Life Technologies Identifiler Direct PCR Amplification Kit User Guide
The Identifiler Direct PCR Amplification Kit is a short tandem repeat (STR) multiplex assay optimized to allow direct amplification of single-source samples including blood and buccal samples on treated paper substrates without the need for sample purification.
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USER GUIDE AmpFlSTR® Identifiler® Direct PCR Amplification Kit for use with: 200 reaction kit (Part no. 4467831) 1000 reaction kit (Part no. 4408580) Publication Part Number 4415125 Rev. H Revision Date June 2012 Information in this document is subject to change without notice. LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. AmpliTaq Gold is a registered trademark of Roche Molecular Systems, Inc. Windows and Windows Vista are registered trademarks of Microsoft Corporation. FTA is a registered trademark of Whatman International Ltd. Whatman is a registered trademark of GE Healthcare Companies. Bode Buccal DNA Collector is a trademark of Bode Technology Group, Inc. NUCLEIC-CARD™ is a trademark and FLOQSwabs is a registered trademark of Copan Italia S.P.A., and used by Life Technologies under their permission. © 2012 Life Technologies Corporation. All rights reserved. Contents About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 ■ CHAPTER 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Substrate examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . About the primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Loci amplified by the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allelic ladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 11 11 11 12 12 13 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Instrument and software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data collection and analysis software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instrument and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . About multicomponent analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How multicomponent analysis works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 15 15 15 15 Materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Standards for samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 ■ CHAPTER 2 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Optimize PCR cycle number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Select samples and prepare plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Determine optimum conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Treated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Sample prep guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Prepare the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Untreated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Sample prep guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Prepare the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Swab substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample prep guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prepare the sample lysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prepare the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Store the sample lysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 25 25 25 27 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 3 Contents ■ CHAPTER 3 Perform Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Allelic ladder requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Section 3.1 3100/3100-Avant and 3130/3130xl instruments . . . . . . . . . . . . . . . . . . . . . . . 31 Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis . . . . . . . . . . . . . . . . 31 Reagents and parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Electrophoresis software setup and reference documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments . . . . . . 32 Section 3.2 3500/3500xL instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Set up the 3500/3500xL instruments for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Reagents and parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Electrophoresis software setup and reference documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Prepare samples for electrophoresis on the 3500/3500xL instruments . . . . . . . . . . . . . . . . . . . . . . 33 Section 3.3 3730 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Set Up the 3730 instrument for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Reagents and parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Electrophoresis software setup and reference documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Prepare samples for electrophoresis on the 3730 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 ■ CHAPTER 4 Analyze Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Section 4.1 GeneMapper® ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Overview of GeneMapper® ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Set up GeneMapper® ID Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . File names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Before using the software for the first time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Import panels and bins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allele tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peak Detector tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peak Quality tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quality Flags tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Create a size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 38 38 38 42 42 43 44 45 46 47 Analyze and edit sample files with GeneMapper® ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Contents Section 4.2 GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Overview of GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Set up GeneMapper® ID-X Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . File names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Before using the software for the first time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Import panels, bins, and marker stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allele tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peak Detector tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peak Quality tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SQ & GQ tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Create a size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 51 51 51 55 56 57 58 59 60 60 Analyze and edit sample files with GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 ■ CHAPTER 5 Experiments and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 1.2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Precision and size windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 66 66 66 67 Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Causes of extra peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stutter products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Addition of 3´ A nucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 75 75 80 81 Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nature of the polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 81 81 82 Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 SWGDAM Guideline 2.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blood on FTA® cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buccal cells on FTA® or Indicating FTA® cards and buccal cells on Bode DNA Collectors . . Effect of DNA quantity on results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 83 83 83 83 84 5 Contents Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA on FTA® cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA on buccal swabs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 85 86 87 Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Population samples used in these studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identifiler® Direct Kit allele frequencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of Hardy-Weinberg equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concordance studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 88 88 88 88 96 99 Mutation rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Additional mutation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Probability of identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Probability of paternity exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 ■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 ■ APPENDIX B Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Equipment and materials not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 ■ APPENDIX C Plate Layouts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Example PCR plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Example electrophoresis plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 ■ APPENDIX D PCR Work Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 PCR setup work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 ■ APPENDIX E Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Specific chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Obtain SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Obtain support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 6 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Contents Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Limited Product Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 7 Contents 8 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide About This Guide IMPORTANT! Before using this product, read and understand the information the “Safety” appendix in this document. Revision history Revision Date Description A May 2009 New document. B August 2009 Add Experiments and Results chapter. C October 2009 Update screen shots for Panel Manager. D September 2010 Change copyright page information. E July 2011 Add 200-reaction kit, Bode Buccal DNA Collector™, Prep-n-Go™ Buffer, and 3100-Avant, 3130, and 3500/3500xL Genetic Analyzer information. F October 2011 Add information for Prep-n-Go™ Buffer to Experiments and Results chapter. G March 2012 Change copyright page information. H May 2012 Add heat protocol for buccal swab lysate preparation. Add results for additional swab types. Purpose The AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide provides information about the Life Technologies instruments, chemistries, and software associated with the AmpFlSTR® Identifiler® Direct PCR Amplification Kit. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 9 About This Guide Purpose 10 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 1 Overview ■ Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 ■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 ■ Instrument and software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 ■ Materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Product overview Purpose The AmpFlSTR® Identifiler® Direct PCR Amplification Kit is a short tandem repeat (STR) multiplex assay optimized to allow direct amplification of single-source: • Blood and buccal samples on treated paper substrates without the need for sample purification. • Blood and buccal samples collected on untreated paper substrates and treated with Applied Biosystems® Prep-n-Go™ Buffer. • Buccal samples collected on swab substrates and treated with Applied Biosystems® Prep-n-Go™ Buffer The Identifiler® Direct Kit amplifies 15 autosomal STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and the sex-determining marker, Amelogenin, in a single PCR reaction. Substrate examples • Treated paper: Copan NUCLEIC-CARD™ system or Whatman FTA® cards • Untreated paper: Bode Buccal DNA Collector™ or 903 paper • Swab: Copan FLOQSwabs™ Product description The Identifiler® Direct Kit contains all the necessary reagents for the amplification of human genomic DNA. The reagents are designed for use with the following Applied Biosystems® instruments: • Applied Biosystems® 3100/3100-Avant Genetic Analyzer • Applied Biosystems® 3130/3130xl Genetic Analyzer • Applied Biosystems® 3500/3500xL Genetic Analyzer • Applied Biosystems® 3730 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 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 11 1 Chapter 1 Overview Product overview About the primers The Identifiler® Direct Kit employs the same primer sequences as used in the AmpFlSTR® Identifiler® PCR Amplification Kit. Degenerate primers for the loci D8S1179, vWA, and D16S539 are included in the AmpFlSTR® Identifiler® Direct Primer Set to address mutations in the primer binding sites. The addition of the degenerate primers allows for the amplification of those alleles in samples containing the mutations without altering the overall performance of the Identifiler® Direct Kit. 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 inter-locus 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 kit Table 1 shows the loci amplified, their chromosomal locations, and the corresponding fluorescent marker dyes. The AmpFlSTR® Identifiler® Direct Allelic Ladder is used to genotype the analyzed samples. The alleles contained in the allelic ladder, and the genotype of the AmpFlSTR® Identifiler® Direct Control DNA 9947A, are also listed in the table. Table 1 AmpFlSTR® Identifiler® Direct PCR Amplification Kit loci and alleles Locus designation Chromosome location Alleles included in Allelic Ladder Dye label Control DNA 9947A 6-FAM™ 13, 13 D8S1179 8 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 D21S11 21q11.2-q21 24, 24.2, 25, 26, 27, 28, 28.2, 29, 29.2, 30, 30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36, 37, 38 30, 30 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 VIC® 14, 15 D3S1358 3p 12, 13, 14, 15, 16, 17, 18, 19 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, 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, 27, 28 19, 23 D19S433 19q12-13.1 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2 vWA 12p12-pter 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 17, 18 TPOX 2p23-2per 6, 7, 8, 9, 10, 11, 12, 13 8, 8 D18S51 18q21.3 7, 9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 15, 19 Amelogenin X: p22.1-22.3; Y: p11.2 X, Y D5S818 5q21-31 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 11, 11 FGA 4q28 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26.2, 27, 28, 29, 30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2 23, 24 12 NED™ PET® 14, 15 X AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 1 Overview Product overview Allelic ladder 1 Figure 1 shows the allelic ladder for the Identifiler® Direct Kit. See “Allelic ladder requirements” on page 30 for information on ensuring accurate genotyping. Figure 1 GeneMapper® ID-X Software plot of the AmpFlSTR® Identifiler® Direct Allelic Ladder AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 13 1 Chapter 1 Overview Workflow Perform PCR Harris Manual Punch Obtain samples BSD600 Semi-Automated Dried Sample Punch Instrument Untreated paper only: Prep-n-Go™ Buffer AmpFlSTR® Identifiler® Direct PCR Amplification Kit GeneAmp® PCR System 9700 Cycler Prepare samples Treated or untreated paper substrates Prepare reactions Prepare reactions Prepare samples Perform PCR Obtain samples Workflow Swab substrates Lyse in Prep-n-Go™ Buffer AmpFlSTR® Identifiler® Direct PCR Amplification Kit Veriti® 96-Well Thermal Cycler Perform electrophoresis 3100/3100-Avant Genetic Analyzer 3130/3130xl Genetic Analyzer 3500/3500xL Genetic Analyzer 3730 Genetic Analyzer Analyze data GeneMapper® ID-X Software 14 GeneMapper® ID Software AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 1 Overview Instrument and software overview 1 Instrument and software overview This section provides information about the data collection and analysis software versions required to run the Identifiler® Direct Kit on specific instruments. Data collection and analysis software Instrument and software compatibility The data collection software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time. As the instrument measures sample fluorescence with its detection system, the data collection software collects the data and stores it. The data collection software stores information about each sample in a sample file (.fsa files for 31xx and 3730 instruments and .hid files for 3500 instruments), which is then analyzed by the analysis software. Instrument 3100/3100-Avant Operating system Windows® NT Data collection software 1.1 (3100) 1.0 (3100-Avant) Windows® 2000 2.0 3130/3130xl† Windows XP 3.0 3730† Windows XP 3.1 3500/3500xL • Windows® XP or 3500 Series Data Collection Software v1.0 • Windows Vista® Analysis software • GeneMapper® ID Software v3.2.1 • GeneMapper® ID-X Software v1.0.1 or higher GeneMapper® ID-X Software v1.2 or higher † We conducted validation studies for the Identifiler® Direct Kit using these configurations. About multicomponent analysis Life Technologies fluorescent multi-color dye technology allows the analysis of multiple loci, including loci that have alleles with overlapping size ranges. Alleles for overlapping loci are distinguished by labeling locus-specific primers with different 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® Direct 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. How multicomponent analysis works Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength. During data collection on Life Technologies instruments, the fluorescence signals are separated by a diffraction grating according to their 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 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 2). The goal of multicomponent analysis is to correct for spectral overlap. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 15 1 Chapter 1 Overview Materials and equipment Figure 2 Emission spectra of the five dyes used in the Identifiler® Direct Kit Dyes Normalized Emission 6-FAM VIC NED PET LIZ 100 80 60 40 20 0 500 550 600 650 700 Wavelength (nm) Materials and equipment Kit contents and storage The Identifiler® Direct Kit contains sufficient quantities of the following reagents for 200 reactions (Part no. 4467831) or 1000 reactions (Part no. 4408580) at 25 µL/reaction. 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. Table 2 Kit Contents and Storage Component Description 200 reaction 1000 reaction Storage –15 to –25°C upon receipt, 2 to 8°C after initial use AmpFlSTR® Identifiler® Direct Master Mix Contains enzyme, salts, dNTPs, carrier protein, and 0.04% sodium azide 2 tubes, 1.25 mL each 1 bottle, 12.5 mL AmpFlSTR® Identifiler® Direct Primer Set Contains forward and reverse primers to amplify human DNA targets. 2 tubes, 1.25 mL each 1 bottle, 12.5 mL AmpFlSTR® Identifiler® Direct Control DNA 9947A Contains 2 ng/μL human female cell line DNA in 0.04% sodium azide and buffer.† 1 tube, 50.0 µL 1 tube, 50.0 µL 1 tube, 50.0 µL 1 tube, 100 µL See Table 1 on page 12 for profile. AmpFlSTR® Identifiler® Direct Allelic Ladder Contains amplified alleles. See Table 1 on page 12 for a list of alleles included in the allelic ladder. † The 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 Control DNA 9947A is not designed to be used as a DNA quantitation control and laboratories may expect to see variation from the labelled concentration when quantitating aliquots of the Control DNA 9947A. 16 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 1 Overview Materials and equipment Standards for samples 1 For the Identifiler® Direct Kit, the panel of standards needed for PCR amplification, PCR product sizing, and genotyping are: • AmpFlSTR® Identifiler® Direct Control DNA 9947A – A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpFlSTR® Identifiler® Direct 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® Direct 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® Direct Allelic Ladder – Developed for accurate characterization of the alleles amplified by the Identifiler® Direct Kit. The Allelic Ladder contains most of the alleles reported for the 15 autosomal loci. Refer to page 12 for a list of the alleles included in the Allelic Ladder. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 17 1 18 Chapter 1 Overview Materials and equipment AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 2 Perform PCR ■ Optimize PCR cycle number. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 ■ Treated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 ■ Untreated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 ■ Swab substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 ■ Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Optimize PCR cycle number Before using the Identifiler® Direct Kit for the first time, perform a single initial sensitivity experiment to determine the appropriate cycle number to use during internal validation studies and operational use of the Identifiler® Direct Kit. This experiment accounts for instrument-to-instrument and sample-to-sample variations. If you are processing multiple sample type and substrate combinations (for example, buccal samples on treated paper and buccal samples on swabs), perform separate sensitivity experiments for each sample type and substrate to be used for testing. The Identifiler® Direct Kit is optimized to amplify unpurified: • Single-source blood samples on treated paper or untreated paper • Buccal samples on treated paper, untreated paper, or swabs When amplifying single-source, unpurified samples using the Identifiler® Direct Kit, you should expect to see greater variation in peak height from sample to sample than is expected with purified samples. Careful optimization of the cycle number will help to minimize this variation. Select samples and prepare plates 1. Select 26 of each sample+substrate type. Ensure the selected samples represent a “typical” range of samples analyzed in your laboratory. IMPORTANT! The number of samples recommended for this study has been chosen to allow you to complete electrophoresis using a single 96-well plate, thus minimizing the impact of run-to-run variation on the results. Examples of PCR and electrophoresis plate layouts are provided on page 109. 2. Prepare the samples and the reactions as described in the protocols later in this chapter. Prepare sufficient PCR reagents to complete amplification of three replicate plates. 3. Create three identical PCR plates (see page 109 for a suggested plate layout). 4. Amplify each plate using a different cycle number to determine the optimum conditions for use in your laboratory. Suggested cycle numbers for different sample type and substrate combinations are listed below: AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 19 2 Chapter 2 Perform PCR Optimize PCR cycle number Sample type Substrate Treated paper Untreated paper Swab Blood 25, 26, 27 cycles 25, 26, 27 cycles N/A Buccal 26, 27, 28 cycles 26, 27, 28 cycles 26, 27, 28 cycles Note: Our testing has not included blood samples on swab substrates. This sample type is not frequently used for the collection of database or casework reference samples. Note: To minimize the effect of instrument-to-instrument variation, use the same thermal cycler to amplify all three plates. To maximize result quality, prepare and amplify Plate 1 then repeat for Plates 2 and 3. Do not prepare all three plates simultaneously. Determine optimum conditions 1. Run the PCR products on the appropriate CE platform using the recommended protocol; see Chapter 3, “Perform Electrophoresis” on page 29. 2. Based on the results of the sensitivity study, select the appropriate PCR cycle number for future experiments. Our studies indicate the optimum PCR cycle number should generate profiles with the following heterozygote peak heights, with no instances of allelic dropout and minimal occurrence of off-scale allele peaks. 20 Instrument Heterozygous peak height 31xx 1000–3000 RFU 3500 Series 3000–12,000 RFU AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 2 Perform PCR Treated paper substrates: prepare reactions 2 Treated paper substrates: prepare reactions Sample prep guidelines • Do not add water to the wells on the reaction plate before adding the punches. If your laboratory is experiencing static issues with the paper discs, you may prepare and dispense the 25 µL reaction mix into the wells of the reaction plate before adding the punches. • Make the punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification. • For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card, hold the barrel of the Harris Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction plate. • For automated punching: Please refer to the User Guide of your automated or semi-automated disc punch instrument for proper guidance. Prepare the reactions 1. Add samples to the reaction plate: Well(s) Add the following to wells of a MicroAmp® Optical 96-Well Reaction Plate... Negative control 1.2 mm blank disc Test samples 1.2 mm sample disc Positive control • For 25 cycles 3 µL of Control DNA 9947A IMPORTANT! Do not add a blank disc to the positive control well. • For 26 and 27 cycles 2 µL of Control DNA 9947A • For 28 cycles 1 µL of Control DNA 9947A Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number. 2. Calculate the volume of each component needed to prepare the reactions, using the table below. Reaction component Volume per reaction Master Mix 12.5 µL Primer Set 12.5 µL Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! The Identifiler® Direct Kit has been optimized for a 25-µL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of Identifiler® Direct Kit chemistry to generate full STR profiles. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 21 2 Chapter 2 Perform PCR Treated paper substrates: prepare reactions 3. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to 8°C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 4. Pipet the required volumes of components into an appropriately sized polypropylene tube. 5. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 6. Dispense 25 µL of the reaction mix into each reaction well of a MicroAmp® Optical 96-Well Reaction Plate. 7. Seal the plate with MicroAmp® Clear Adhesive Film or MicroAmp® Optical Adhesive Film. 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, 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. 8. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 9. Amplify the samples in a GeneAmp® PCR System 9700 with the silver or gold-plated silver 96-well block or a Veriti® 96-well Thermal Cycler as described in “Perform PCR” on page 28. IMPORTANT! The Identifiler® Direct 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® Direct Kit. 22 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 2 Perform PCR Untreated paper substrates: prepare reactions 2 Untreated paper substrates: prepare reactions Sample prep guidelines • Make the punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification. • If you are using a Bode Buccal DNA Collector™, make the punch as close as possible to the tip of the DNA collector to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification. Bode Buccal DNA Collector™ Take punch as close to the tip as possible • For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card, hold the barrel of the Harris Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction plate. • For automated punching: Please refer to the User Guide of your automated or semi-automated disc punch instrument for proper guidance. Prepare the reactions 1. Add 2 µL of Prep-n-Go™ Buffer (Part no. 4467079) to the sample and negative control wells in a 96-well plate. Do not add Prep-n-Go™ Buffer to the positive control wells. 2. Add samples to the reaction plate: Well(s) Add the following to wells of a MicroAmp® Optical 96-Well Reaction Plate... Negative control 1.2 mm blank disc Test samples 1.2 mm sample disc Positive control • For 25 cycles 3 µL of Control DNA 9947A IMPORTANT! Do not add a blank disc to the positive control well. • For 26 and 27 cycles 2 µL of Control DNA 9947A • For 28 cycles 1 µL of Control DNA 9947A Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number. 3. Centrifuge the plate to ensure the punches are immersed in the Prep-n-Go™ Buffer. 4. Calculate the volume of each component needed to prepare the reactions, using the table below. Reaction component Volume per reaction Master Mix 12.5 µL Primer Set 12.5 µL Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 23 2 Chapter 2 Perform PCR Untreated paper substrates: prepare reactions IMPORTANT! The Identifiler® Direct Kit has been optimized for a 25-µL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of Identifiler® Direct Kit chemistry to generate full STR profiles. 5. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to 8°C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 6. Pipet the required volumes of components into an appropriately sized polypropylene tube. 7. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 8. Dispense 25 µL of the reaction mix into each reaction well of a MicroAmp® Optical 96-Well Reaction Plate. 9. Seal the plate with MicroAmp® Clear Adhesive Film or MicroAmp® Optical Adhesive Film. 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, 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. 10. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 11. Amplify the samples in a GeneAmp® PCR System 9700 with the silver or gold-plated silver 96-well block or a Veriti® 96-well Thermal Cycler as described in “Perform PCR” on page 28. IMPORTANT! The Identifiler® Direct 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 AmpFlSTR® Identifiler® Direct PCR Amplification Kit. 24 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 2 Perform PCR Swab substrates: prepare reactions 2 Swab substrates: prepare reactions Note: Performance verification and optimization experiments for this protocol were conducted using Copan 4N6 FLOQSwabs®, OmniSwabs, and Puritan swabs air-dried and stored at room temperature for up to three months. Sample prep guidelines • Detach buccal swab heads from the swab shaft for lysis. • Lysis is performed under heated conditions using Prep-n-Go™ Buffer (Part no. 4471406 for buccal swabs) in either of the following formats: – 1.5 mL tubes with a heat block (VWR Scientific Select dry heat b lock or similar) – 96-well deep-well plate (Part no. 4392904) with an oven and a metal plate adaptor (Robbins Scientific Model 400 Hybridization Incubator or similar, Agilent Benchtop Rack for 200 µl Tubes/V Bottom Plates (metal) Part no. 410094 or similar) IMPORTANT! Do not use a plastic plate adaptor. • For optimum performance, lysis of a whole swab is recommended. To preserve the sample, evaluate lysis of a half swab. Prepare the sample lysate 1. Preheat the heat block to 90°C or the oven with metal plate adaptor to 99°C. 2. Add 400 µL Prep-n-Go™ Buffer (for buccal swabs, Part. no. 4471406) to 1.5 mL tubes or the appropriate wells of a 96-well deep-well plate (Part no. 4392904). 3. Into each tube or well, put the entire head of each swab. If you are using tubes, cap the tubes. Let the tubes or plate stand for 20 minutes in the preheated heat block or oven to lyse the sample. 4. After 20 minutes, remove the tubes or the deep-well plate from the heat block or oven. Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate. 5. Let the lysate stand at room temperature for at least 15 minutes to cool the lysate (for accurate pipetting). 6. Transfer the sample lysate out of the sample plate into tubes or plates for storage, then discard the deep-well plate containing the swab heads. 7. Proceed to the next section to prepare the reactions or see “Store the sample lysate” on page 27. Prepare the reactions 1. Calculate the volume of each component needed to prepare the reactions, using the table below. Reaction component Volume per reaction Master Mix 12.5 µL Primer Set 12.5 µL AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 25 2 Chapter 2 Perform PCR Swab substrates: prepare reactions Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! This kit has been optimized for a 25-µL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of Identifiler® Direct Kit chemistry to generate full STR profiles. 2. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to 8°C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 3. Pipet the required volumes of components into an appropriately sized polypropylene tube. 4. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 5. Dispense 25 µL of the reaction mix into each reaction well of a MicroAmp® Optical 96-Well Reaction Plate. 6. Add samples to the reaction plate: Well(s) Add the following to wells of a MicroAmp® Optical 96-Well Reaction Plate... Negative control 3 µL of Prep-n-Go™ Buffer Test samples 3 µL of lysate Positive control • For 25 cycles 3 µL of Control DNA 9947A • For 26 and 27 cycles 2 µL of Control DNA 9947A • For 28 cycles 1 µL of Control DNA 9947A Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number. 7. Seal the plate with MicroAmp® Clear Adhesive Film or MicroAmp® Optical Adhesive Film. 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, 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. 8. Vortex the reaction mix at medium speed for 3 seconds. 9. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 26 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 2 Perform PCR Swab substrates: prepare reactions 2 10. Amplify the samples in a GeneAmp® PCR System 9700 with the silver or gold-plated silver 96-well block or a Veriti® 96-well Thermal Cycler as described in “Perform PCR” on page 28. IMPORTANT! The Identifiler® Direct 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® Direct Kit. Store the sample lysate Cap the sample lysate storage tubes or seal the sample lysate storage plate with MicroAmp® Clear Adhesive Film. Store the sample lysate as needed: If you are storing the sample lysate... Then place at... <2 weeks 2 to 8°C >2 weeks –15 to –25°C These storage recommendations are preliminary pending the results of ongoing stability studies. The effects of multiple freeze-thaw cycles on the lysate have not been fully evaluated. Therefore, multiple freeze-thaw cycles are not recommended. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 27 2 Chapter 2 Perform PCR Perform PCR Perform PCR 1. Program the thermal cycling conditions. IMPORTANT! When using the GeneAmp PCR System 9700 with either 96-well silver or gold-plated silver block, select 9600 Emulation Mode. Initial incubation step Optimum cycle number† Denature HOLD 95°C 11 min Anneal Extend Final extension Final hold HOLD HOLD 60°C 25 min 4°C ∞ CYCLE 94°C 20 sec 59°C 2 min 72°C 1 min † Determine the optimum cycle number for your laboratory according to the instructions on page 19. 2. Load the plate into the thermal cycler and close the heated cover. 3. Start the run. 4. On completion of the run, store the amplified DNA. If you are storing the DNA... Then place at... <2 weeks 2 to 8°C >2 weeks –15 to –25°C IMPORTANT! Protect the amplified products from light. 28 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 3 Perform Electrophoresis Allelic ladder requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30 ■ Section 3.1 3100/3100-Avant and 3130/3130xl instruments . . . . . . . . . . . . . . . . . . 31 Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis . . . 31 Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 ■ Section 3.2 3500/3500xL instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Set up the 3500/3500xL instruments for electrophoresis . . . . . . . . . . . . . . . . . . . . . 33 Prepare samples for electrophoresis on the 3500/3500xL instruments. . . . . . . . . . 33 ■ Section 3.3 3730 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Set Up the 3730 instrument for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Prepare samples for electrophoresis on the 3730 instrument . . . . . . . . . . . . . . . . . 35 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 29 3 Chapter 3 Perform Electrophoresis Allelic ladder requirements Allelic ladder requirements To accurately genotype samples, you must run an allelic ladder sample along with the unknown samples. Instrument Number of allelic ladders to run One injection 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 3730 2 per injection 48 samples 46 samples + 2 allelic ladders Number of samples per allelic ladder(s) 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. 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. 30 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 3.1 3100/3100-Avant and 3130/3130xl instruments Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis 3 Section 3.1 3100/3100-Avant and 3130/3130xl instruments Reagents and parts Appendix B, “Ordering Information” on page 105 lists the required materials not supplied with the Identifiler® Direct Kit. IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Keep freeze-thaw cycles to a minimum. Electrophoresis software setup and reference documents Genetic Analyzer The following table lists data collection software and the run modules that can be used to analyze Identifiler® Direct Kit PCR products. For details on the procedures, refer to the documents listed in the table. Data Collection Software Applied Biosystems® 3100-Avant 1.0 Applied Biosystems® 3100 2.0 Operating System Run modules and conditions References Windows® NT • GeneScan36Avb_DyeSetG5Module Injection condition: 3 kV/5sec 3100/3100-Avant Genetic Analyzers Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products User Bulletin (Part no. 4332345) • GS600v2.0Analysis.gsp Windows® 2000 • HIDFragmentAnalysis36_POP4_1 Injection condition: 3kV/10 sec • Dye Set G5 1.1 Windows® NT • GeneScan36vb_DyeSetG5Module Injection condition: 3kV/10 sec • GS600v2.0Analysis.gsp Applied Biosystems® 3130/3130xl 3.0† Windows® XP • HIDFragmentAnalysis36_POP4_1 Injection conditions: – 3130 = 3 kV/5 sec – 3130xl = 3 kV/10 sec • Dye Set G5 3100/3100-Avant Genetic Analyzers Using Data Collection Software v2.0, Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products User Bulletin (Part no. 4350218) 3100/3100-Avant Genetic Analyzers Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products User Bulletin (Part no. 4332345) Applied Biosystems 3130/3130xl Genetic Analyzers Using Data Collection Software v3.0, Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products User Bulletin (Part no. 4363787) † We conducted validation studies for the Identifiler® Direct Kit using this configuration. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 31 3100 and 3130 Instruments Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis 3 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments Prepare the samples for electrophoresis immediately before loading. 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 0.3 µL Hi-Di™ Formamide 8.7 µL Reagent OR Volume per reaction GeneScan™ 600 LIZ® Size Standard v2.0 0.5 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. Pipet 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 briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and 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 on the autosampler. 9. Start the electrophoresis run. 32 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 3 3500/3500xL Instruments Set up the 3500/3500xL instruments for electrophoresis 3 Section 3.2 3500/3500xL instruments Set up the 3500/3500xL instruments for electrophoresis Appendix B, “Ordering Information” on page 105 lists the required materials not supplied with the Identifiler® Direct Kit. IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Keep freeze-thaw cycles to a minimum. Electrophoresis software setup and reference documents Genetic Analyzer Applied Biosystems® 3500 Applied Biosystems® 3500xL The following table lists data collection software and the run modules that you can use to analyze Identifiler® Direct Kit PCR products. For details on the procedures, refer to the documents listed in the table. Data Collection Software 3500 Data Collection Software v1.0 Operating System Run modules and conditions Windows® XP • HID36_POP4 Injection conditions: 1.2kV/15 sec or • Dye Set G5 Windows Vista® • HID36_POP4 Injection conditions: 1.2kV/24 sec References Applied Biosystems® 3500/ 3500xL Genetic Analyzer User Guide (Part no. 4401661) 3500 and 3500xL Genetic Analyzers Quick Reference Card (Part no. 4401662) • Dye Set G5 Prepare samples for electrophoresis on the 3500/3500xL instruments Prepare the samples for electrophoresis immediately before loading. 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™ 8.5 µL Formamide 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. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 33 3500/3500 xL Instruments Reagents and parts 3 Chapter 3 3500/3500xL Instruments Prepare samples for electrophoresis on the 3500/3500xL instruments 2. Pipet 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 briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the plate in a thermal cycler for 3 minutes at 95°C. 7. Immediately place the plate on ice for 3 minutes. 8. Place the sample tray on the autosampler. 9. Start the electrophoresis run. 34 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 3 3730 Instrument Set Up the 3730 instrument for electrophoresis 3 Section 3.3 3730 instrument Set Up the 3730 instrument for electrophoresis Reagents and parts Appendix B, “Ordering Information” on page 105 lists the required materials not supplied with the Identifiler® Direct Kit. Electrophoresis software setup and reference documents Operating system Windows XP The following table lists data collection software and the run modules that you can use to analyze Identifiler® Direct Kit PCR products. For details on the procedures, refer to the documents listed in the table. Data collection software 3.0† Run module • GeneMapper_36_POP7 • Dye Set G5_RCT References Applied Biosystems® 3730 DNA Analyzer Human Identification Validation Report‡ † We conducted concordance studies for the Identifiler® Direct Kit using this configuration. ‡ Contact your sales or support representative to obtain a copy of the 3730 DNA Analyzer Human Identification Validation Report. Prepare samples for electrophoresis on the 3730 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: Reagent Volume per reaction GeneScan™ 500 LIZ® Size Standard 0.3 µL Hi-Di™ Formamide 8.7 µL Reagent OR Volume per reaction GeneScan™ 600 LIZ® Size Standard v2.0 0.5 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. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 35 3730 Instruments 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. 3 Chapter 3 3730 Instrument Prepare samples for electrophoresis on the 3730 instrument 2. Pipet 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 briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the plate in a thermal cycler for 3 minutes at 95°C. 7. Immediately place the plate on ice for 3 minutes. 8. Place the sample tray on the autosampler. 9. Start the electrophoresis run. 36 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 4 Analyze Data ■ Section 4.1 GeneMapper® ID Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Overview of GeneMapper® ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Set up GeneMapper® ID Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . 38 Analyze and edit sample files with GeneMapper® ID Software. . . . . . . . . . . . . . 48 Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 For more information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 ■ Section 4.2 GeneMapper® ID-X Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Overview of GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Set up GeneMapper® ID-X Software for data analysis . . . . . . . . . . . . . . . . . . . . . . 51 Analyze and edit sample files with GeneMapper® ID-X Software. . . . . . . . . . . . 62 Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 For more information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 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 an .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 15 for a list of compatible instruments. 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. Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis. 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. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 37 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for data analysis 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, verify the sample result according to your 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. Before using the software for the first time To analyze sample files (.fsa) using GeneMapper® ID Software v3.2.1 for the first time: • Import panels and bins into the Panel Manager, as explained in “Import panels and bins” on page 38. • Create an analysis method, as explained in “Create an analysis method” on page 42. • Create a size standard, as explained in “Create a size standard” on page 47. • Define custom views of analysis tables. Refer to the GeneMapper® ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial (Part no. 4335523) for more information. • Define custom views of plots. Refer to the GeneMapper® ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial (Part no. 4335523) for more information. Import panels and bins To import the Identifiler® Direct Kit panel and bin set from the Life_Technologies web site into the GeneMapper® ID Software v3.2.1 database: 1. Download and open the file containing panels and bins: a. From the Support menu of www.lifetechnologies.com, select SupportSoftware Downloads, Patches & UpdatesGeneMapper® ID Software v 3.2 Updates & Patches, and download the file Identifiler Direct Analysis Files GMID. b. Unzip the file. 38 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis 4 2. Start the GeneMapper® ID Software, then log in with the appropriate user name and password. IMPORTANT! For logon instructions, refer to the GeneMapper® ID Software Version 3.1 Human Identification Analysis User Guide (Part no. 4338775). 3. Select ToolsPanel Manager. GeneMapper® ID Software 4. Find, then open the folder containing the panels and bins: a. Select Panel Manager in the navigation pane. b. Select FileImport Panels to open the Import Panels dialog box. c. Navigate to, then open the Identifiler Direct Analysis Files GMID folder that you unzipped in step 1 on page 38. 5. Select IdentifilerDirect_GS500_Panels_v1.txt, then click Import. Note: Importing this file creates a new folder in the navigation pane of the Panel Manager, IdentifilerDirect_GS500_v1. This folder contains the panel and associated markers. 6. Import IdentifilerDirect_GS500_Bins_v1.txt: a. Select the IdentifilerDirect_GS500_v1 folder in the navigation pane. b. Select FileImport Bin Set to open the Import Bin Set dialog box. c. Navigate to, then open the Identifiler Direct Analysis Files GMID folder. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 39 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for data analysis d. Select IdentifilerDirect_GS500_Bins_v1.txt, then click Import. Note: Importing this file associates the bin set with the panels in the IdentifilerDirect_GS500_Panels_v1 folder. 7. View the imported panels in the navigation pane: a. Double-click the IdentifilerDirect_GS500_v1 folder to view the IdentifilerDirect_GS500_Panels_v1 folder. b. Double-click the IdentifilerDirect_GS500_Panels_v1 folder to display the panel information in the right pane. 40 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis 4 8. Select D8S1179 to display the Bin view for the marker in the right pane. GeneMapper® ID Software 9. Click Apply, then OK to add the Identifiler® Direct Kit 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. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 41 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for data analysis Create an analysis method To create an HID analysis method for the Identifiler® Direct 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 shown in the figures on the following pages. Note: The Analysis Method Editor closes when you save your settings. To complete this step quickly, do not save the analysis method until you finish entering settings in all of the tabs. 5. After you enter settings in all tabs, click Save. General tab settings 42 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis 4 In the Name field, either type the name as shown for consistency with files supplied with other AmpFlSTR® kits, or enter a name of your choosing. The Description and Instrument fields are optional. Allele tab settings GeneMapper® ID Software • In the Bin Set field, select the IdentifilerDirect_GS500_Bins_v1 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 IdentifilerDirect_GS500_Panels_v1 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. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 43 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for data 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® Direct 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® Direct Kit has been validated using the Local Southern sizing method. Before using alternative sizing methods, perform the appropriate internal validation studies. 44 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis 4 3500/3500xL and 3730 data • 3500/3500xL and 3730 data: Overall peak heights for the data are approximately 3 times higher than peak heights obtained for samples run on the 31xx series instruments. Evaluate validation data carefully to determine the appropriate Peak Amplitude Thresholds for reliable analysis. • For more information: – Refer to User Bulletin: Applied Biosystems® 3500/3500xL Genetic Analyzer: Protocols for Analysis of AmpFlSTR® PCR Amplification Kit PCR Products and Validation Summary (Part no. 4469192) – Contact your sales or support representative to obtain a copy of the 3730 DNA Analyzer Human Identification Validation Report 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 that allow for reliable interpretation of Identifiler® Direct Kit data. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 45 GeneMapper® ID Software • 3730 data only: Due to differences in the resolution of peaks using POP-7™ polymer versus POP-4® polymer, reduce the Peak Window Size setting in GeneMapper® ID Software from 15 pts to 11 pts to obtain accurate genotyping results. 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for 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 to use in your laboratory. 46 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis Create a size standard 4 The size standards for the Identifiler® Direct Kit use the following size standard peaks in their definitions: GeneScan™ 600 LIZ® Size Standard v2.0 peak sizes 75, 100, 139, 150, 160, 200, 300, 350, 400, and 450 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 300, 314, 320, 340, 360, 380, 400, 414, 420, 440 and 460 Note: The 250-nt and the 340-nt peak in the GeneScan™ 500 LIZ® Size Standard are not included in the size standard definition. These peaks can be used as an indicator of precision within a run. To create the size standard for the Identifiler® Direct Kit: 1. Select ToolsGeneMapper Manager to open the GeneMapper Manager. 2. Select the Size Standards tab, then click New. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 47 GeneMapper® ID Software GeneScan™ 500 LIZ® Size Standard peak sizes 4 Chapter 4 Analyze Data Analyze and edit sample files with GeneMapper® ID Software 3. Enter a name as shown below or enter a name of your choosing. In the Size Standard Dye field, select Orange. In the Size Standard Table, enter the peak sizes specified in on page 47. The example below is for the GeneScan™ 500 LIZ® Size Standard. 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. The names of the settings shown are the names suggested in the sections above. If you named the settings differently, select the names you specified. Parameter Settings Sample Type Select the sample type. Analysis Method IdentifilerDirect_AnalysisMethod_v1 (or the name of the analysis method you created) Panel IdentifilerDirect_GS500_Panels_v1 Size Standard CE_G5_IdentifilerDirect_GS500 (or the name of the size standard you created) For more information about how the Size Caller works, refer to the ABI PRISM® GeneScan® Analysis Software for the Windows NT® Operating System Overview of the Analysis Parameters and Size Caller User Bulletin (Part no. 4335617). For additional information about size standards, refer to the GeneMapper® ID Software Version 3.1 Human Identification Analysis User Guide (Part no. 4338775). 48 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Examine and edit a project 4 3. Click (Analyze), enter a name for the project (in the Save Project dialog box), then click OK to start analysis. During a run: • The status bar displays the progress of analysis as both: – A completion bar extending to the right with the percentage completed indicated – With text messages on the left • The Genotypes tab becomes available 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 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 (Part no. 4335523) • GeneMapper® ID Software Version 3.1 Human Identification Analysis User Guide (Part no. 4338775) • Installation Procedures and New Features for GeneMapper® ID Software Software Version v3.2 User Bulletin (Part no. 4352543) AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 49 GeneMapper® ID Software • The table displays the row of the sample currently being analyzed in green (or red if analysis failed for the sample). 4 Chapter 4 GeneMapper® ID-X Software 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 or .hid file. Using GeneMapper® ID-X Software, you can then analyze and interpret the data from the .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 15 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. Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis. 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, verify the sample result according to your laboratory protocol. 50 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 Set up GeneMapper® ID-X Software for data analysis 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.appliedbiosystems.com. Before using the software for the first time Before you use GeneMapper® ID-X Software to analyze data files (GeneMapper® ID-X Software v1.0.1 or higher for .fsa files, GeneMapper® ID-X Software v1.2 or higher for .hid files): • Import panels, bins, and marker stutter into the Panel Manager, as explained in “Import panels, bins, and marker stutter” on page 51. • Create an analysis method, as explained in “Create an analysis method” on page 55. • Create a size standard, as explained in “Create a size standard” on page 60. • Define custom views of analysis tables. Refer to the GeneMapper® ID-X Software Version 1.0 Getting Started Guide (Part no. 4375574) for more information. • Define custom views of plots. Refer to the GeneMapper® ID-X Software Version 1.0 Getting Started Guide (Part no. 4375574) for more information. Import panels, bins, and marker stutter To import the Identifiler® Direct Kit panel, bin set, and marker stutter from the Life Technologies web site into the GeneMapper® ID-X Software database: 1. Download and open the file containing panels, bins, and marker stutter: a. From the Support menu of www.lifetechnologies.com, select SupportSoftware Downloads, Patches & UpdatesGeneMapper® ID-X Software Updates & Patches, and download the file Identifiler Direct 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 (Part no. 4375574). 3. Select ToolsPanel Manager. 4. Find, then open the folder containing the panels, bins, and marker stutter: a. Select Panel Manager in the navigation pane. b. Select FileImport Panels to open the Import Panels dialog box. c. Navigate to, then open the Identifiler Direct Analysis Files GMIDX folder that you unzipped in step 1. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 51 GeneMapper® ID-X Software File names 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 5. Select IdentifilerDirect_GS500_v1X, then click Import. Note: Importing this file creates a new folder in the navigation pane of the Panel Manager “IdentifilerDirect_GS500_Panels_v1X”. This folder contains the panel and associated markers. 6. Import IdentifilerDirect_GS500_Bins_v1X.txt: a. Select the IdentifilerDirect_GS500_Panels_v1X 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 Identifiler Direct Analysis Files GMIDX folder. d. Select IdentifilerDirect_GS500_Bins_v1X.txt, then click Import. Note: Importing this file associates the bin set with the panels in the IdentifilerDirect_GS500_v1X folder. 52 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 7. View the imported panels in the navigation pane: a. Double-click the IdentifilerDirect_GS500_v1X folder. b. Double-click the IdentifilerDirect_GS500_Panels_v1X folder to display the panel information in the right pane and the markers below it. GeneMapper® ID-X Software 8. Select D8S1179 to display the Bin view for the marker in the right pane. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 53 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 9. Import IdentifilerDirect_GS500_Stutter_v1X.txt: a. Select the IdentifilerDirect_GS500_v1 folder in the navigation panel. b. Select FileImport Marker Stutter to open the Import Marker Stutter dialog box. c. Navigate to, then open the Identifiler Direct Analysis Files GMIDX folder. d. Select IdentifilerDirect_GS500_Stutter_v1X.txt, then click Import. Note: Importing this file associates the marker stutter ratio with the bin set in the IdentifilerDirect_GS500_Bins_v1X folder. 10. View the imported marker stutters in the navigation pane: a. Select the IdentifilerDirect_GS500_v1X folder to display its list of markers in the right pane. b. Double-click the IdentifilerDirect_GS500_v1X folder to display its list of markers below it. 54 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 c. Double-click D16S539 to display the Stutter Ratio & Distance view for the marker in the right pane. GeneMapper® ID-X Software 11. Click Apply, then OK to add the Identifiler® Direct 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 method Use the following procedure to create an analysis method for the Identifiler® Direct Kit. 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 Software 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. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 55 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 2. Select the Analysis Methods tab, then click New to open the Analysis Method Editor with the General tab selected. 3. Enter the settings shown in the figures on the following pages. Note: The Analysis Method Editor closes when you save your settings. To complete this step quickly, do not save the analysis method until you finish entering settings in all of the tabs. 4. After you enter the settings on all tabs, click Save. 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 drop-down list. The Description and Instrument fields are optional. 56 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 Allele tab settings GeneMapper® ID-X Software • In the Bin Set field, select the IdentifilerDirect_GS500_Bins_v1X bin set imported previously and configure the parameters as shown. • GeneMapper® ID-X Software 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 IdentifilerDirect_GS500_Stutter_v1X.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. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 57 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis Peak Detector tab settings Perform internal validation studies to determine settings IMPORTANT! Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of Identifiler® Direct 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 – This kit has been validated using the Local Southern sizing method. Before using alternative sizing methods, perform the appropriate internal validation studies. • Normalization (v1.2 or higher) – For use with 3500 data. Perform internal validation studies to determine whether to use the Normalization feature for analysis of Identifiler® Direct Kit data. 58 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 Peak Quality tab settings GeneMapper® ID-X Software 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, maximum peak height threshold, and the minimum peak height ratio threshold for interpretation of Identifiler® Direct Kit data. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 59 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for 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. Create a size standard The size standards for the Identifiler® Direct Kit uses the following size standard peaks in their definitions: GeneScan™ 500 LIZ® Size Standard peak sizes GeneScan™ 600 LIZ® Size Standard v2.0 peak sizes 75, 100, 139, 150, 160, 200, 300, 350, 400, and 450 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 300, 314, 320, 340, 360, 380, 400, 414, 420, 440 and 460 Note: The 250-nt and the 340-nt peaks in the GeneScan™ 500 LIZ® Size Standard are not included in the size standard definition. These peaks can be used as an indicator of precision within a run. Use the following procedure to create the size standard for the Identifiler® Direct Kit. 1. Select ToolsGeneMapper® ID-X Manager to open the GeneMapper® ID-X Manager. 60 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 2. Select the Size Standards tab, then click New. Security Group field, select the Security Group appropriate to your software configuration from the drop-down list. In the Size Standard Dye field, select Orange. In the Size Standard Table, enter the peak sizes specified on page 60. The example below is for the GeneScan™ 500 LIZ® Size Standard. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 61 GeneMapper® ID-X Software 3. Complete the Name field as shown below or with a name of your choosing. In the 4 Chapter 4 GeneMapper® ID-X Software Analyze and edit sample files with GeneMapper® ID-X Software 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. The names of the settings shown are the names suggested in the sections above. If you named the settings differently, select the names you specified. Parameter Settings Sample Type Select the sample type. Analysis Method IdentifilerDirect_AnalysisMethod_v1X (or the name of the analysis method you created) Panel IdentifilerDirect_GS500_Panels_v1X Size Standard CE_G5_IdentifilerDirect_GS500 (or the name of the size standard you created) For more information about how the Size Caller works, or about size standards, refer to the GeneMapper® ID-X Software v1.2 Reference Guide (Part no. 4426481). 3. Click (Analyze), enter a name for the project (in the Save Project dialog box), then click OK to start analysis. During a run: • The status bar displays the progress of analysis as a completion bar extending to the right with the percentage completed 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 upon completion of the analysis. The figure below shows the analysis summary window after analysis. 62 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Examine and edit a project 4 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). GeneMapper® ID-X Software For more information For more information about any of these tasks, refer to: • GeneMapper® ID-X Software Version 1.0 Getting Started Guide (Part no. 4375574) • GeneMapper® ID-X Software Version 1.0 Quick Reference Guide (Part no. 4375670) • GeneMapper® ID-X Software Version 1.0 Reference Guide (Part no. 4375671) • GeneMapper® ID-X Software Version 1.1(Mixture Analysis) Getting Started Guide (Part no. 4396773) • GeneMapper® ID-X Software Version 1.2 Reference Guide (Part no. 4426481) • GeneMapper® ID-X Software Version 1.2 Quick Reference Guide (Part no. 4426482) AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 63 4 64 Chapter 4 GeneMapper® ID-X Software For more information AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 5 Experiments and Results ■ Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 ■ Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 ■ Extra peaks in the electropherogram. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 ■ Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 ■ Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 ■ Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 ■ Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 ■ Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 ■ Mutation rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 ■ Probability of identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ■ Probability of paternity exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Overview This chapter provides results of the developmental validation experiments we performed using the Identifiler® Direct Kit for samples punched from FTA® cards. Importance of validation Validation of a DNA typing procedure for human identification applications is an 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 conditions We performed experiments to evaluate the performance of the Identifiler® Direct Kit. 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. Additional validation was performed according to the revised guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM, July 10, 2003). Based on these guidelines, we conducted experiments that comply with guidelines 1.0 and 2.0 and its associated subsections. This DNA methodology is not novel. (Moretti et al., 2001; Frank et al., 2001; Wallin et al., 2002; and Holt et al., 2000). AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 65 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility This chapter discusses many of the experiments we performed and provides examples of results obtained. We chose conditions that produced optimum PCR product yield and that met reproducible performance standards. It is our opinion that while these experiments are not exhaustive, they are appropriate for a manufacturer of STR kits intended for forensic and/or parentage testing use. Each laboratory using the Identifiler® Direct Kit should perform their own internal validation studies. Validation studies included testing on the following sample+substrate combinations: • Unpurified, single-source blood or buccal samples on FTA® paper (treated paper substrate) • Buccal samples on a Bode Buccal DNA Collector™ (untreated paper substrate) Additional performance verification studies included testing on Copan 4N6 FLOQSwabs® (swab substrate). We did not perform mixture or inhibition studies during the developmental validation of the Identifiler® Direct Kit because these tests are not relevant for the intended use of this chemistry. Accuracy, precision, and reproducibility SWGDAM guideline 1.2.1 “Developmental validation is the demonstration of the accuracy, precision, and reproducibility of a procedure by the manufacturer, technical organization, academic institution, government laboratory, or other party.” (SWGDAM, July 2003) SWGDAM guideline 2.9 “The extent to which a given set of measurements of the same sample agree with their mean and the extent to which these measurements match the actual values being measured should be determined.” (SWGDAM, July 2003) Accuracy Laser-induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology (Holt et al., 2000; and Wallin et al., 2002). However, accuracy and reproducibility of Identifiler® Direct Kit profiles have been determined from various sample types. Figure 3 illustrates the size differences that are typically observed between sample alleles and allelic ladder alleles on the Applied Biosystems® 3130xl Genetic Analyzer with POP-4→ polymer. The x-axis in Figure 3 represents the nominal nucleotide sizes for the AmpFlSTR® Identifiler® Direct Allelic Ladder. The dashed lines parallel to the x-axis represent ±0.25-nt windows. The y-axis represents the deviation of each sample allele size from the corresponding allelic ladder allele size. All sample alleles are within ±0.5 nt from a corresponding allele in the allelic ladder. 66 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Figure 3 Size deviation of 200 blood samples on FTA® card analyzed on the Applied Biosystems 3130xl Genetic Analyzer Precision and size windows Sizing precision allows for determining accurate and reliable genotypes. Sizing precision was measured on the Applied Biosystems® 3130xl Genetic Analyzer. The recommended method for genotyping is to employ a ±0.5-nt “window” around the size obtained for each allele in the AmpFlSTR® Identifiler® Direct Allelic Ladder. A ±0.5-nt window allows for the detection and correct assignment of alleles. Any sample allele that sizes outside the specified window could be: • An “off-ladder” allele, that is, an allele of a size that is not represented in the AmpFlSTR® Identifiler® Direct Allelic Ladder or • An allele that corresponds to an allelic ladder allele, but whose size falls just outside a window because of measurement error The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times. Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument. Table 3 on page 68 shows typical precision results obtained from five runs (16 capillaries/run) of the AmpFlSTR® Identifiler® Direct Allelic Ladder on the Applied Biosystems® 3130xl Genetic Analyzer (36-cm capillary and POP-4® polymer) sized using the GeneScan™ 500 LIZ® Size Standard. The results were obtained within a consecutive set of injections on a single capillary array. Sample alleles may occasionally size outside of the ±0.5-nt window for a respective allelic ladder allele because of measurement error. The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing. Figure 3 illustrates the tight clustering of allele sizes obtained on the Applied AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 67 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Biosystems® 3130xl Genetic Analyzer, where the standard deviation in sizing is typically less than 0.15 nt. The instance of a sample allele sizing outside the ±0.5-nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0.15 nt or less (Smith, 1995). For sample alleles that do not size within a ±0.5-nt window, the PCR product must be rerun to distinguish between a true off-ladder allele versus measurement error of a sample allele that corresponds 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 flag sample alleles that do not size within the prescribed window around an allelic ladder allele by labelling the allele as OL (Off-ladder). Maximum precision is obtained with a set of capillary injections on each of the supported platforms however the determined allele sizes will vary between the different platforms. Cross-platform sizing differences occur from a number of factors including type and concentration of polymer, run temperature, and electrophoresis conditions. Variations in sizing can also occur between runs on the same instrument and between runs on different instruments of the same platform type because of these factors. We recommend strongly that the allele sizes obtained should be compared to the sizes obtained for known alleles in the AmpFlSTR® Identifiler® Direct Allelic Ladder from the same run and then converted to genotypes (as described in “Before you start” on page 37 and 50). Refer to Table 3 for the results of five runs of the AmpFlSTR® Identifiler® Direct Allelic Ladder. For more information on precision and genotyping, see Lazaruk et al., 1998 and Mansfield et al., 1998. In Table 3, the mean sizes for all the alleles in each run (16 capillaries) were calculated. The mean range shown in the table represents the lowest- and highest-mean size values obtained across all five runs. Similarly, the standard deviation for the allele sizing was calculated for all the alleles in each run. The standard deviation range shown in Table 3 represents the lowest and highest standard deviation values obtained across all five runs. Table 3 Precision results of five runs (16 capillaries/run) of the AmpFlSTR® Identifiler® Direct Allelic Ladder Applied Biosystems® 3130xl Genetic Analyzer Allele Mean Standard deviation X 106.26–106.43 0.033–0.044 Y 111.92–112.06 0.032–0.046 6 304.04–304.20 0.038–0.053 7 308.09–308.26 0.033–0.052 8 312.15–312.32 0.038–0.047 9 316.20–316.37 0.033–0.048 10 320.24–320.42 0.027–0.051 11 324.30–324.45 0.033–0.055 Amelogenin CSF1PO 68 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Applied Biosystems® 3130xl Genetic Analyzer Allele Mean Standard deviation 12 328.34–328.49 0.036–0.053 13 332.37–332.52 0.033–0.047 14 336.42–336.57 0.038–0.052 15 340.46–340.60 0.036–0.045 8 216.56–216.75 0.033–0.050 9 220.55–220.72 0.020–0.051 10 224.53–224.70 0.035–0.043 11 228.52–228.70 0.037–0.048 12 232.58–232.76 0.037–0.049 13 236.48–236.66 0.031–0.051 14 240.40–240.60 0.037–0.044 15 244.40–244.59 0.038–0.048 5 252.22–252.42 0.040–0.050 8 264.17–264.35 0.030–0.052 9 268.18–268.35 0.040–0.051 10 272.15–272.33 0.031–0.048 11 276.16–276.33 0.034–0.047 12 280.15–280.34 0.039–0.050 13 284.16–284.33 0.032–0.052 14 288.17–288.33 0.029–0.058 15 292.17–292.36 0.037–0.055 7 261.88–261.98 0.028–0.045 9 269.99–270.12 0.039–0.058 10 274.08–274.20 0.031–0.045 10.2 276.08–276.20 0.029–0.054 11 278.15–278.28 0.040–0.047 12 282.22–282.35 0.036–0.049 13 286.27–286.40 0.038–0.053 13.2 288.28–288.42 0.040–0.050 14 290.37–290.50 0.033–0.049 14.2 292.39–292.50 0.037–0.053 15 294.47–294.60 0.038–0.050 16 298.55–298.70 0.041–0.053 D13S317 D16S539 D18S51 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 69 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Applied Biosystems® 3130xl Genetic Analyzer Allele Mean Standard deviation 17 302.68–302.82 0.034–0.052 18 306.82–306.99 0.042–0.053 19 310.96–311.11 0.043–0.060 20 315.10–315.25 0.031–0.048 21 319.23–319.38 0.031–0.049 22 323.42–323.57 0.038–0.054 23 327.48–327.63 0.043–0.055 24 331.59–331.74 0.031–0.052 25 335.69–335.83 0.029–0.052 26 339.81–339.96 0.044–0.052 27 343.92–344.04 0.037–0.051 9 101.38–101.46 0.032–0.039 10 105.28–105.36 0.030–0.036 11 109.20–109.28 0.027–0.042 12 113.14–113.22 0.028–0.038 12.2 115.15–115.21 0.032–0.038 13 117.11–117.17 0.030–0.045 13.2 119.11–119.17 0.028–0.038 14 121.07–121.14 0.022–0.045 14.2 123.10–123.17 0.035–0.047 15 125.09–125.13 0.031–0.048 15.2 127.12–127.16 0.026–0.045 16 129.11–129.16 0.034–0.044 16.2 131.17–131.20 0.028–0.044 17 133.17–133.22 0.033–0.044 17.2 135.24–135.27 0.022–0.043 24 184.40–184.51 0.035–0.042 24.2 186.39–186.50 0.023–0.043 25 188.35–188.44 0.025–0.040 26 192.30–192.39 0.029–0.043 27 196.27–196.33 0.026–0.042 28 200.14–200.21 0.039–0.043 28.2 202.11–202.18 0.028–0.042 29 204.08–204.16 0.031–0.041 D19S433 D21S11 70 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Applied Biosystems® 3130xl Genetic Analyzer Allele Mean Standard deviation 29.2 206.14–206.21 0.031–0.041 30 208.10–208.17 0.024–0.039 30.2 210.08–210.15 0.019–0.040 31 212.09–212.16 0.028–0.036 31.2 214.06–214.13 0.025–0.041 32 216.07–216.15 0.032–0.045 32.2 218.04–218.12 0.030–0.038 33 220.06–220.14 0.022–0.042 33.2 222.01–222.07 0.029–0.045 34 224.13–224.21 0.020–0.041 34.2 226.02–226.11 0.030–0.042 35 228.10–228.18 0.027–0.047 35.2 230.02–230.10 0.036–0.052 36 232.01–232.10 0.032–0.046 37 236.07–236.15 0.030–0.040 38 240.00–240.09 0.036–0.045 15 306.40–306.56 0.032–0.056 16 310.49–310.64 0.036–0.049 17 314.56–314.72 0.034–0.048 18 318.62–318.77 0.038–0.040 19 322.69–322.84 0.025–0.044 20 326.74–326.89 0.035–0.049 21 330.81–330.95 0.030–0.042 22 334.87–335.00 0.029–0.047 23 338.90–339.05 0.039–0.052 24 342.94–343.08 0.039–0.047 25 346.99–347.13 0.031–0.050 26 350.99–351.13 0.040–0.051 27 354.94–355.06 0.031–0.050 28 359.08–359.21 0.031–0.054 D2S1338 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 71 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Applied Biosystems® 3130xl Genetic Analyzer Allele Mean Standard deviation 12 111.35–111.49 0.034–0.052 13 115.45–115.58 0.034–0.046 14 119.44–119.58 0.034–0.047 15 123.37–123.49 0.035–0.053 16 127.55–127.67 0.033–0.051 17 131.74–131.86 0.029–0.048 18 135.85–135.96 0.035–0.050 19 139.96–140.07 0.036–0.056 7 133.85–133.95 0.037–0.048 8 137.96–138.06 0.040–0.046 9 142.31–142.42 0.032–0.045 10 146.78–146.89 0.033–0.044 11 151.13–151.26 0.032–0.043 12 155.36–155.50 0.027–0.042 13 159.51–159.67 0.020–0.045 14 163.57–163.73 0.032–0.044 15 167.60–167.76 0.030–0.055 16 171.63–171.77 0.036–0.049 6 255.09–255.23 0.031–0.047 7 259.11–259.25 0.038–0.048 8 263.13–263.27 0.036–0.049 9 267.16–267.29 0.029–0.041 10 271.20–271.32 0.041–0.048 11 275.23–275.37 0.032–0.051 12 279.26–279.40 0.037–0.047 13 283.28–283.43 0.035–0.049 14 287.32–287.45 0.043–0.052 15 291.35–291.49 0.037–0.053 8 122.84–122.95 0.030–0.046 9 126.91–127.01 0.027–0.053 10 131.01–131.10 0.031–0.052 11 135.14–135.24 0.037–0.051 D3S1358 D5S818 D7S820 D8S1179 72 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Applied Biosystems® 3130xl Genetic Analyzer Allele Mean Standard deviation 12 139.33–139.43 0.029–0.059 13 143.90–144.02 0.027–0.045 14 148.36–148.48 0.034–0.045 15 152.70–152.82 0.022–0.044 16 156.93–157.09 0.026–0.041 17 161.08–161.24 0.026–0.046 18 165.14–165.33 0.035–0.056 19 169.22–169.40 0.035–0.056 17 214.31–214.49 0.035–0.046 18 218.33–218.5 0.037–0.046 19 222.38–222.56 0.020–0.047 20 226.40–226.58 0.036–0.046 21 230.42–230.60 0.032–0.046 22 234.46–234.65 0.033–0.047 23 238.49–238.69 0.038–0.048 24 242.54–242.73 0.038–0.054 25 246.57–246.78 0.033–0.050 26 250.62–250.82 0.039–0.059 26.2 252.63–252.82 0.040–0.045 27 254.63–254.82 0.035–0.053 28 258.69–258.89 0.038–0.051 29 262.75–262.95 0.045–0.053 30 266.81–267.04 0.033–0.054 30.2 268.66–268.85 0.042–0.062 31.2 272.72–272.93 0.039–0.052 32.2 276.78–277.01 0.037–0.055 33.2 280.85–281.07 0.044–0.053 42.2 317.96–318.20 0.042–0.057 43.2 322.08–322.31 0.051–0.056 44.2 326.18–326.43 0.039–0.059 45.2 330.33–330.55 0.046–0.060 46.2 334.34–334.56 0.039–0.047 47.2 338.43–338.65 0.047–0.056 48.2 342.59–342.80 0.047–0.064 50.2 350.71–350.91 0.040–0.053 FGA AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 73 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Applied Biosystems® 3130xl Genetic Analyzer Allele Mean Standard deviation 51.2 354.67–354.87 0.048–0.058 4 162.79–162.92 0.034–0.054 5 166.84–166.97 0.034–0.048 6 170.88–171.00 0.030–0.047 7 174.88–175.01 0.028–0.046 8 178.89–179.01 0.031–0.045 9 182.87–182.98 0.031–0.042 9.3 185.90–186.02 0.025–0.049 10 186.83–186.94 0.035–0.047 11 190.79–190.89 0.025–0.046 12 201.62–201.71 0.034–0.045 6 221.96–222.07 0.030–0.043 7 225.93–226.04 0.035–0.044 8 229.90–230.01 0.027–0.043 9 233.86–233.98 0.032–0.039 10 237.86–237.98 0.023–0.049 11 241.83–241.96 0.028–0.037 12 245.84–245.95 0.032–0.043 13 249.83–249.93 0.027–0.044 11 154.16–154.27 0.025–0.044 12 158.30–158.44 0.029–0.054 13 162.40–162.54 0.034–0.045 14 166.62–166.78 0.029–0.048 15 170.56–170.70 0.028–0.046 16 174.57–174.71 0.028–0.045 17 178.56–178.71 0.028–0.045 18 182.51–182.66 0.032–0.044 19 186.48–186.60 0.031–0.045 20 190.41–190.53 0.026–0.043 21 194.29–194.43 0.032–0.044 22 198.21–198.33 0.025–0.043 23 202.06–202.18 0.034–0.040 24 206.38–206.48 0.031–0.040 TH01 TPOX vWA 74 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra peaks in the electropherogram 5 Extra peaks in the electropherogram Causes of extra peaks Peaks other than the target alleles may be detected on the electropherogram. Causes for the appearance of extra peaks include stutter products, incomplete 3´ A nucleotide addition (at the n-1 position), dye artifacts, and mixed DNA samples (see DAB Standard 8.1.2.2). Stutter products A stutter is a well-characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller (or less frequently, one repeat larger) than the major STR product (Butler, 2005; Mulero et al., 2006). 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. Peak heights were measured for amplified samples at the loci used in the Identifiler® Direct Kit: • Treated paper workflow: 370 blood samples on FTA® card and 299 buccal samples on Indicating FTA® cards • Untreated paper workflow: 370 buccal samples on Bode Buccal DNA Collectors All data were generated on the Applied Biosystems 3130xl Genetic Analyzer. Some conclusions from these measurements and observations are: • For each Identifiler® Direct Kit locus, the percent stutter generally increases with allele length, as shown in – Treated paper workflow: Figure 4 through Figure 7 on page 76 through page 77 – Untreated paper workflow: Figure 8 through Figure 11 on page 78 through page 79 • Smaller alleles display a lower level of stutter relative to the longer alleles within each locus. • Each allele within a locus displays a percent stutter that is consistent. • The stutter value for each locus shown for the treated paper workflow in Table 4 on page 80 was determined by taking the mean plus three times the standard deviation. These values are the stutter filter percentages in the Identifiler® Direct stutter file and will be used during the filtering step in GeneMapper® ID Software or GeneMapper® ID-X Software. Peaks in the stutter position that are above the stutter filter percentage will not be filtered. Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated. • Stutter percentages generated using the untreated paper workflow were calculated on a different, smaller data set than was used for the original stutter calculations. We used the stutter values of the most common alleles at each locus to compare the data sets. There was no significant difference in the stutter values (mean plus three times the standard deviation) for the individual loci with the exception of D3S1358 (0.7%), D7S820 (0.4%), D16S539 (0.2%) and FGA (1.2%). For D3S1358, D7S820, and FGA, the stutter values were slightly lower than the original stutter values calculated for punches from FTA® cards processed with the AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 75 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram Identifiler® Direct Kit. The D16S539 stutter percentage (mean plus three times the standard deviation) was slightly higher than the original stutter value. You should evaluate the impact of sample type on stutter percentages when implementing a direct amplification system. • The measurement of percent stutter for allele peaks that are off-scale may be unusually high. Off-scale peaks were not included in the evaluation of stutter characterized here. Figure 4 Treated paper workflow: FTA® card sample stutter percentages for D8S1179, D21S11, D7S820, and CSF1PO loci (red = blood samples; blue = buccal samples) Figure 5 Treated paper workflow: FTA® card sample stutter percentages for D3S1358, TH01, D13S317, D16S539, and D2S1338 loci (red = blood samples; blue = buccal samples) 76 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra peaks in the electropherogram 5 Figure 6 Treated paper workflow: FTA® card sample stutter percentages for D19S433, vWA, TPOX, and D18S51 loci (red = blood samples; blue = buccal samples) Figure 7 Treated paper workflow: FTA® card sample stutter percentages for D5S818 and FGA loci (red = blood samples; blue = buccal samples) AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 77 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram Figure 8 Untreated paper workflow: Bode Buccal DNA Collector™ sample stutter percentages for D8S1179, D21S11, D7S820, and CSF1PO loci Figure 9 Untreated paper workflow: Bode Buccal DNA Collector™ sample stutter percentages for D3S1358, TH01, D13S317, D16S539, and D2S1338 loci 78 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra peaks in the electropherogram 5 Figure 10 Untreated paper workflow: Bode Buccal DNA Collector™ sample stutter percentages for D19S433, vWA, TPOX, and D18S51 loci Figure 11 Untreated paper workflow: Bode Buccal DNA Collector™ sample stutter percentages for D5S818 and FGA loci (red = blood samples; blue = buccal samples) AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 79 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram Table 4 Treated paper workflow: FTA® card sample marker-specific stutter filter percentages for Identifiler® Direct Kit loci † Locus % Stutter† CSF1PO 8.48 D13S317 9.39 D16S539 9.42 D18S51 12.89 D19S433 11.15 D21S11 10.42 D2S1338 11.77 D3S1358 11.45 D5S818 9.89 D7S820 8.60 D8S1179 9.54 FGA 11.62 TH01 4.76 TPOX 5.27 vWA 11.99 These percentages are used as stutter filters in GeneMapper® ID Software IdentifilerDirect_GS500_Panels_v1 and GeneMapper® ID-X Software IdentifilerDirect_GS500_Stutter_v1X.txt. Addition of 3´ A nucleotide AmpliTaq Gold® enzyme, like many other DNA polymerases, can catalyze the addition of a single nucleotide (predominately adenosine) to the 3´ ends of doublestranded PCR products (Clark, 1988; Magnuson et al., 1996). This nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence. The PCR product with the extra nucleotide is referred to as the “+A” form. The efficiency of +A addition is related to the particular sequence of the DNA at the 3´ end of the PCR product. The AmpFlSTR® Identifiler® Direct PCR Amplification 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 25 min. The final extension step gives the AmpliTaq Gold® DNA polymerase additional time to complete +A addition to all double-stranded PCR products. STR systems (where each allele is represented by two peaks that are one nucleotide apart) that have not been optimized for +A addition may have “split peaks.” 80 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Characterization of loci 5 Figure 12 Omitting the final extension step results in split peaks due to incomplete A nucleotide addition. Data are from an Applied Biosystems 3130xl Genetic Analyzer using the AmpFlSTR® Identifiler® Direct Kit. Artifacts Artifacts and anomalies are seen in all molecular biological systems. Artifacts are typically reproducible while anomalies are non-reproducible, intermittent occurrences that are not observed consistently in a system (for example, spikes and baseline noise). Reproducible artifacts have not been seen in data produced on the genetic analyzers used during developmental validation of the Identifiler® Direct Kit. Characterization of loci SWGDAM guideline 2.1 “The basic characteristics of a genetic marker must be determined and documented.” (SWGDAM, July 2003) This section describes basic characteristics of the 15 loci and the sex-determining marker, Amelogenin that are amplified with the Identifiler® Direct Kit. These loci have been extensively characterized by other laboratories. Nature of the polymorphisms The primers for the Amelogenin locus flank a 6-nucleotide deletion within intron 1 of the X homologue. Amplification results in 107-nt and 113-nt products from the X and Y chromosomes, respectively. (Sizes are the actual nucleotide size according to sequencing results, including 3´ A nucleotide addition.) The remaining Identifiler® Direct 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-nt repeat units. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 81 5 Chapter 5 Experiments and Results Species specificity We have subjected to sequencing all the alleles in the AmpFlSTR® Identifiler® Direct Allelic Ladder. In addition, other groups in the scientific community have sequenced alleles at some of these loci. Among the various sources of sequence data on the Identifiler® Direct Kit loci, there is consensus on the repeat patterns and structure of the STRs. Mapping The Identifiler® Direct Kit loci have been mapped, and the chromosomal locations have been published (Nakahori et al., 1991; Edwards et al., 1992; Kimpton et al., 1992; Mills et al., 1992; Sharma and Litt, 1992; Li et al., 1993; Straub et al., 1993; Barber and Parkin, 1996). Species specificity SWGDAM Guideline 2.2 “For techniques designed to type human DNA, the potential to detect DNA from forensically relevant nonhuman species should be evaluated.” (SWGDAM, July 2003) The Identifiler® Direct Kit provides the required specificity for detecting primate alleles. Other species do not amplify for the loci tested. Nonhuman studies Nonhuman DNA may be present in forensic casework samples. The data from Identifiler® Direct Kit experiments on nonhuman DNA sources are shown in Figure 13. Figure 13 Representative electropherograms from a species-specificity study including positive and non-template controls (NTC) Control DNA 9947A Chimpanzee Pig Cat Microbial pool NTC 82 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Sensitivity 5 Figure 13 shows amplification for: Control DNA 9947A (1 ng, panel 1), chimpanzee (1 ng, panel 2), pig (10 ng, panel 3), cat (10 ng, panel 4), microbial DNA pool (equivalent to 105 copies of Candida albicans, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Lactobacillus casei, Staphylococcus aureus, Streptococcus mitis, Streptococcus mutans, Streptococcus salivarius, and Streptococcus viridans, panel 5), and the non-template control (panel 6). The extracted DNA samples were amplified with the Identifiler® Direct Kit and analyzed using the Applied Biosystems 3130xl Genetic Analyzer. • Primates: gorilla, chimpanzee, orangutan, and macaque (1 ng each) • Non-primates: mouse, dog, pig, cat, horse, hamster, rat, chicken, and cow (10 ng each) • Microorganisms: Candida albicans, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Lactobacillus casei, Staphylococcus aureus, Streptococcus mitis, Streptococcus mutans, Streptococcus salivarius, and Streptococcus viridans (equivalent to 105 copies). These microorganisms are commonly found in the oral cavity (Suido et al., 1986; Guthmiller et al., 2001). All the primate DNA samples amplified, producing fragments within the 100 to 350 base pair region (Lazaruk, et al., 2001; Wallin, et al., 1998). The microorganisms, chicken, cat, hamster, rat, rabbit, and mouse samples did not yield detectable product. Horse, cow, dog, and pig samples produced a 104-bp fragment near the Amelogenin locus in PET® dye. Sensitivity SWGDAM guideline 2.3 “When appropriate, the range of DNA quantities able to produce reliable typing results should be determined.” (SWGDAM, July 2003) Blood on FTA® cards The Identifiler® Direct Kit has been optimized at 25 µL PCR reaction volume to overcome the PCR inhibition expected when amplifying blood samples directly from unpurified 1.2 mm FTA® discs. Depending on the volume of blood spotted onto the FTA® card, DNA quantities present on the 1.2 mm disc may vary from laboratory to laboratory. It is essential for your laboratory to optimize the PCR conditions based on the types of blood samples received or based on your standard operating protocol used in the spotting of blood onto FTA® cards. Refer to page 19 for instructions on PCR optimization. Buccal cells on FTA® or Indicating FTA® cards and buccal cells on Bode DNA Collectors The Identifiler® Direct Kit has been optimized at 25 µL PCR reaction volume to overcome the PCR inhibition expected when amplifying buccal cells directly from unpurified 1.2 mm FTA® discs or Indicating FTA® discs. Depending on the collecting devices used, the collection methods applied, and the swab-to-FTA® transfer protocol employed, DNA quantities present on the 1.2 mm disc may vary from sample to sample and from laboratory to laboratory. It is essential for your laboratory to optimize the PCR conditions based on the types of buccal samples received or based on your standard operating protocol used in transferring saliva from a buccal swab onto an FTA® card or Indicating FTA® cards. Refer to page 19 for instructions on PCR optimization. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 83 5 Chapter 5 Experiments and Results Sensitivity Effect of DNA quantity on results If too much DNA is added to the PCR reaction, the increased amount of PCR product that is generated can result in: • Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument (“off-scale” data). Off-scale data is a problem because: – Quantitation (peak height and area) for off-scale peaks is not accurate. For example, an allele peak that is off-scale can cause the corresponding stutter peak to appear higher in relative intensity, thus increasing the calculated percent stutter. – Multicomponent analysis of off-scale data is not accurate. This inaccuracy results in poor spectral separation (“pull-up”). • Incomplete +A nucleotide addition. To ensure minimal occurrence of offscale data when using the Identifiler® Direct Kit, optimize PCR cycle number according to instructions on page 19. When the total number of allele copies added to the PCR is extremely low, unbalanced amplification of the alleles may occur because of stochastic fluctuation. 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. 84 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Stability 5 Figure 14 Effect of amplifying varying amounts of white blood cells (WBCs) spotted onto Indicating FTA® discs Note that the y-axis scale is magnified for the lower amounts of DNA, analyzed using the Applied Biosystems® 3130xl Genetic Analyzer. The amount of DNA on the Indicating FTA® cards were calculated based on the assumptions of 100% cell lysis efficiency and that each cell contain 6 pg of DNA. The results from white blood cells spotted onto Bode DNA Collectors were comparable to the results shown here obtained using the Identifiler® Direct Kit with white blood cells spotted onto FTA® Indicating Cards (data not shown). Stability SWGDAM guideline 2.4 “The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected to various environmental and chemical insults has been extensively documented. In most instances, assessment of the effects of these factors on new forensic DNA procedures is not required. However, if substrates and/ or environmental and/or chemical insults could potentially affect the analytical process, then the process should be evaluated using known samples to determine the effects of such factors.” (SWGDAM, July 2003) AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 85 5 Chapter 5 Experiments and Results Stability DNA on FTA® cards Aged blood on FTA® cards and aged buccal cells on Indicating FTA® cards were prepared to examine the sample-on-substrate stability. Finger-prick blood spotted onto FTA® card and buccal samples swabbed and transferred using the EasiCollect™ devices were collected on three individuals over the course of 30 weeks. The Identifiler® Direct Kit was used to amplify the aged FTA® samples in a GeneAmp® PCR System 9700 with the gold-plated silver 96-well block and were electrophoresed and detected using an Applied Biosystems® 3130xl Genetic Analyzer. The results of the aged blood on FTA® card are shown in Figure 15 and the results of the aged buccal cells on Indicating FTA® card are shown in Figure 16. The analysis revealed that the age of the FTA® samples did not impact the performance of the AmpFlSTR® Identifiler® Direct Kit. Figure 15 Amplification of blood on FTA® card stored for various amounts of time at room temperature Figure 16 Amplification of buccal cells on Indicating FTA® card stored for various amounts of time at room temperature 86 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Stability DNA on buccal swabs 5 Aged buccal cell samples on Copan 4N6 FLOQSwabs®, Whatman OmniSwabs, and Puritan swabs were also prepared to verify their respective sample-on-substrate stability. Buccal swabs were collected from 40 individuals on each swab type over the course of three months. The aged swab samples were processed with Prep-n-Go™ Buffer, amplified using the Identifiler® Direct Kit in a GeneAmp® PCR System 9700 with the gold-plated silver 96-well block, and were electrophoresed and detected using an Applied Biosystems® 3130xl Genetic Analyzer. Figure 17 shows the results of the aged buccal samples collected on each swab type and lysed at 90°C for 20 minutes. For comparison, Figure 18 shows the results of fresh buccal samples collected on Copan 4N6 FLOQSwabs® and lysed at room temperature. The analysis revealed that buccal samples on the swab types tested, air-dried immediately after collection, and aged up to three months at room temperature produce acceptable profiles when amplified with the Identifiler® Direct Kit. Figure 17 Amplification of buccal cells on aged Copan 4N6 FLOQSwabs®, OmniSwabs, and Puritan swabs stored for 3 months at room temperature and lysed with Prep-n-Go™ Buffer at 99°C for 20 minutes Copan 4N6 FLOQSwabs® OmniSwabs Puritan swabs Figure 18 Amplification of buccal cells on fresh Copan 4N6 FLOQSwabs® and lysed with Prep-n-Go™ Buffer at room temperature AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 87 5 Chapter 5 Experiments and Results Population data Population data SWGDAM guideline 2.7 “The distribution of genetic markers in populations should be determined in relevant population groups.” (SWGDAM, July 2003) Overview To interpret the significance of a match between genetically typed samples, you must know the population distribution of alleles at each locus in question. If the genotype of the relevant evidence sample is different from the genotype of a suspects reference sample, then the suspect is excluded as the donor of the biological evidence that was tested. An exclusion is independent of the frequency of the two genotypes in the population. 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 samples used in these studies The Identifiler® Kit, prior to the addition of the D8S1179 degenerate primer, was used to generate the population data provided in this section. Samples were collected from individuals throughout the United States with no geographical preference. Population Number of samples African-American 357 U.S. Caucasian 349 U.S. Hispanic 290 Native American 191 Samples provided by Kentucky State Police and the Federal Bureau of Investigation Minnesota Bureau of Criminal Apprehension/Memorial Blood Center of Minneapolis In addition to the alleles that were observed and recorded in the Life Technologies databases, other alleles have been published or reported to Life Technologies by other laboratories (see the STRBase at www.cstl.nist.gov/div831/strbase). Identifiler® Direct Kit allele frequencies Table 5 shows the Identifiler® Direct Kit allele frequencies in four populations, listed as percentages. Table 5 Identifiler® Direct Kit allele frequencies AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) 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 31.91 28.28 21.99 Allele CSF1PO 88 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Population data Allele AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) 11.3 0.14† † † † 12 29.13 32.81 39.66 32.72 13 5.32 7.31 6.38 4.71 0.79† 14 0.98 1.43 0.86† 15 † 0.29† † † 15 0.14† † † † 16 5.32 4.73 2.41 2.62 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 2.38 22 13.17 4.01 7.41 15.18 23 10.78 11.46 11.36 11.78 24 9.80 11.75 8.45 7.85 25 8.12 10.60 5.17 3.14 0.79† 5 D2S1338 26 1.96 2.72 0.69† 27 0.14† 0.14† † † 28 † † † † <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† † D3S1358 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 89 5 Chapter 5 Experiments and Results Population data AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) 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† † 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 † † † † 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 † † † † Allele D5S818 7 D7S820 D8S1179 90 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Population data AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) 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† 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† † † 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 Allele 5 D13S317 D16S539 D18S51 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 91 5 Chapter 5 Experiments and Results Population data Allele AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) 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 † † † † 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 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† 24 † † † † 24.2 0.14† 0.43† 0.17† † 24.3 † † † † 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† D19S433 D21S11 92 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Population data Allele AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) 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† † † † 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 22 21.57 16.91 17.24 10.47 22.2 0.28† 1.29 0.34† 0.26† 22.3 0.14† 0.14† † † 23 14.99 15.19 11.90 15.97 23.2 0.14† 0.14† 0.86† 0.26† 24 17.51 13.75 15.34 15.71 5 FGA AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 93 5 Chapter 5 Experiments and Results Population data Allele AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) 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 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† † † † 47.2 † † † † 48.2 0.14† † † † 50.2 † † † † 51.2 † † † † 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 † † † † 9 † † † 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† † † † 6 6.72 0.14† 0.34† † 7 2.24 † 0.34† 0.26† 8 36.13 53.30 49.66 37.96 TH01 TPOX 94 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Population data Allele AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) 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 † † † † 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† † † 5 vWA † 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. Low-frequency alleles Some alleles of the Identifiler® Direct Kit loci occur at a low frequency. For these alleles, a minimum frequency (5 divided by 2n, where n equals the number of individuals in the database) was assigned for the Identifiler® Direct Kit AfricanAmerican, Asian, U.S. Caucasian, and U.S. Hispanic databases, as suggested in the 1996 report of the Committee on DNA Forensic Science (National Research Council, 1996). These databases are summarized in Table 8 on page 101. The minimum reportable genotype frequency at each locus is: 1.19 ✕ 10–4 for the African-American database; 1.19 ✕ 10–4 for the U.S. Caucasian database; 1.70 ✕ 10–4 for the U.S. Hispanic database; and 2.97 ✕ 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: 1.36 ✕ 10–59 for the African-American database; 1.36 ✕ 10–59 for the U.S. Caucasian database; 2.86 ✕ 10–57 for the U.S. Hispanic database; and 1.23 x 10-53 for the Native American database. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 95 5 Chapter 5 Experiments and Results Population data Evaluation of Hardy-Weinberg equilibrium Estimates of expected heterozygosity (HExp) were computed as described by Nei, M. (1973) using the program PopGene 1.32. Possible divergence from Hardy-Weinberg expectations (HWE) was tested using various methods: by calculating the unbiased estimate of the expected homozygote/heterozygote frequencies (Levene, H., Nei, M. 1978) and using chi-square (HW X2p) and likelihood ratio (HW G2p) tests (as implemented in the program PopGene 1.32): and with an exact test (HW Exact p), which is a Markov chain method, based on 1000 shuffling experiments, to estimate without bias the exact P-value of the Hardy-Weinberg test with multiple alleles (Guo, S.W. 1992), as implemented in the program GenePop 3.4. An inter-class correlation test analysis (Burrows' composite measure of linkage disequilibria between pairs of loci and X2 tests for significance [Weir, B. 1990]) was performed separately in each population to detect any correlations between alleles at any of the pair-wise comparisons of the 15 loci, using the program PopGene 1.32. Observed heterozygosity (Ho), expected heterozygosity, information content, and tests for detecting departures from Hardy-Weinberg equilibrium are shown for each population in Table 6. While a number of the chi-square tests gave seemingly significant p-values (putatively indicating departures from Hardy-Weinberg equilibrium), chi-squared tests are very sensitive to small expected values (as in the case of multiple rare alleles where the expected number of certain genotypes is 1 or fewer, such as with some of these markers), and can greatly inflate the test statistic in this situation (Weir, B. 1990). With the exact test, the number of tests with p-value < 0.05 were 0 in the African American and U.S. Caucasian populations, 1 in the U.S. Hispanic population (D8S1179; p=0.0304) and 2 in the Native Americans (D21S11, p=0.0118; D5S818, p=0.0205). These are no more than would be expected by chance. No more alleles were observed to be in linkage disequilibrium than would be expected by chance alone. The average observed heterozygosity across the 15 STR loci was 0.804 in the African American population, 0.792 in the U.S. Caucasian sample population, 0.793 in the Hispanic sample population, and 0.757 in the Native Americans. The most heterozygous locus was FGA (mean observed heterozygosity across all populations of 0.875), and the least heterozygous STR locus was TPOX (mean observed heterozygosity across all populations of 0.677). Table 6 Heterozygosity and p-values for Hardy-Weinberg tests of the 15 Identifiler STR loci in four U.S. populations† AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) CSF1P0 HW X2 p 0.13649 0.926431 0.951476 0.839278 HW G2 p 0.08902 0.894972 0.918038 0.728023 HW Exact p 0.0762 0.2688 0.5456 0.6148 CSF1P0 (continued) HExp 0.7829 0.7267 0.7051 0.7398 Ho 0.7703 0.7421 0.7138 0.7958 D2S1338 † HW X2 p, probability value of X2 test for Hardy-Weinberg equilibrium; HW G2 p, probability value of the G-statistic of the Likelihood Ratio test for multinomial proportions; HW Exact p; A Markov chain unbiased exact test to estimate the P-value of the Hardy-Weinberg test with multiple alleles; Hexp, Expected heterozygosity; Ho, observed heterozygosity 96 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Population data AfricanAmerican (n = 357) HW X2 p U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) 0.409878 0.537758 0.975972 0.722543 0.962501 0.407932 0.973054 0.760953 HW Exact p 0.7838 0.3488 0.9794 0.5825 HExp 0.8936 0.8823 0.8529 0.8428 Ho 0.8768 0.8653 0.8379 0.801 0.947371 0.670787 0.681659 0.087223 0.907905 0.654776 0.852278 0.175807 HW Exact p 0.2967 0.2814 0.4684 0.0614 HExp 0.7681 0.7986 0.7361 0.7028 Ho 0.7955 0.8166 0.7414 0.7382 HW X2 p 0.993751 0.859805 0.944725 0.073002 HW G2 p 0.989776 0.520417 0.979044 0.08025 HW Exact p 0.958 0.462 0.4662 0.0205 HExp 0.7476 0.6931 0.7351 0.7378 Ho 0.7479 0.7077 0.7586 0.6806 0.987668 0.571989 0.336834 0.324754 0.969887 0.44694 0.687948 0.289733 HW Exact p 0.9818 0.2286 0.4028 0.1276 HExp 0.7758 0.8117 0.7822 0.7858 Ho 0.7955 0.7908 0.7862 0.7487 0.067164 0.545414 0.047783 0.446248 0.568837 0.275218 0.302937 0.760077 HW Exact p 0.2176 0.3264 0.0304 0.1656 HExp 0.7925 0.8047 0.7853 0.7403 Ho 0.7899 0.8424 0.8 0.6806 HW G2 p 5 D3S1358 HW X2 p HW G2 p D5S818 D7S820 HW X2 p HW G2 p D8S1179 HW X2 p HW G2 p AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 97 5 Chapter 5 Experiments and Results Population data AfricanAmerican (n = 357) U.S. Caucasian (n = 349) Native American (n = 191) U.S. Hispanic (n = 290) D13S317 HW X2 p 0.014379 0.711127 0.353995 0.813948 0.609389 0.871173 0.190736 0.814681 HW Exact p 0.3818 0.667 0.2415 0.6851 HExp 0.6977 0.7797 0.8251 0.8222 Ho 0.6695 0.7364 0.8207 0.8168 0.433216 0.67702 0.058631 0.996396 0.482435 0.594871 0.37601 0.981384 HW Exact p 0.3753 0.4328 0.3068 0.9986 HExp 0.7939 0.7632 0.7747 0.7766 Ho 0.8263 0.7822 0.7828 0.7853 HW X2 p 0.999844 0.628334 0.999203 0.343027 HW G2 p 1 0.872113 0.999492 0.798859 HW Exact p 0.978 0.0982 0.9152 0.2265 HExp 0.8694 0.8769 0.8761 0.8463 Ho 0.8824 0.8682 0.8862 0.8377 HW X2 p 0.91703 0.806717 0.731222 0.810711 HW G2 p 0.83419 0.999765 0.975476 0.898389 HW Exact p 0.4517 0.69 0.3475 0.4301 HExp 0.8364 0.7659 0.8310 0.8430 Ho 0.8011 0.7622 0.8414 0.822 0.985687 0.936146 0 0 1 0.999757 0.999794 0.712937 HW Exact p 0.7627 0.7861 0.6476 0.0118 HExp 0.8585 0.8427 0.8290 0.8003 Ho 0.8711 0.8567 0.7931 0.801 0 0.904953 0.263223 0.999686 1 0.999812 0.960137 0.999946 HW Exact p 0.9761 0.4459 0.0891 0.9161 HExp 0.8659 0.8686 0.8751 0.8746 Ho 0.8824 0.8854 0.8724 0.8482 HW G2 p D16S539 HW X2 p HW G2 p D18S51 D19S433 D21S11 HW X2 p HW G2 p FGA HW X2 p HW 98 G2 p AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Mutation rate AfricanAmerican (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) 5 Native American (n = 191) TH01 HW X2 p 0.961911 0.997905 0.649467 0.329461 0.940414 0.99169 0.617212 0.318591 HW Exact p 0.8286 0.9716 0.4495 0.1377 HExp 0.7323 0.7866 0.7666 0.7016 Ho 0.7395 0.7822 0.8103 0.6492 0.765163 0.801518 0.875348 0.333914 0.611014 0.757735 0.913091 0.229017 HW Exact p 0.7247 0.5775 0.8356 0.0647 HExp 0.7643 0.6311 0.6607 0.6765 Ho 0.7563 0.6304 0.6759 0.6178 HW X2 p 0.925176 0.005048 0.641684 0.994248 HW G2 p 0.964308 0.218817 0.934427 0.997184 HW Exact p 0.7033 0.0564 0.7066 0.8845 HExp 0.8141 0.8081 0.7818 0.7457 Ho 0.8571 0.8138 0.7759 0.7277 HW G2 p TPOX HW X2 p HW G2 p vWA Concordance studies We compared allele calls between the Identifiler® and Identifiler® Direct Kits. The genotype data from the 200 analyzed treated paper workflow samples showed 100% concordance between the Identifiler® and Identifiler® Direct Kits. The genotype data from 84 buccal samples processed using Prep-n-Go™ Buffer and the Identifiler® Direct Kit showed 100% concordance to allele calls generated for purified DNA samples analyzed with the Identifiler® kit. Mutation rate Estimation of spontaneous or induced germline mutation at genetic loci can be achieved by comparing the genotypes of offspring to those of their parents. From such comparisons the number of observed mutations are counted directly. In previous studies, genotypes of ten STR loci that were 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; mutations were not detected at any of the other nine STR loci. The D18S11 mutation was represented by an increase of one 4-nt repeat unit, allele 17 was inherited as allele 18 (single-step mutation). The maternal/paternal source of this mutation could not be distinguished. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 99 5 Chapter 5 Experiments and Results Probability of identity Additional mutation studies Additional studies (Edwards et al., 1991; Edwards et al., 1992; Weber and Wong, 1993; 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® Direct Kit loci. • Methods for modifications of these mutation rates (to infer mutation rates indirectly for those loci where the 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® Direct Kit loci individually and combined. Table 7 Probability of Identity values for the Identifiler® Direct Kit STR loci AfricanAmerican 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 vWA 0.066 0.066 0.080 0.103 Locus Combined 1.31 ✕ 10−18 5.01 ✕ 10−18 7.65 ✕ 10−18 3.62 ✕ 10−17 The PI value is the probability that two individuals selected at random will have an identical Identifiler® Direct Kit genotype (Sensabaugh, 1982). The PI values for the populations described in this section are then approximately 1/7.64 ✕ 1017 (AfricanAmerican), 1/2.00 ✕ 1017 (U.S. Caucasian), 1/1.31 ✕ 1017 (U.S. Hispanic), and 1/2.76 ✕ 1016 (Native American). 100 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results 5 Probability of paternity exclusion Probability of paternity exclusion Table 8 shows the Probability of Paternity Exclusion (PE) values of the Identifiler® Direct Kit STR loci individually and combined. Table 8 Probability of Paternity Exclusion values for the Identifiler® Direct Kit loci AfricanAmerican U.S. Caucasian U.S. Hispanic Native American 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 0.9999996 0.9999992 0.9999990 0.9999527 Locus Combined The PE value is the probability, averaged over all possible mother-child pairs, that a random alleged father will be excluded from paternity after DNA typing of the Identifiler® Direct Kit STR loci (Chakraborty and Stivers, 1996). AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 101 5 102 Chapter 5 Experiments and Results Probability of paternity exclusion AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide A Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis. Table 9 Troubleshooting Observation Faint or no signal from both the AmpFlSTR® Identifiler® Direct Control DNA 9947A and the DNA test samples at all loci Possible causes Recommended actions Incorrect volume or absence of Identifiler® Direct Master Mix or Identifiler® Direct Primer Set Repeat amplification. No activation of AmpliTaq Gold® DNA Polymerase Repeat amplification, making sure to hold reactions initially at 95°C for 11 minutes. Master Mix not vortexed thoroughly before aliquoting Vortex the Master Mix thoroughly. Identifiler® Direct Primer Set exposed to too much light Store the Primer Set protected from light. PCR System malfunction Refer to the thermal cycler user’s manual and check instrument calibration. Use of incorrect thermal cycling parameters Check the protocol for correct thermal cycling parameters. MicroAmp® Base used with tray/ retainer set and tubes in GeneAmp® 9700 Remove MicroAmp Base from tray/retainer set and repeat test. Insufficient PCR product electrokinetically injected Prepare PCR product as described in Chapter 3, “Perform Electrophoresis” on page 29. Degraded formamide Check the storage of formamide; do not thaw and refreeze multiple times. Try Hi-Di™ Formamide. Sample punch location was not optimal For blood samples on treated paper, punch in the center of the blood stain. For buccal samples on treated paper, punch in the center of the buccal transfer or punch in the optimal spot based on past experiences. For buccal samples collected with the Bode Buccal DNA Collector™, punch from near the tip of the collector. Insufficient lysis of the swab head AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Ensure swab heads are incubated for 20 minutes in 400 µL Prep-N-Go™ Buffer. 103 A Appendix A Troubleshooting Observation More than two alleles present at a locus Possible causes Recommended actions Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling. Amplification of stutter product (n-4 nt position) See “Stutter products” on page 75. Incomplete 3´ A base addition (n-1 nt position) See “Addition of 3´ A nucleotide” on page 80. Be sure to include the final extension step of 60°C for 10 minutes in the PCR. Signal exceeds dynamic range of instrument (off-scale data) Ensure cycle number is optimized according to instructions on page 19. Repeat PCR amplification using fewer PCR cycles or use your laboratory’s SOP to analyze off-scale data. Poor spectral separation (bad matrix) Follow the steps for creating a spectral file. Confirm that Filter Set G5 modules are installed and used for analysis. Some but not all loci visible on electropherogram of DNA Test Samples STR profiles contain many off-scale alleles 104 Contamination carried over from the disc punching tool Clean the disc punching tool thoroughly. If necessary, include a blank punch step in between the sample punches. Incomplete denaturation of double stranded DNA Use recommended amount of Hi-Di™ Formamide and perform heat denaturation step according to the instructions in Chapter 3, “Perform Electrophoresis”. Disc size used in the amplification reaction was greater than 1.2 mm Repeat amplification using a use 1.2 mm punch size. Insufficient volume of swab lysate added to the reaction Repeat amplification using the recommended lysate input volume. Less than 25 µL of PCR reaction volume was used Repeat amplification using the recommended PCR reaction volume of 25 µL. PCR cycle number was too high Perform sensitivity experiment (page 19) to determine the optimal PCR cycle number based on the sample type. For blood samples: Too much liquid blood was spotted onto paper substrate Spot <100 µL of liquid blood per sample area. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide B Ordering Information Equipment and materials not included Table 10 and Table 11 list required and optional equipment and materials not supplied with the Identifiler® Direct Kit. Unless otherwise noted, many of the items are available from major laboratory suppliers (MLS). Table 10 Equipment Equipment 3100/3100-Avant Genetic Analyzer Applied Biosystems® 3500/3500xL Genetic Analyzer Applied Biosystems® 3130/3130xl Genetic Analyzer Applied Biosystems® 3730 Genetic Analyzer GeneAmp® Source Contact your local Life Technologies sales representative PCR System 9700 with the Silver 96-Well Block N8050001 GeneAmp® PCR System 9700 with the Gold-plated Silver 96-Well Block 4314878 Silver 96-Well Sample Block N8050251 Gold-plated Silver 96-Well Sample Block 4314443 Veriti® 4375786 96-Well Thermal Cycler Tabletop centrifuge with 96-Well Plate Adapters (optional) MLS Harris Manual Punch, 1.2 mm MLS BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a 1.2 mm punch head BSD1000-GenePunch Automated Dried Sample Punch Instrument with a 1.2 mm punch head Bode Buccal DNA Collector™ Contact your local Life Technologies support representative for information. 4467893 This part number is not available for sale in the US. Copan FLOQSwabs® Contact your local Life Technologies support representative for information. Copan NUCLEIC-CARD™ system Contact your local Life Technologies support representative for information. This product is not available for sale in the US. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 105 B Appendix B Ordering Information Equipment and materials not included Equipment Source 96-well, deep-well plate 4392904 Table 11 User-supplied materials Item† AmpFlSTR® Identifiler® Source Direct PCR Amplification Kit, 200 reaction 4467831 AmpFlSTR® Identifiler® Direct PCR Amplification Kit, 1000 reaction 4408580 Prep-n-Go™ Buffer (untreated paper substrate) 4467079 Prep-n-Go™ Buffer (buccal swab) 4471406 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™ 4311320 Formamide DS-33 Matrix Standard Kit (Dye Set G5) MicroAmp® 4345833 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 instrument, refer to Appendix B of the 3100 Genetic Analyzer and 3100-Avant Genetic Analyzer User Reference Guide (Part 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 GeneScan™ 500 LIZ® Size Standard 4322682 OR OR GeneScan™ 600 LIZ® Size Standard v2.0 4408399 Running Buffer, 10✕ 402824 DS-33 Matrix Standard Kit (Dye Set G5) 4345833 MicroAmp® 106 Optical 96-Well Reaction Plate N8010560 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Appendix B Ordering Information Equipment and materials not included Item† Hi-Di™ Formamide B Source 4311320 For a complete list of parts and accessories for the 3130xl instrument, refer to Appendix A of the Applied Biosystems 3130/ 3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide (Part 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® 4393715 polymer (384 samples) for 3500/3500xL Genetic Analyzers DS-33 Matrix Standard Kit (Dye Set G5) 4345833 GeneScan™ 4408399 600 LIZ® Size Standard v2.0 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 (PN 4401661). 3730 Analyzer materials 3730 DNA Analyzer Capillary Array, 36-cm 4331247 GeneScan™ 500 LIZ® Size Standard 4322682 OR OR GeneScan™ 600 LIZ® Size Standard v2.0 4408399 Hi-Di™ 4311320 Formamide Running Buffer, 10✕ 4335613 DS-33 Matrix Standard Kit (Dye Set G5) 4345833 96-Well Plate Septa 4315933 MicroAmp® Optical 96-Well Reaction Plate POP-7™ Polymer for the 3730 Genetic Analyzer N8010560 4332241 For a complete list of parts and accessories for the 3730 instrument, refer to Appendix A of the Applied Biosystems® 3730/ 3730xl DNA Analyzer Getting Started Guide (Part no. 4359476). 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 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 107 B Appendix B Ordering Information Equipment and materials not included Item† MicroAmp® 96-Well Tray/Retainer Set MicroAmp® 96-Well Base Source 403081 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 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. 108 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide C Plate Layouts Example PCR plate layout The following layout is recommended for use with the sensitivity experiment on page 19. Create 3 identical plates for amplification at 3 different cycle numbers. Example electrophoresis plate layout The following layout is recommended for use with the sensitivity experiment on page 19. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 109 C 110 Appendix C Plate Layouts Example electrophoresis plate layout AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide D PCR Work Areas ■ Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 ■ PCR setup work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 ■ Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 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® Direct 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® Direct 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 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 111 D Appendix D PCR Work Areas 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® Direct 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® Direct Kit. • Veriti® 96-Well Thermal Cycler 112 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide E 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. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 113 E Appendix E Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, 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 handling CAS 26628-22-8 Chemical Sodium Azide Phrase Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. 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. 114 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Appendix E Safety Biological hazard safety E 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/ AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 115 E 116 Appendix E Safety Biological hazard safety AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Bibliography Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994. 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Legal Med. 114:285–287. 120 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Documentation and Support Related documentation Part number Document title 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 User Bulletin AmpFlSTR® PCR Amplification Kit PCR Products Applied Biosystems® 3130/3100xl Genetic Analyzers Using Data Collection Software v3.0 User Bulletin Applied Biosystems® 3130/3130xl Genetic Analyzers Getting Started Guide 4332345 4363787 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 Applied Biosystems® 3500/3500xL Genetic Analyzer Quick Reference Card Applied Biosystems® 4331468 4401662 3500/3500xL Genetic Analyzer User Guide, Data Collection v1.0 Applied Biosystems® 3500/3500xL Genetic Analyzer User Bulletin: Solutions to issues related to software, data, 4401661 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 Quantifiler® N805-0200 Quantifiler® Kits: Human DNA Quantification Kit and Quantification Kit User’s Manual Quantifiler® Y Human Male DNA 4344790 AmpFlSTR® Identifiler® PCR Amplification Kit User’s Manual 4323291 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® GeneMapper® ID Software v3.2 User Bulletin ID-X Software Version 1.0 Getting Started Guide 4352543 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 GeneMapper® ID-X Software Version 1.1 (Mixture Analysis) Quick Reference Guide 4402094 GeneMapper® ID-X Software Version 1.2 Reference Guide 4426481 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 121 Documentation and Support Obtain SDSs Part number Document title 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 our web site, use the Adobe® Acrobat® Reader® software available from www.adobe.com. Obtain SDSs Safety Data Sheets (SDSs) are available from www.lifetechnologies.com/support. 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 [email protected], 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 122 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Notices 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.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support. AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 123 Notices Limited Product Warranty 124 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Index Symbols .fsa sample files 38, 51 .hid sample file 50 +A nucleotide addition defined 80 efficiency of 80 Numerics 3100/3100-Avant instruments 31 3500/3500xL instruments 33 3730 instrument 33 A accuracy and reproducibility 66 alleles low frequency 95 off-ladder 67 allelic ladder figure 13 requirements for accurate genotyping 30 volume per reaction 32, 34, 36 analysis method 42, 55 analysis method, create 42 analyze a project 48, 62 artifacts in data 81 B bins import 51 bins, import 38, 51 biohazard safety 114 blood samples 11, 19, 21 Bode Buccal DNA Collector 11, 19, 23 buccal samples 11, 19, 21, 23 buccal swabs 25 C chemical safety 114 concordance studies 99 contents of kit 16 control DNA, about 17 Copan FLOQSwabs 87 treated cards 11 D data collection software 15 data, accuracy, precision, and reproducibility of 66 data, artifacts 81 data, for different populations 88 developmental validation 66 DNA effect of quantity, figure 85 negative control sample preparation 21, 23, 26 positive control sample preparation 21, 23, 26 sensitivity 83 your sample preparation 21, 23, 26 documentation, related 121 E electropherogram causes of extra peaks 75 extra peaks 75 species specificity 82, 86, 87 electrophoresis Data Collection Software 31, 33 data collection software 35 prepare samples 32, 33, 35 references 31, 33, 35 run module 31, 33, 35 set up of 3100/3100-Avant instruments 31 set up of 3500/3500xL instruments 33 set up of 3730 instrument 33, 35 emission spectra 16 equipment, not included with kit 105 extra peaks, causes 75 characterization of loci, validation 81 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide 125 Index F instruments for use with 11 loci amplified 12 master mix 16 primers 12, 16 purpose 11 reagents 16 storage 16 thermal cyclers for use with 112 five-dye fluorescent system 12 fluorescent dyes 15 FSA sample files 38, 51 FTA cards 11, 19, 21 G GeneMapper ID Software analyze a project 48 analyze files 48 data analysis 38 edit a project 49 examine a project 49 import panels and bins 38 size standard, create 47 GeneMapper ID-X Software analyze a project 62 analyze files 62 data analysis 51 marker stutter 51 panels, bins, and stutter, import 51 size standard, create 60 GeneScan size standard about 17 dye label 15 volume per reaction 32, 33, 35 GS 500 17 GS 600 17 L Limited Product Warranty 123 LIZ size standard about 17 volume per reaction 32, 33, 35 loci characterization 81 chromosomal location 12 combined genotype frequency 95 dye label 12 genotype frequency in population 95 mapping 82 lysate, prepare 25 M Hi-Di formamide, volume per reaction 32, 33, 35 marker stutter, import 51 master mix, volume per reaction 21, 23, 25 materials and equipment 16 materials, not included with kit 105 multicomponent analysis 15 mutation studies 100 mutation, STR 99 I N import bins 38 panels 38 panels, bins, stutter 51 size standard 47, 60 Instrument and software compatibility 15 Instrument and special software compatibility 15 normalization 58 H K kit contents 16 description 11 fluorescent dyes 15 126 O off-ladder alleles 67 operating systems 15, 31, 33, 35 P panels, import 38, 51 PCR optimize cycle number 19 perform 28 setup 111 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Index PCR work areas 105, 111 percent stutter off-scale peaks 76 relation to allele length 75 precision and size windows 67 precision, sizing 67 Prep-n-Go Buffer 11, 19, 23 primers about 12 Amelogenin 81 volume per reaction 21, 23, 25 probability of identity definition 100 values 100 project examination and editing 63 punches, size 21, 23 R reaction mix, for PCR 22, 24, 26 reactions, prepare for PCR 21, 23, 25 reagents not included with kit 105 run module, electrophoresis 31, 33, 35 S safety biohazard 114 chemical 114 Safety Data Sheets (SDSs), obtaining 122 sample discs, size 21, 23 sample files, .fsa 38, 51 size deviation, sample alleles and ladder alleles 66 size standard create 47, 60 GS 50 and GS 600 17 size standard, create 47, 60 sizing precision 67 species specificity 82 split peaks, +A nucleotide addition 80 standards for samples 17 STRBase 88 stutter products 75 substrates swab 25 treated paper 21 untreated paper 23 support, obtaining 122 swab AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide PCR 28 prepare reactions 25 sample preparation guidelines 25 samples 11 types 11 validation 87 T technical support 122 Terms and Conditions 123 thermal cyclers for use with kit 112 programming 28 training, information on 122 treated paper 21 PCR 28 prepare reactions 21 sample preparation guidelines 21 U untreated paper 23 PCR 28 prepare reactions 23 sample preparation guidelines 23 V validation characterization of loci 81 developmental 66 effect of DNA quantity 84 experiments to evaluate 65 importance of 65 mutation rate 99 population data 88 probability of identity 100 probability of paternity exclusion 101 sensitivity 83 size deviation, sample and ladder alleles 66 species specificity 82 swabs 87 W warranty 123 work area amplified DNA 109, 112 PCR setup 111 setup and lab design 111 127 Index workflow overview 14 128 AmpFlSTR® Identifiler® Direct PCR Amplification Kit User Guide Headquarters 5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288 For support visit www.lifetechnologies.com/support www.lifetechnologies.com ">

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Key features
- Direct amplification of single-source samples
- Blood and buccal samples on treated paper substrates
- No need for sample purification
- 15 autosomal STR loci
- Sex-determining marker Amelogenin
- Compatible with Applied Biosystems instruments
- Multicomponent analysis for separating fluorescent dye colors
Frequently asked questions
The kit is optimized to amplify single-source blood and buccal samples on treated paper substrates without the need for sample purification.
The kit amplifies 15 autosomal STR loci and the sex-determining marker, Amelogenin.
The kit is designed for use with Applied Biosystems instruments such as the 3100/3100-Avant Genetic Analyzer, 3130/3130xl Genetic Analyzer, 3500/3500xL Genetic Analyzer, 3730 Genetic Analyzer, GeneAmp PCR System 9700, and Veriti 96-Well Thermal Cycler.
Multicomponent analysis is a process that separates the five different fluorescent dye colors into distinct spectral components, allowing for the simultaneous amplification and efficient separation of multiple loci with overlapping size ranges. The dyes in the Identifiler Direct Kit are 6-FAM™, VIC®, NED™, PET®, and LIZ®.