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Improved Analysis of DNA Short Tandem Repeats with Time-Of-Flight Mass Spectrometry Final Report

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Improved Analysis of DNA Short Tandem Repeats with Time-Of-Flight Mass Spectrometry Final Report The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report: Document Title: Improved Analysis of DNA Short Tandem Repeats with Time-of-Flight Mass Spectrometry Final Report Author(s): John M. Butler ; Christopher H. Becker Document No.: 179419 Date Received: November 30, 1999 Award Number: 97-LB-VX-0003 This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this Federally- funded grant final report available electronically in addition to traditional paper copies. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. FINAL REPORT on NIJ Grant 97-LB-VX-0003 Improved Analysis of DNA Short Tandem Repeats with Time-of-FlightMass Spectrometry John M. Butler and Christopher H. Becker GeneTrace Systems Inc. Research performed from June 1997 to April 1999 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. NIJ Grant 97-LB-VX-0003 Final Report Improved Analysis of DNA Short Tandem Repeats with Time-of-Flight Mass Spectrometry John M . Butler and Christopher H . Becker TABLEOF CONTENTS I . Executive Summary A . Introduction ................................................................................. 1 B . GeneTrace Systems and Mass Spectrometry.......................................... 4 C . Short Tandem Repeats (STRs) ........................................................... 5 D . Single Nucleotide Polymorphisms (SNPs) ............................................ 8 E . Papers Published ......................................................................... 10 F . Presentations Made ..................................................................... 11 II . Project Description A . STR Grant ................................................................................ 13 B . SNP Grant ................................................................................ 15 m . Scope and Methodology A . Assay Development and Primer Testing ............................................. 18 B . Sample Cleanup and Mass Spectrometry ............................................ 26 C . Sample Genotyping ..................................................................... 30 D . Comparison Tests with AB1 3 10 ...................................................... 32 IV . Results and Discussion for STR Analysis by Mass Spectrometry A . Marker Selection and Feasibility Studies ............................................ 34 1 . Caveats for DNA Mass Spectrometry............................................ 36 2 . Size Reduction Methods ............................................................ 37 B . Mu!t:p!ex ST?_ Work ................................................................... 40 C . Comparison Tests Between AB1 310 and Mass Spec Results ..................... 43 D . PCR Issues ............................................................................... 44 E . Analytical Capabilities of Mass Spec ................................................ 52 V . Results and Discussion for Multiplex SNPs A . Mitochondrial DNA Work ............................................................. 60 B . Y Chromosome Work ................................................................... 64 VI . Conclusions and Implications of Findings ................................................ 68 VI1. Endnotes A . Works Cited ............................................................................... 70 B . Biographical Note about Authors ...................................................... 74 Vm. Exhibits (Graphs. Tables. and Charts) ..................................................... 75 1 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. NIJ Grant 97-LB-VX-0003 Final Report ACKNOWLEDGMENTS The project described in this final grant report could not have happened without the hard work and support of a number of people at GeneTrace Systems Inc. First and foremost was Jia Li, who did some of the early primer design and STR work to demonstrate that STRs could be effectively analyzed by mass spectrometry. Jia taught me a lot about PCR and was always encouraging of my work. Likewise, Tom Shaler was important in the early phases of this research with his expert advice in mass spectrometry and data processing. The first GeneTrace STR mass spectra were carefully collected by Tom and thus he along with Jia deserves credit for helping obtain the funding for this grant. Dan Pollart synthesized numerous cleavable primers for this project especially in the first year of our work. David Joo and Wendy Lam also prepared PCR and SNP primers for the later part of this work. A number of people assisted in robotic sample preparation and sample cleanup including Mike Abbott, Jon Marlowe, David Wexler, and Rebecca Turincio. Joanna Hunter, Vera Delgado, and Can Nhan ran many of the STR samples on the automated mass spectrometers. Their hard work made it possible to focus on experimental design and data analysis rather than routine sample handling. Having talented and supportive coworkers was a great blessing throughout the course of this project. Kathy Stephens, Jia Li, Tom Shaler, Yuping Tan, Christine Loehrlein, Joanna Hunter, Hua Lin, Gordy Haupt, and Nathan Hunt provided useful discussions on a number of issues and helped develop assay parameters and tackle automation issues among other things. Nathan Hunt was especially important to the success of this project as he developed the STR genotyping algorithm and CallSSR software as well as the multiplex SNP primer design software. Kevin Coopman developed the SNP genotyping algorithm and calling software and was always eager to analyze my multiplex SNP samples. Joe Monforte and Roger Walker served as my supervisors for the first year and second year of this project, respectively, allowed me the opportunity to devote sufficient time to doing the work described in this project. Last but not least, Debbie Krantz served as an able administrator of these two NU grants and took care of the financial end of the grants. We were also supported with samples and sequence information from a number of scientific collabcratcrs. Sieve Lee and John Tonkyn from the California Department of JusticeDNA z Laboratory provided genomic DNA samples and STR allelic ladders. Dibang Liu from Northwestern University provided the D3S1358 DNA sequence used for improved primer design purposes. Peter Oefner and Peter Underhill from the Department of Genetics at Stanford University provided male population samples and Y chromosome SNP sequences. The encouragement and support of Lisa Forman and Richard Rao from the Office of Justice Programs at the National Institute of Justice propelled this work from an idea to a worlung product. In addition, Dennis Reeder from the National Institute of Standards and Technology was always a constant source of encouragement at scientific meetings. This project was supported under award number 97-LB-VX-0003 from the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. Points of view in this document are those of the authors and do not necessarily represent the official position of the U.S. Department of Justice. For further information, contact: John Butler, GeneTrace Systems Inc., 1401 Harbor Bay Parkway, Alameda, CA 94025; (510) 748-6124; butler 0genetrace .corn. This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. NIJ Grant 97-LB-VX-0003 Final Report EXECUTIVE SUMMARY A. Introduction The advent of DNA typing and its use for human identity testing has revolutionized law enforcement in recent years by allowing forensic laboratories to match suspects with miniscule amounts of biological evidence from a crime scene. Equally important is the use of DNA to exclude suspects who were not involved in a crime or to identify human remains in an accident. The last d5cade has seen numerous advances in the DNA testing procedures, most notably among them is the development of PCR (polymerase chain reaction)-based DNA typing methods. Technologies for measuring DNA variation, both length and sequence polymorphisms, have also advanced rapidly in the last decade. The time for the determination of a sample’s DNA profile has dropped from 6-8 weeks to 1-2 days with the possibility of being able to process samples in only a few minutes or hours due to more recent advancements. Simultaneous with the evolution of DNA markers and technologies embraced by the forensic community has been the acceptaxe zix! LISP, of DNA typing information. The courtroom battles over statistical issues that were common in the late 1980s and early 1990s have subsided as DNA evidence has become more widely accepted. In the past five years, DNA databases have
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