in the PET™ (Red) dye channel is below the analysis threshold of 50
RFU. (Y-scale 300 RFU)
Figure 29 Sensitivity study: electropherogram of 125 pg Sample 3 amplified with Control B

mix. One allele at the FGA locus in the PET™ (Red) dye channel is below the analysis
threshold of 50 RFU. (Y-scale 300 RFU)

5 Inhibition study
Genotype Genotypes for Test and Control mixes were 100% concordant (Table 9).
concordance Table 9 Sensitivity study: genotype concordance
DNA Input Amount Reagent Mix Genotype Concordance

125 pg Test A 100%
Test B 100%
Test C 100%
Control A 100%
Control B 100%
250 pg Test A 100%
Test B 100%
Test C 100%
Control A 100%
Control B 100%
500 pg Test A 100%
Test B 100%
Test C 100%
Control A 100%
Control B 100%
1 ng Test A 100%
Test B 100%
Test C 100%
Control A 100%
Control B 100%
Inhibition study
An inhibition series of 1 ng control DNA 007 (consisting of uninhibited control, Humic
Acid at a final concentration of 15.25 ng/µL, and Hematin at a final concentration of
34 µM in replicates of five) was amplified using each of Test and Control mixes. The
amount of each inhibitor tested was titrated to cause an approximate 50% reduction in
overall peak height of the samples. Results were evaluated for mean referenced peak
height, minimum referenced peak height, intracolor balance, and levels of allelic
dropout.
Mean referenced Uninhibited Control DNA 007 Test and Control mixes displayed no significant
peak height, difference in mean referenced peak height, minimum referenced peak height, and
intracolor balance. For the Humic Acid-inhibited and Hematin-inhibited DNA,
minimum
however, the Test mixes showed slightly improved performance compared to the
referenced peak Control mixes for mean referenced peak height, intracolor balance, and minimum
height, and referenced peak height (Figure 30, 31, and 32). All results obtained for all Test and
intracolor balance Control mixes fall within the expected range of performance for the Identifiler™ Kit.

Inhibition study 5
Figure 30 Inhibition study: mean referenced peak height. Inhibitors: HA = Humic Acid,
HE = Hematin, PRI = Pristine or Uninhibited DNA

Figure 31 Inhibition study: minimum referenced peak height. Inhibitors: HA = Humic Acid,
HE = Hematin, PRI = Pristine or Uninhibited DNA

5 Inhibition study
Figure 32 Inhibition study: intracolor balance. (Y-axis intracolor balance percentage versus
X-axis dye color. Inhibitors: HA = Humic Acid, HE = Hematin, PRI = Pristine or Uninhibited DNA
Representative electropherograms from the inhibition study are shown in Figure 33,
34, and 35.
Figure 33 Inhibition study: representative electropherograms using uninhibited Control DNA
007 (Y-scale 4000 RFU)
Control A
Control B
Test A
Test B
Test C

Inhibition study 5
Figure 34 Inhibition study: representative electropherograms using Control DNA 007 inhibited
with 34 μM Hematin (Y-scale 4000 RFU)

Control A
Control B
Test A
Test B
Test C
Figure 35 Inhibition study: representative electropherograms using Control DNA 007 inhibited
with15.25 ng/μL Humic Acid (Y-scale 4000 RFU)
Control A
Control B
Test A
Test B
Test C
Allelic dropout No allelic dropout events were seen for any Test or Control mixes tested on
uninhibited Control DNA 007 and Control DNA 007 inhibited with Hematin or Humic
Acid.

5 Conclusions
Conclusions
Laboratories can expect to obtain equivalent quality profiles across a wide range of
forensic samples when using the Identifiler™ Kit containing the AmpliTaq Gold™
enzyme and 10✕ PCR Buffer II manufactured by Life Technologies as compared to
the original Identifiler™ Kit containing AmpliTaq Gold™ enzyme and 10✕ PCR Buffer
II manufactured by Roche Molecular Systems.

A Troubleshooting
Follow the actions recommended in this appendix to troubleshoot problems that occur
during analysis.
Table 10 Troubleshooting
Observation Possible causes Recommended actions

Faint or no signal from Incorrect volume or absence of PCR Repeat amplification.
both the AmpFlSTR™ Master Mix or Identifiler™ Primer Set
Control DNA 9947A and
No activation of AmpliTaq Gold™ DNA Repeat amplification, making sure to hold
the DNA test samples at
Polymerase reactions initially at 95°C for 11 minutes.
all loci
Master Mix not vortexed thoroughly Vortex the Master Mix thoroughly.
before aliquoting
Identifiler™ Primer Set exposed to Store the Primer Set protected from light.
too much light
GeneAmp™ PCR System malfunction Refer to the thermal cycler user’s manual and
check instrument calibration.
Use of incorrect thermal cycling Check the protocol for correct thermal cycling
parameters parameters.
Tubes not seated tightly in the Push reaction tubes firmly into contact with block
thermal cycler during amplification after first cycle. Repeat test.
Wrong PCR reaction tube Use Applied Biosystems MicroAmp™ Reaction
Tubes with Caps for the GeneAmp™ PCR
System 9700.
MicroAmp™ Base used with tray/ Remove MicroAmp™ Base from tray/retainer
retainer set and tubes in set and repeat test.
GeneAmp™ 9700
Insufficient PCR product Prepare PCR product as described in Chapter 3,
electrokinetically injected “Perform Electrophoresis” on page 25.
Degraded formamide Check the storage of formamide; do not thaw and
refreeze multiple times. Try Hi-Di™ Formamide.

Appendix A Troubleshooting
A
Observation Possible causes Recommended actions

Positive signal from Quantity of test DNA sample is below Quantitate DNA and add 1.0 ng of DNA. Repeat
AmpFlSTR™ Control assay sensitivity test.
DNA 9947A but partial or
Test sample contains high Quantitate DNA and add minimum necessary
no signal from DNA test
concentration of PCR inhibitor (for volume. Repeat test.
samples
example, heme compounds, certain
Wash the sample in a Centricon™-100 centrifugal
dyes)
filter unit. Repeat test.
Test sample DNA is severely If possible, evaluate the quality of DNA sample by
degraded running an agarose gel. If DNA is degraded,
reamplify with an increased amount of DNA or
use the AmpFlSTR™ MiniFiler™ Kit.
Dilution of test sample DNA in water Redilute DNA using low-TE Buffer (with 0.1 mM
or wrong buffer (for example, TE EDTA).
formula with incorrect EDTA
concentration)
More than two allele Presence of exogenous DNA Use appropriate techniques to avoid introducing
present at a locus foreign DNA during laboratory handling.
Amplification of stutter product See “Stutter products” on page 77.
Mixed sample
Some but not all loci Test-sample DNA is severely If possible, evaluate the quality of DNA sample by
visible on degraded running an agarose gel. If DNA is degraded,
electropherogram of reamplify with an increased amount of DNA or
DNA test samples use the AmpFlSTR™ MiniFiler™ Kit.
Test sample contains high Quantitate DNA and add minimum necessary
concentrations of a PCR inhibitor (for volume. Repeat test.
example, heme compounds, certain
Wash the sample in a Centricon™-100 centrifugal
dyes)
filter unit. Repeat test.
Poor peak height Incorrect thermal cycler parameters Check the protocol for correct thermal cycler
balance parameters.
GeneAmp™ PCR System 9700 Use Applied Biosystems GeneAmp™ PCR System
with Aluminum 96-Well block or 9700 with silver or gold-plated silver blocks only, or
third-party thermal cyclers the Veriti™ 96-Well Thermal Cycler.

B Ordering Information
Equipment and materials not included

Table 11 and Table 12 list required and optional equipment and materials not supplied
with the Identifiler™ Kit. Unless otherwise noted, many of the items are available
from major laboratory suppliers (MLS).
Table 11 Equipment
Equipment Source
3100/3100-Avant Genetic Analyzer Contact your local
Life Technologies
Applied Biosystems 3130/3130xl Genetic Analyzer
sales representative
Applied Biosystems 3500/3500xL Genetic Analyzer for Human Identification
Applied Biosystems 310 Genetic Analyzer
GeneAmp™ PCR System 9700 with the Silver 96-Well Block N8050001
GeneAmp™ PCR System 9700 with the gold-plated silver 96-well block 4314878
Veriti™ 96-Well Thermal Cycler 4375786
Silver 96-well sample block N8050251
Gold-plated silver 96-well sample block 4314443
Tabletop centrifuge with 96-well plate adapters (optional) MLS
Table 12 User-supplied materials
Item† Source
AmpFlSTR™ Identifiler™ PCR Amplification Kit 4322288
3100 Analyzer materials
96-well plate septa 4315933
Reservoir septa 4315932
3100/3130xl Genetic Analyzer capillary array, 36-cm 4315931
POP-4™ polymer for 3100/3100-Avant Genetic Analyzers 4316355
3100/3100-Avant Genetic Analyzer Autosampler Plate Kit, 96-well 4316471
GeneScan™ 500 LIZ™ Size Standard 4322682
OR OR
GeneScan™ 600 LIZ™ Size Standard v2.0 4408399
Running Buffer, 10✕ 402824
Hi-Di™ Formamide 4311320

Appendix B Ordering Information
B Equipment and materials not included
Item† Source
DS-33 Matrix Standard Kit (Dye Set G5) 4345833
MicroAmp™ Optical 96-well reaction plate N8010560
250-µL glass syringe (array-fill syringe) 4304470
5.0-mL glass syringe (polymer-reserve syringe) 628-3731
For a complete list of parts and accessories for the 3100/3100-Avant instrument, refer to Appendix B of the 3100
Genetic Analyzer and 3100-Avant Genetic Analyzer User Reference Guide (Pub. No. 4335393).
3130xl Analyzer materials
96-well plate septa 4315933
Reservoir septa 4315932
3100/3130xl Genetic Analyzer capillary array, 36-cm 4315931
POP-4™ polymer for 3130/3130xl Genetic Analyzers 4352755
3100/3100-Avant Genetic Analyzer Autosampler Plate Kit, 96-well 4316471
OR OR
MicroAmp™ Optical 96-well reaction plate N8010560
Hi-Di™ Formamide 4311320
For a complete list of parts and accessories for the 3130/3130xl instrument, refer to Appendix A of the Applied
Biosystems 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide (Pub. No. 4352716).
3500/3500xL Analyzer materials
Anode buffer container (ABC) 4393927
Cathode buffer container (CBC) 4408256
POP-4™ polymer (960 samples) for 3500/3500xL Genetic Analyzers 4393710
POP-4™ polymer (384 samples) for 3500/3500xL Genetic Analyzers 4393715
Conditioning reagent 4393718
8-Capillary array, 36 cm for 3500 Genetic Analyzers 4404683
24-Capillary array, 36 cm for 3500xL Genetic Analyzers 4404687
96-well retainer & base set (Standard) 3500/3500xL Genetic Analyzers 4410228
8-Tube retainer & base set (Standard) for 3500/3500xL Genetic Analyzers 4410231
8-Strip Septa for 3500/3500xL Genetic Analyzers 4410701
96-Well Septa for 3500/3500xL Genetic Analyzers 4412614
Septa Cathode Buffer Container, 3500 series 4410715
For a complete list of parts and accessories for the 3500/3500xL instrument, refer to the Applied Biosystems
3500/3500xL Genetic Analyzer User Guide (Pub. No. 4401661)

Equipment and materials not included B
Item† Source
310 Analyzer materials
310 DNA Analyzer capillary array, 47-cm 402839
0.5 mL sample tray 5572
96-well tray adaptor (for 9700 thermal cycler trays) 4305051
OR OR
Genetic analyzer septa retainer clips for 96-tube sample tray 402866
Genetic analysis sample tubes (0.5-mL) 401957
Septa for 0.5-mL sample tubes 401956
DS-33 Matrix Standard Set (6-FAM™, VIC™, NED™, PET™, and LIZ™ dyes) for 310/377 systems 4318159
MicroAmp™ 8-tube strip, 0.2-mL N8010580
MicroAmp™ 96-well base (holds 0.2-mL reaction tubes) N8010531
MicroAmp™ 96-well full plate cover N8010550
MicroAmp™ 96-well tray/retainer set 403081
POP-4™ polymer for the 310 Genetic Analyzer 402838
For a complete list of parts and accessories for the 310 instrument, refer to Appendix B of the 310 Genetic Analyzer
User Guide (Pub. No. 4317588).
PCR Amplification
MicroAmp™ 96-well tray N8010541
MicroAmp™ reaction tube with cap, 0.2-mL N8010540
MicroAmp™ 8-tube strip, 0.2-mL N8010580
MicroAmp™ 8-cap strip N8010535
MicroAmp™ 96-well tray/retainer set 403081
MicroAmp™ 96-well base N8010531
MicroAmp™ clear adhesive film 4306311
MicroAmp™ optical adhesive film 4311971
MicroAmp™ optical 96-well reaction plate N8010560
Other user-supplied materials
Hi-Di™ Formamide, 25-mL 4311320
Aerosol resistant pipette tips MLS
Microcentrifuge tubes MLS
Pipettors MLS
Tape, labeling MLS
Tube, 50-mL Falcon MLS
Tube decapper, autoclavable MLS
Deionized water, PCR grade MLS

B Equipment and materials not included
Item† Source
Tris-HCL, pH 8.0 MLS
EDTA, 0.5 M MLS
Vortex MLS
† For the Safety Data Sheet (SDS) of any chemical not distributed by Life Technologies, contact the chemical manufacturer. Before handling any
chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions.

C PCR Work Areas
■ Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

■ PCR setup work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
■ Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Work area setup and lab design

Many resources are available for the appropriate design of a PCR laboratory. If you
are using the AmpFlSTR™ Identifiler™ PCR Amplification Kit for:
• Forensic DNA testing, refer to “Forensic Laboratories: Handbook for Facility
Planning, Design, Construction and Moving,” National Institute of Justice, 1998)
• Parentage DNA testing, refer to the “Guidance for Standards for Parentage
Relationship Testing Laboratories,” American Association of Blood Banks, 7th
edition, 2004
The sensitivity of the Identifiler™ 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).
Also take care while handling and processing samples 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
IMPORTANT! These items should never leave the PCR Setup Work Area.
• Calculator
• Gloves, disposable
• Marker pen, permanent
• Microcentrifuge
• Microcentrifuge tubes, 1.5-mL, or 2.0-mL, or other appropriate clean tube (for
Master Mix preparation)
• Microcentrifuge tube rack
• Pipette tips, sterile, disposable hydrophobic filter-plugged
• Pipettors

Appendix C PCR Work Areas
C Amplified DNA work area
• Tube decapper, autoclavable

• Vortex
Amplified DNA work area
IMPORTANT! Place the thermal cyclers in the Amplified DNA Work Area.
You can use the following systems:

• GeneAmp™ PCR System 9700 with the Silver 96-Well Block
• GeneAmp™ PCR System 9700 with the Gold-plated Silver 96-Well Block
IMPORTANT! The Identifiler™ Kit is not validated for use with the GeneAmp™
PCR System 9700 with the Aluminium 96-Well Block. Use of this thermal cycling
platform may adversely affect performance of the Identifiler™ Kit.
• Veriti™ 96-Well Thermal Cycler

D 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.

Appendix D Safety
D 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, and 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.
Specific chemical
CAS Chemical Phrase
handling
26628-22-8 Sodium Azide Sodium azide may react with lead and copper
plumbing to form highly explosive metal azides.

Appendix D Safety
Biological hazard safety D
Biological hazard safety
WARNING! Potential Biohazard. Depending on the samples used on this

instrument, the surface may be considered a biohazard. Use appropriate
decontamination methods when working with biohazards.
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. Follow all applicable local, state/provincial, and/or
national regulations. Wear appropriate protective equipment, which includes
but is not limited to: protective eyewear, face shield, clothing/lab coat, and
gloves. All work should be conducted in properly equipped facilities using the
appropriate safety equipment (for example, physical containment devices).
Individuals should be trained according to applicable regulatory and company/
institution requirements before working with potentially infectious materials.
Read and follow the applicable guidelines and/or regulatory requirements in
the following:
In the U.S.:
• U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories found at:
www.cdc.gov/biosafety
• Occupational Safety and Health Standards, Bloodborne Pathogens
(29 CFR§1910.1030), found at: www.access.gpo.gov/nara/cfr/waisidx_01/
29cfr1910a_01.html
• Your company’s/institution’s Biosafety Program protocols for working with/
handling potentially infectious materials.
• Additional information about biohazard guidelines is available at:
www.cdc.gov
In the EU:
Check local guidelines and legislation on biohazard and biosafety precaution
and refer to the best practices published in the World Health Organization
(WHO) Laboratory Biosafety Manual, third edition, found at: www.who.int/
csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/

Appendix D Safety
D Biological hazard safety

Documentation and Support
Related documentation
Document title Pub. No.

AmpFlSTR™ Identifiler™ PCR Amplification Kit: Human Identification: Application Note 040302
3100/3100-Avant Data Collection v2.0 User Guide 4347102
3100/3100-Avant Genetic Analyzers Using Data Collection Software v2.0 User Bulletin 4350218
3100 Genetic Analyzer User Manual (Data Collection v1.1) 4315834
3100/3100-Avant Genetic Analyzers Protocols for Processing AmpFlSTR™ PCR Amplification Kit PCR 4332345
Products User Bulletin
Applied Biosystems 3130/3100xl Genetic Analyzers Using Data Collection Software v3.0 User Bulletin 4363787
Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide 4352715
Applied Biosystems 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide 4352716
Applied Biosystems 3130/3130xl Genetic Analyzers Quick Reference Card 4362825
Applied Biosystems 3130/3130xl Genetic Analyzers AB Navigator Software Administrator Guide 4359472
Applied Biosystems 3130/3100xl DNA Analyzers User Guide 4331468
Applied Biosystems 3500/3500xL Genetic Analyzer Quick Reference Card 4401662
Applied Biosystems 3500/3500xL Genetic Analyzer User Guide, Data Collection v1.0 4401661
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin: Solutions to issues related to software, data, 4445098
hardware, and consumables
Note: Additional user bulletins may be available at www.lifetechnologies.com
Applied Biosystems 3730/3730xl Genetic Analyzer Getting Started Guide 4359476
GeneAmp™ PCR System 9700 Base Module User’s Manual N805-0200
Veriti™ 96-Well Thermal Cycler AmpFlSTR™Kit Validation User Bulletin 4440754
Quantifiler™ Kits: Quantifiler™
Human DNA Quantification Kit and Quantifiler™ Y Human Male DNA 4344790
Quantification Kit User’s Manual
PrepFiler™ Forensic DNA Extraction Kit User Guide 4390932
GeneMapper™ ID Software Version 3.1 Human Identification Analysis User Guide 4338775
GeneMapper™ ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial 4335523
Installation Procedures and New Features for GeneMapper™ ID Software v3.2 User Bulletin 4352543
GeneMapper™ ID-X Software Version 1.0 Getting Started Guide 4375574
GeneMapper™ ID-X Software Version 1.0 Quick Reference Guide 4375670
GeneMapper™ ID-X Software Version 1.0 Reference Guide 4375671
GeneMapper™ ID-X Software Version 1.1 (Mixture Analysis) Getting Started Guide 4396773

Documentation and Support
Obtain SDSs
Document title Pub. No.

GeneMapper™ ID-X Software Version 1.1 (Mixture Analysis) Quick Reference Guide 4402094
GeneMapper™ ID-X Software Version 1.2 Reference Guide 4426481
GeneMapper™ ID-X Software Version 1.2 Quick Reference Guide 4426482
Portable document format (PDF) versions of this guide and the documents listed
above are available at www.lifetechnologies.com.
Note: To open the user documentation available from the Applied Biosystems
web site, use the Adobe™ Acrobat™ Reader™ software available from
www.adobe.com.Safety Data Sheets (SDSs) are available from
www.lifetechnologies.com/support.
Obtain SDSs
Note: For the SDSs of chemicals not distributed by Life Technologies, contact the
chemical manufacturer.
Obtain support
For HID support:

• In North America – Send an email to HIDTechSupport@lifetech.com, or call
888-821-4443 option 1.
• Outside North America – Contact your local support office.
For the latest services and support information for all locations, go to:
www.lifetechnologies.com
At the website, you can:
• Access worldwide telephone and fax numbers to contact Technical Support and
Sales facilities
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Search for user documents, SDSs, vector maps and sequences, application notes,
formulations, handbooks, certificates of analysis, citations, and other product
support documents
• Obtain information about customer training
• Download software updates and patches
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.

Bibliography
Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994.
Identification of the heme compound copurified with deoxyribonucleic acid (DNA)
from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.
J. Forensic Sci. 39:362–372.
Barber, M.D., Piercy, R.C., Andersen, J.F., and Parkin, B.H. 1995. Structural variation of
novel alleles at the Hum vWA and Hum FES/FPS short tandem repeat loci. Intl. J. Legal
Med. 108:31–35.
Barber, M.D., McKeown, B.J., and Parkin, B.H. 1996. Structural variation in the alleles
of a short tandem repeat system at the human alpha fibrinogen locus. Intl. J. Legal Med.
108:180–185.
Barber, M.D. and Parkin, B.H. 1996. Sequence analysis and allelic designation of the
two short tandem repeat loci D18S51 and D8S1179. Intl. J. Legal Med. 109:62–65.
Baron, H., Fung, S., Aydin, A., Bahrig, S., Luft, F.C., and Schuster, H. 1996.
Oligonucleotide ligation assay (OLA) for the diagnosis of familial
hypercholesterolemia. Nat. Biotechnol. 14:1279–1282.
Begovich A.B., McClure G.R., Suraj V.C., Helmuth R.C., Fildes N., Bugawan T.L.,
Erlich H.A., and Klitz W. 1992. Polymorphism, recombination, and linkage
disequilibrium within the HLA class II region. J. Immunol. 148:249–58.
Bender, K., Farfan, M.J., and Schneider, P.M. 2004. Preparation of degraded human
DNA under controlled conditions. Forensic Sci. Int. 139:134–140.
Brinkmann, B., Moller, A. and Wiegand, P. 1995. Structure of new mutations in 2 STR
systems. Intl. J. Legal Med. 107:201–203.
Brinkmann, B., Klintschar, M., Neuhuber, F., Huhne, J. and Rolf, B. 1998. Mutation rate
in human microsatellites: Influence of the structure and length of the tandem repeat.
Am. J. Hum. Genet. 62:1408–1415.
Budowle, B. et al. 1998. CODIS and PCR Based Short Tandem Repeat Loci: Law
Enforcement Tools. Second European Symposium on Human Identification. 73–88.
Butler, J.M. 2005. Forensic DNA Typing. Burlington, MA:Elsevier Academic Press.
Chakraborty, R., Stivers, D., and Zhong, Y. 1996. Estimation of mutation rates from
parentage exclusion data: applications to STR and VNTR loci. Mutat. Res. 354:41–48.
Chakraborty, R. and Stivers, D.N. 1996. Paternity exclusion by DNA markers: effects of
paternal mutations. J. Forensic Sci. 41:671–677.
Chakraborty, R. Kimmel, M., Stivers, D., Davison, L., and Deka, R. 1997. Relative
mutation rates at di-, tri-, and tetranucleotide microsatellite loci. Proc. Natl. Acad. Sci.
USA 94:1041–1046.
Clark J.M. 1988. Novel non-templated nucleotide addition reactions catalyzed by
procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16:9677–9686.

Bibliography
DeFranchis, R., Cross, N.C.P., Foulkes, N.S., and Cox, T.M. 1988. A potent inhibitor of
Taq DNA polymerase copurifies with human genomic DNA. Nucleic Acids Res.
16:10355.
DNA Advisory Board, Federal Bureau of Investigation, U.S. Department of Justice.
1998. Quality assurance standards for forensic DNA testing laboratories.
Edwards, A., Civitello, A., Hammond, H., and Caskey, C. 1991. DNA typing and
genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet.
49:746–756.
Edwards, A., Hammond, H.A., Lin, J., Caskey, C.T., and Chakraborty, R. 1992. Genetic
variation at five trimeric and tetrameric tandem repeat loci in four human population
groups. Genomics 12:241–253.
Frank, W., Llewellyn, B., Fish, P., et al. 2001. Validation of the AmpFlSTR™ Profiler
Plus™ PCR Amplification Kit for use in forensic casework. J. Forensic Sci. 46:642–646.
Grossman, P.D., Bloch, W., Brinson, E., Chang, C.C., Eggerding, F.A., Fung, S.,
Iovannisci, D.M., Woo, S., and Winn-Deen, E.S. 1994. High-density multiplex detection
of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded
separation. Nucleic Acids Res. 22:4527–4534.
Guo S.W., and Thompson, E.A. 1992. Performing the exact test of Hardy-Weinberg
proportion for multiple alleles. Biometrics 48:361–372.
Hammond, H., Jin, L., Zhong, Y., Caskey, C., and Chakraborty, R. 1994. Evaluation of
13 short tandem repeat loci for use in personal identification applications. Am J. Hum.
Genet. 55:175–189.
Holt, C., Stauffer, C., Wallin, J., Lazaruk, L., Nguyen, T., Budowle, B., and Walsh, P.
2000. Practical applications of genotypic Surveys for forensic STR testing. Forensic Sci.
Int. 112:91–109.
Kimpton, C., Walton, A., and Gill, P. 1992. A further tetranucleotide repeat
polymorphism in the vWF gene. Hum. Mol. Genet. 1:287.
Kong, X., Murphy, K., Raj, T., He, C., White, P.S., and Matise, T.C. 2004. A combined
linkage-physical map of the human genome. Am. J. Hum. Genet. 75:1143–1148.
Kwok, S., and Higuchi, R. 1989. Avoiding false positives with PCR. Nature 339:237–238.
Lazaruk, K., Walsh, P.S., Oaks, F., Gilbert, D., Rosenblum, B.B., Menchen, S., Scheibler,
D., Wenz, H.M., Holt, C., Wallin, J. 1998. Genotyping of forensic short tandem repeat
(STR) systems based on sizing precision in a capillary electrophoresis instrument.
Electrophoresis 19:86–93.
Li, H. Schmidt, L., Wei, M-H., Hustad, T. Leman, M.I., Zbar, B., and Tory, K. 1993.
Three tetranucleotide polymorphisms for loci:D3S1352; D3S1358; D3S1359. Hum. Mol.
Genet. 2:1327.
Magnuson, V.L., Ally, D.S., Nylund, S.J., Karanjawala, Z.E., Rayman, J.B., Knapp, J.I.,
Lowe, A.L., Ghosh, S., and Collins, F.S. 1996. Substrate nucleotide-determined non-
templated addition of adenine by Taq DNA polymerase: implications for PCR-based
genotyping and cloning. Biotechniques 21:700–709.
Mansfield, E.S., Robertson, J.M., Vainer, M., Isenberg, A.R., Frazier, R.R., Ferguson, K.,
Chow, S., Harris, D.W., Barker, D.L., Gill, P.D., Budowle, B., and McCord, B.R. 1998.
Analysis of multiplexed short tandem repeat (STR) systems using capillary array
electrophoresis. Electrophoresis 19:101–107.

Bibliography
Mills, K.A., Even, D., and Murrau, J.C. 1992. Tetranucleotide repeat polymorphism at
the human alpha fibrinogen locus (FGA). Hum. Mol. Genet. 1:779.
Möller, A., Meyer, E., and Brinkmann, B. 1994. Different types of structural variation in
STRs: HumFES/FPS, HumVWA, and HumD21S11. Intl. J. Legal Med. 106:319–323.
Momhinweg, E., Luckenbach, C., Fimmers, R., and Ritter, H. 1998. D3S1358: sequence
analysis and gene frequency in a German population. Forensic Sci. Int. 95:173–178.
Moretti, T., Baumstark, A., Defenbaugh, D., Keys, K., Smerick, J., and Budowle, B.
2001. Validation of short tandem repeats (STRs) for forensic usage: Performance testing
of fluorescent multiplex STR systems and analysis of authentic and simulated forensic
samples. J. Forensic Sci. 46(3):647–660.
Mulero, J.J., Chang, C.W., and Hennessy, L.K. 2006. Characterization of N+3 stutter
product in the trinucleotide repeat locus DYS392. J. Forensic Sci. 51:826–830.
Nakahori, Y., Takenaka, O., and Nakagome, Y. 1991. A human X-Y homologous region
encodes amelogenin. Genomics 9:264–269.
National Research Council. 1996. The evaluation of forensic DNA evidence. National
Academy Press, Washington, D.C.
Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small
number of individuals. Genetics 89:583–590.
Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad.
Sci. USA 70:3321–3323.
Revised Validation Guidelines-Scientific Working Group on DNA Analysis Methods
(SWGDAM). Forensic Science Communications (July 2004) Volume 6 (3). Available at
www.fbi.gov/hq/lab/fsc/current/standards/2004_03_standards02.htm
Puers, C., Hammond, H., Jin, L., Caskey, C., and Schumm, J. 1993. Identification of
repeat sequence heterogeneity at the polymorphic short tandem repeat locus
HUMTH01 [AATG]n and reassignment of alleles in population analysis using a locus-
specific allelic ladder. Am. J. Hum. Genet. 53:953–958.
Sensabaugh, G.F. 1982. Biochemical markers of individuality. In: Saferstein, R., ed.
Forensic Science Handbook. Prentice-Hall, Inc., New York, pp. 338–415.
Sharma, V., and Litt, M. 1992. Tetranucleotide repeat polymorphism at the D21S11
locus. Hum Mol. Genet. 1:67.
Smith, R.N. 1995. Accurate size comparison of short tandem repeat alleles amplified by
PCR. Biotechniques 18:122–128.
Sparkes, R., Kimpton, C., Watson, S., Oldroyd, N., Clayton, T., Barnett, L., Arnold, J.,
Thompson, C., Hale, R., Chapman, J., Urquhart, A., and Gill, P. 1996a. The validation
of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing,
degradation and species studies. Int. J. Legal Med. 109:186–194.
Sparkes, R., Kimpton, C., Gilbard, S., Carne, P., Andersen, J., Oldroyd, N., Thomas, D.,
Urquhart, A., and Gill, P. 1996b. The validation of a 7-locus multiplex STR test for use
in forensic casework. (II), Artifacts, casework studies and success rates. Int. J. Legal
Med. 109:195–204.
Straub, R.E., Speer, M.C., Luo, Y., Rojas, K., Overhauser, J., Ott, J., and Gilliam, T.C.
1993. A microsatellite genetic linkage map of human chromosome 18. Genomics 15:48–
56.

Bibliography
Szibor, R., Lautsch, S., Plate, I., Bender, K., and Krause, D. 1998. Population genetic
data of the STR HumD3S1358 in two regions of Germany. Int. J. Legal Med. 111:160–
161.
Wallin, J.M., Buoncristiani, M.R., Lazaruk, K.D., Fildes, N., Holt, C.L., Walsh, P.S. 1998.
SWGDAM validation of the AmpFlSTR blue PCR amplification kit for forensic
casework analysis. J. Forensic Sci. 43:854–870.
Wallin, J.M., Holt, C.L., Lazaruk, K.D., Nguyen, T.H., and Walsh, P.S. 2002.
Constructing universal multiplex PCR systems for comparative genotyping. J. Forensic
Sci. 47:52–65.
Walsh, P.S., Fildes, N.J., and Reynolds, R. 1996. Sequence analysis and characterization
of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Res. 24:2807–
2812.
Watson, S., Kelsey, Z., Webb, R., Evans, J., and Gill, P. 1998. The development of a third
generation STR multiplex system (TGM). In: Olaisen, B., Brinkmann, B., and Lincoln,
P.J., eds. Progress in Forensic Genetics 7: Proceedings of the 17th International ISFH
Congress, Oslo 2-6 September 1997. Elsevier, Amsterdam, pp. 192–194.
Weber, J. and Wong, C. 1993. Mutation of human short tandem repeats. Hum. Mol.
Genet. 2:1123–1128.
Weir, B.S. 1996. Genetic data analysis II. Sunderland, MA: Sinauer Associates, Inc.

Index
Numerics developmental validation 66

310 instrument 31 differential amplification of loci 88
3130/3130xl instrument 27 DNA
control, about 18
3500/3500xL instrument 29
effect of DNA quantity on results 85–86
how degraded DNA affects which loci
A amplify 88–89
A nucleotide, addition by AmpliTaq Gold to 3´ end of mixed samples causing extra peaks 91
amplicon 81 negative-control reaction 21
agarose gel, using to examine DNA 88 positive-control reaction 21
allele frequencies in the population databases 95–104 quantification 19
quantification methods 20
allelic ladder
sample preparation 21
about 18
test sample 21
precision 69
using agarose gel analysis to examine the
profile 13
DNA 88
requirements for accurate genotyping 25
documentation, related 131
volume per reaction 28, 30, 32
amplification
differential amplification of loci 88 E
loci 12 effect of inhibitors 87
using bloodstained FTA cards 22 electropherogram
AmpliTaq Gold DNA Polymerase, catalyzing the ad- addition of a 3´ A nucleotide 81
dition of a 3´ A nucleotide 81 causes for extra peaks 92–93
DNA from more than one individual 91
B stutter peak 77
bins electrophoresis
check version 50 Data Collection Software 27, 29, 31
import 35, 50 preparing samples on the 310 instrument 31
biohazard safety 129 preparing samples on the 3100/3100-Avant or
3130/3130xl instrument 28
buffer, new 108
preparing samples on the 3500/3500xL
instrument 29
C reagents and parts 27, 29, 31
chemical safety 128 references 27, 29, 31
contents of kit 18 run module 27, 29, 31
set up 27, 29, 31
control DNA 9947A 14, 18
emission spectra 17
enzyme, new 108
D equipment, not included with kit 121
Data Collection Software, overview 16 evidence, exclusion of suspects 95
degraded DNA 88–89
AmpFl STR™ Identifiler™ PCR Amplification Kit User Guide 137

Index
F instrumentation
310 genetic analyzer 16, 31
fluorescent dyes 16
3100/3100-Avant genetic analyzer 16, 27
FTA cards
3130/3130xl genetic analyzer 16, 27
amplification 22
3500/3500xL genetic analyzer 16, 27, 29
bloodstained 22
software compatibility 16
G K
gels 88
kit
GeneMapper ID Software allelic ladder 18
analyze project 46
amplification 11
create analysis method 39 contents 18
create size standard 44 control DNA 18
examine and edit project 47
description 11
import panels and bins 35 DNA polymerase 18, 21
overview 16, 33
fluorescent dyes 16
set up 34
loci amplification 12
GeneMapper ID-X Software PCR reaction mix 18, 21
analyze project 61 primers 11, 18, 20
check version of panels, bins, and stutter 49, 50 reagents 18
create analysis method 55 supported instruments 11
examine and edit project 62 thermal cyclers for use with 126
overview 16, 48
set up 49
GeneScan size standard L
about 18 limited product warranty 132
dye label 16 LIZ size standard
volume per reaction 28, 29, 31 about 18
genetics 94–104 volume per reaction 28, 29, 31
allele frequencies 95–104 loci
populations and samples used in studies 95 allele frequencies in the population
probability of identity 105 databases 95–104
probability of paternity exclusion 106 chromosomal location 12
genotype differential amplification 88
determining 69 dye label 12
exclusion of suspects 95 lack of amplification, effect of DNA quantity on
resolving in mixed samples 92 results 85–86
population data, allele frequencies 95–104
population data, probability of identity 105
H population data, probability of paternity
hematin, effect on DNA samples 87 exclusion 106
Hi-Di formamide, volume per reaction 28, 29, 31 population data, samples used in studies 95
locus. See loci
I low-TE buffer 19
import
HID size standard 44 M
panels and bins 35 materials and equipment
panels, bins, and stutter 50 included in kit 18
138 AmpFl STR™ Identifiler™ PCR Amplification Kit User Guide

Index
not included with kit 121 S

mixed samples 91 safety
multicomponent analysis 16, 17 biohazard 129
chemical 128
N Safety Data Sheets (SDSs), obtaining 132
negative control, sample preparation 21 sample preparation 21
DNA negative control 21
DNA positive control 21
O standards 18
operating systems 16, 27, 29, 31 samples, DNA from more than one individual 91
detecting 91–92
P detection limit 92–94
resolving genotypes in mixed samples 92
panels
software, instrument compatibility 16
check version 49, 50
STRBase 104
import 35, 50
PCR stutter
check version 49, 50
amplification of tetranucleotide STR loci (stutter
peak) 77 import 50
inhibitor causing lack of amplification 87 stutter peak or product 77
performing 22 support, obtaining 132
setup 125
thermal cycling conditions, programming 22 T
PCR work areas 121, 125
technical support 132
population genetics 94–104
thermal cyclers
allele frequencies 95–104
for use with kit 126
populations and samples used in the studies 95
programming conditions 22
Probability of Exclusion (PE) from Paternity 106
training, information on 132
Probability of Identity (PI) 105
See Also allele troubleshooting 119
positive control, sample preparation 21
precision results 71–75 U
primer manufacturing process improvements 107 user-supplied reagents 19
primers, volume per reaction 21
V
Q validation
quantification, DNA 19 accuracy, precision, reproducibility 69
characterization of loci 82
developmental 66
R
experiments to evaluate 66
reaction mix, volume per reaction 21 importance of 66
reactions, preparing for PCR 21 minimum sample requirement 94
reagents mixture studies 91
not included with kit 121 mutation rate 105
user supplied 19 population data 94
references 133 probability of identity 105
run module, electrophoresis 27, 29, 31 probability of paternity exclusion 106
sensitivity 85
AmpFl STR™ Identifiler™ PCR Amplification Kit User Guide 139

Index
species specificity 83
stability 86
thermal cycler parameters 67
W
warranty 132
work area
amplified DNA 126
PCR setup 125
setup and lab design 125
workflow overview 15
140 AmpFl STR™ Identifiler™ PCR Amplification Kit User Guide

thermofisher.com/support | thermofisher.com/askaquestion
thermofisher.com
24 August 2018

cms_041201

Uploaded by

Copyright:

Available Formats

cms_041201

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

cms_041201

Uploaded by

Copyright:

Available Formats

AmpFℓSTR™ Identifiler™ PCR Amplification Kit

for use with:

Catalog Number 4322288

For Forensic or Paternity Use Only.

The information in this guide is subject to change without notice.

Revision history: Pub. No. 4323291

G March 2012 Change to limited licensing information.

About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

■ CHAPTER 2 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 3

■ CHAPTER 3 Perform Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Section 3.1 3100/3100-Avant and 3130/3130xl instruments . . . . . . . . . . . . . . . . . . . . . . . 27

Section 3.2 3500/3500xL Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Section 3.3 310 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

■ CHAPTER 4 Analyze Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

4 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

Section 4.2 GeneMapper™ ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

■ CHAPTER 5 Experiments and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 5

Section 5.2 Performance Verification After Primer Manufacturing Process

Section 5.3 Performance Validation After Buffer and Enzyme Component

6 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

Allelic dropout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

■ APPENDIX B Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

■ APPENDIX C PCR Work Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

■ APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 7

8 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 9

10 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 11

Chromosome Alleles included in AmpFlSTR™ Dye Control DNA

12 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 13

14 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

AutoMate Express™ System + PrepFiler™ Express Kit

Quantifiler™ Duo DNA Quantification Kit

AmpFlSTR™ Identifiler™ PCR Amplification Kit

GeneAmp™ PCR System 9700 Cycler Veriti™ 96-Well Thermal Cycler

3100/3100-Avant 3130/3130xl 3500/3500xL 310 Genetic

GeneMapper™ ID-X or GeneMapper™ ID Software

AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 15

Instrument and software overview

Instrument and Table 2 Software specific to each instrument

3500/3500xL • Windows™ XP 3500 Series GeneMapper™ ID-X

3130/3130xl Windows™ XP 3.0 • GeneMapper™ ID

310 Windows XP 3.1

Note: We conducted validation studies for the AmpFlSTR™ Identifiler™ PCR

16 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 17

Materials and equipment

Component Description 200✕ Volume Storage

18 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

■ Required user-supplied reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Required user-supplied reagents

2. Aliquot and autoclave the solutions.

AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide 19

20 AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide

b. Select FileImport Panels to open the Import Panels dialog box.