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DNA Short Tandem Repeats with Time-Of-Flight Mass Spectrometry

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DNA Short Tandem Repeats with Time-Of-Flight Mass Spectrometry U.S. Department of Justice Office of Justice Programs National Institute of Justice Improved Analysis of DNA Short Tandem Repeats With Time-of-Flight Mass Spectrometry science and technology research report U.S. Department of Justice Office of Justice Programs 810 Seventh Street N.W. Washington, DC 20531 John Ashcroft Attorney General Office of Justice Programs National Institute of Justice World Wide Web Site World Wide Web Site http://www.ojp.usdoj.gov http://www.ojp.usdoj.gov/nij IMPROVED ANALYSIS OF DNA SHORT TANDEM REPEATS WITH TIME-OF-FLIGHT MASS SPECTROMETRY John M. Butler and Christopher H. Becker Science and Technology Research Report October 2001 NCJ 188292 Sarah V. Hart Director, National Institute of Justice Lois Tully Project Monitor John M. Butler, Ph.D., is currently a research chemist at the National Institute of Standards and Technology and principle investigator on an NIJ-funded project to further develop multiplex PCR and time-of-flight mass spectrometry for future forensic DNA typing assays. He was the first to demonstrate that short tandem repeat typing could be performed with capillary electrophoresis. Christopher H. Becker, Ph.D., is currently senior director of proteomics technology at Thermo Finnigan in San Jose, California. During the span of this project, he was president and chief operations officer of GeneTrace Systems, Inc. This project was supported under grant 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 or policies of the U.S. Department of Justice. This document is not intended to create, does not create, and may not be relied upon to create any rights, substantive or procedural, enforceable at law by any party in any matter, civil or criminal. For further information, contact John M. Butler, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899; phone 301–975–4049; e-mail [email protected]. The National Institute of Justice is a component of the Office of Justice Programs, which also includes the Bureau of Justice Assistance, the Bureau of Justice Statistics, the Office of Juvenile Justice and Delinquency Prevention, and the Office for Victims of Crime. ACKNOWLEDGMENTS he project described in this ran many of the STR samples on the described in this project. Last but not report could not have happened automated mass spectrometers. Their least, Debbie Krantz served as an able Twithout the hard work and sup- hard work made it possible to focus on administrator of these two NIJ grants port of a number of people at GeneTrace experimental design and data analysis and took care of the financial aspects. Systems, Inc. First and foremost, Jia Li rather than routine sample handling. We also were supported with samples did some of the early primer design It was a great blessing to have talented and sequence information from a num- and STR work to demonstrate that and supportive coworkers throughout ber of scientific collaborators. Steve Lee STRs could be effectively analyzed by the course of this project. Kathy and John Tonkyn from the California mass spectrometry. Jia taught us a lot Stephens, Jia Li, Tom Shaler, Yuping Department of Justice DNA Laboratory about PCR and was always encourag- Tan, Christine Loehrlein, Joanna provided genomic DNA samples and ing of our work. Likewise, Tom Shaler Hunter, Hua Lin, Gordy Haupt, and STR allelic ladders. Debang Liu from was important in the early phases of Nathan Hunt provided useful discus- Northwestern University provided this research with his expert advice in sions on a number of issues and helped the D3S1358 DNA sequence used for mass spectrometry and data processing. develop assay parameters and tackle improved primer design purposes. The first GeneTrace STR mass spectra automation issues, among other things. Peter Oefner and Peter Underhill from were carefully collected by Tom, and Nathan Hunt was especially important the Department of Genetics at Stanford thus he and Jia deserve credit for help- to the success of this project because University provided male population ing obtain the funding for this study. he developed the STR genotyping algo- samples and Y-chromosome SNP Dan Pollart synthesized numerous rithm and CallSSR software as well as sequences. The encouragement and cleavable primers for this project, espe- the multiplex SNP primer design soft- support of Lisa Forman and Richard cially in the first year of our work. ware. Kevin Coopman developed the Rau from the Office of Justice Programs David Joo and Wendy Lam also pre- SNP genotyping algorithm and calling at the National Institute of Justice pro- pared PCR and SNP primers for the software and was always eager to ana- pelled this work from an idea to a later part of this work. A number of lyze our multiplex SNP samples. Joe working product. In addition, Dennis people assisted in robotic sample prepa- Monforte and Roger Walker served as Reeder from the National Institute of ration and sample cleanup, including our supervisors for the first year and Standards and Technology was always Mike Abbott, Jon Marlowe, David second year of this project, respectively, a constant source of encouragement Wexler, and Rebecca Turincio. Joanna which allowed us the opportunity to at scientific meetings. Hunter, Vera Delgado, and Can Nhan devote sufficient time to doing the work iii CONTENTS Acknowledgments ........................................................................................................iii Executive Summary ......................................................................................................1 Introduction ............................................................................................................1 Purpose of the Report ..............................................................................................1 Short Tandem Repeats..............................................................................................2 Single Nucleotide Polymorphisms............................................................................5 Conclusions and Implications ..................................................................................6 Project Description ........................................................................................................9 STR Grant ................................................................................................................9 SNP Grant..............................................................................................................10 Scope and Methodology ..............................................................................................21 Assay Development and Primer Testing..................................................................22 Sample Cleanup and Mass Spectrometry................................................................29 Sample Genotyping ................................................................................................31 Comparison Tests With ABI 310 Genetic Analyzer ................................................33 Results and Discussion of STR Analysis by Mass Spectrometry ....................................35 Marker Selection and Feasibility Studies With STR Loci ........................................35 Multiplex STR Work ..............................................................................................40 Comparison Tests Between ABI 310 and Mass Spectrometry Results......................40 PCR Issues..............................................................................................................41 Analytical Capabilities of This Mass Spectrometry Method ....................................51 Results and Discussion of Multiplex SNPs ..................................................................63 Mitochondrial DNA Work......................................................................................64 Y-Chromosome Work ............................................................................................66 References ....................................................................................................................73 Published Papers and Presentations ............................................................................75 v List of exhibits included with this report: Exhibit 1. Schematic of GeneTrace automated time-of-flight mass spectrometer......2 Exhibit 2. PCR product sizes with newly designed primers for commonly used STR loci compared with commercially available primers used in multiplex sets for fluorescence-based assays ....................................3 Exhibit 3. Fluorescent multiplex STR result with AmpF1STR® COfilerTM kit and ABI 310 Genetic Analyzer ........................................................4 Exhibit 4. Schematic of expected allele masses for a CSF1PO-TPOX-THO1 (CTT) multiplex involving overlapping allele size ranges......................4 Exhibit 5. Mass spectrum of an STR triplex involving TPOX, THO1, and CSF1PO ........................................................................................5 Exhibit 6. Mass spectrum of SNP 10-plex assay for screening polymorphic sites in the mtDNA control region ................................................................6 Exhibit 7. Overlay of CE electropherograms demonstrating male-specific amplification with the 17-plex set of PCR primers................................7
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