THE DESIGN, OPTIMIZATION AND TESTING OF Y CHROMOSOME SHORT TANDEM REPEAT MEGAPLEXES by Richard Schoske submitted to the Faculty of the College of Arts and Sciences of American University in Partial Fulfillment of the requirements for the Degree of Doctor of Philosophy in Chemistry Chair: ________________________ Dr. James Girard ________________________ Dr. Albert Cheh ________________________ Dr. John M. Butler ______________________________ Dean of the College _________________ Date 2003 American University Washington, D.C. 20016 THE DESIGN, OPTIMIZATION AND TESTING OF Y CHROMOSOME SHORT TANDEM REPEAT MEGAPLEXES BY Richard Schoske ABSTRACT A multiplex polymerase chain reaction (PCR) assay capable of the simultaneous amplifying 20 Y chromosome short tandem repeat (STR) markers has been developed and tested to aid human testing and population studies. These markers include all of the Y-STR markers that make up the “extended haplotype” used in Europe (DYS19, DYS385 a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, and YCAII a/b) plus the additional polymorphic Y-STR markers (DYS437, DYS438, DYS439, DYS447, DYS448, DYS388, DYS460, and GATA H4). The Y-STR 20plex is the first to include a simultaneous amplification of all the markers within the European “minimal” and “extended haplotype.” A subset of the Y-STR 20plex primers, the Y-STR 9plex was also developed and tested. The Y-STR 9plex contains only the markers within the European minimal haplotype. Lastly, a Y-STR 11plex was designed and tested. The markers within the Y-STR 11plex are DYS385 a/b, DYS447, DYS448, DYS450, DYS456, DYS458 and DYS 464 a/b/c/d. ii Validation experiments were performed in order to assess the reliability of the haplotypes generated by these newly designed Y-STR multiplexes. The validation experiments included concordance, precision, specificity and sensitivity studies. Additionally, a total of 647 male samples from three different U.S. populations were analyzed for all the loci included in the Y-STR 20plex and Y-STR 11plex. Allelic frequencies for all of the Y-STR markers were tabulated as well as haplotype diversity data for various combinations of markers. Y-STR multiplexes were tested against samples from forensic cases that have already been closed. Analysis of these samples demonstrate that the Y-STR multiplexes developed here can handle real casework samples. Finally, the prototype Y Standard Reference Material (SRM) 2395 was characterized. SRMs are often used to validate a laboratory’s measurement capability. SRM 2395 is a Y-STR profiling standard that consists of five components, four male (A-D) and one female (E). The characterization included the DNA sequencing of 18 different Y-STR loci for each component (including all of the markers in the European minimal haplotype), presenting a suggested nomenclature for allele designations, and running these components against commercially available Y-STR kits and the Y-STR multiplexes designed in this study. iii ACKNOWLEDGMENTS This work is a culmination of a lengthy process that first started in 1999. There are some people that have been crucial to my success. Below I would like to acknowledge each of the individuals who afforded me the opportunity to study at American University, and complete my graduate studies in chemistry. I would first like to thank the United States Air Force, specifically the leadership at the Department of Chemistry at United States Air Force Academy. If people like Colonel Hans J. Mueh, Colonel Clifford M. Utermoehlen, Lt. Colonel Ronald P. Furstneau, and Major Dennis Tuthill did not show faith in me I would not have even had the opportunity to study at American University. Also, I would like to thank the United States Air Force for funding me for the last three years. I would not have been able at this age and with a family gone back to pursue my doctoral degree with out the Air Forces generous support. My wholehearted thanks goes to my academic advisor, Dr. James Girard. I first met Dr. Girard in 1999, a full year before I officially enrolled at American University. He has been helpful in every facet of my graduate studies. He was both an excellent teacher in the classroom and advisor in many of the one on one meetings we had. I would also like to thank Dr. Albert Cheh for taking the time out of his busy schedule to be on my doctoral committee. He has also offered me expert council when I needed it. Furthermore, I would like to thank Dr. Girard for sending me to do my research with Dr. John Butler at the National Institute of Standards and Technology. iv Deep appreciation goes to my research advisor, Dr. John Butler. Dr. Butler has expertly guided me through my research from the day I started with him in November of 2000. Besides expert guidance, thanks to his funding from the National Institute of Justice, I have wanted for nothing. I have enjoyed unfettered access to top-notch equipment, a limitless supply of reagents and an opportunity to learn from Dr. Butler’s colleagues. These colleagues are Dr. Peter Vallone, Ms. Margaret Kline and Ms. Jan Redman. Each of these people offered expert advice, guidance and were always available to help me out. I would also like to thank Mr. Clem Smetana, Mr. Raymond Meeks, Mr. Del Price, and Mr. Phil Mills. These four gentlemen are forensic biologists at the United States Army Criminal Investigation Laboratory (USACIL) in Forest Park Georgia. It was because of their curiosity in Y-STR assays that I got the opportunity to go down to their laboratory and test my Y-STR assays on samples from forensic cases that have already been closed. Next, I would like to express my gratitude to my mother, Dorothea Schoske and my aunt Irmgard O’Brien. Both have always believed in me and I draw strength from those beliefs. I know they are proud of me, but I want to let them know that I am equally proud of them. Finally, I would like to thank my wife Sherry for her undying patience and support during this entire process. There were many nights when I had to work very late or some weekends were I had to work. She always believed in me and without her and the support of my other children Lauren, Connor and Gunnar I would not be were I am today. Also, I would like to thank Sherry’s parents Gerard and Barbara Elphick for helping out when we needed them because I had matters to attend to. v TABLE OF CONTENTS ABSTRACT …………………………………………………………………………..… ii ACKNOWLEDGMENTS ……………………………………………………………… iv LIST OF FIGURES …………………………………………………………………...… x LIST OF TABLES ……………………………………………………………………. xiii LIST OF APPENDICES ………………………………………………………….…… xv CHAPTER 1 INTRODUCTION ……………………………………………………………… 1 DNA Polymorphisms and Short Tandem Repeats (STRs)….…………... 3 Polymerase Chain Reaction (PCR) and Multiplex PCR ………………... 5 Y-Chromosome STRs and Y-STR Multiplex Assays ………………… 10 Standard Reference Materials (SRMs) ………………………………… 14 Capillary Electrophoresis ………………………………………………. 15 Fluorescence Detection …………………………………….…………... 17 CHAPTER 2 STATEMENT OF PURPOSE ……….………………………………………… 21 CHAPTER 3 MATERIALS AND METHODS ……………………………………………… 23 DNA Samples ………………………………………………………….. 23 Development of References Sequences …………………………….….. 25 vi Primer Design …………………………………………………...……... 25 Primer Modification …………………………………………………… 27 Primer Quality Control ………………………………………………… 28 PCR Amplification Conditions ………………………………………… 29 Singleplex PCR ………………………………………………… 29 Y-STR 20plex, Y-STR9plex, Y-STR 11plex ………………….. 29 Y-PLEXTM 6 Kit ……………………………………………….. 30 Detection and Analysis of PCR Products in Y-STR Analysis ………… 31 DNA Sequencing ………………………………………………………. 32 Statistical Analysis of Population Data ………………………..…….… 37 CHAPTER 4 RESULTS AND DISCUSSION Multiplex PCR Primer Design, Optimization and Testing …………….. 39 Y-STR 20plex Multiplex Design ………………………………. 41 Selection of Loci to be Included in the Y-STR 20plex…………………………………………... 41 Allele and Size Range Determination for Loci in the Y-STR 20plex ……………………………… 46 Primer Design Issues for Y-STR 20plex Primers ……… 50 Initial Testing of the Y-STR 20plex …………………… 62 Y-STR 11plex Multiplex Design ………………………………. 66 vii Selection of Loci to be Included in the Y-STR 11plex…………………………………………... 66 Allele and Size Range Determination for Loci in the Y-STR 11plex ……………………………… 67 Primer Design Issues for Y-STR 11plex Primers ……… 68 Initial Testing of the Y-STR 11plex …………………… 77 Y-STR Multiplex Assay Performance …………………………. 81 GeneScan® and Genotyper®: Software Used for Measurement of STR Systems ……………………... 82 Y-STR 20plex and Y-STR 11plex Genotyper® Macro Construction ……………………………………. 88 Concordance Studies …………………………………... 96 Precision Studies ………………………………………. 99 Precision of the Y-STR 20plex ……………….. 101 Precision of the Y-STR 11plex ……………….. 109 Minimum Sensitivity …………………………………. 114 Specificity and Male:Female Mixtures ……………….. 116 Population Studies ……………………………………………. 122 Prototype Standard Reference Material® 2395 Human Y-Chromosome DNA Profiling Standard …………………..... 137 Sequence Analysis ……………………………………. 141 Y-STR Nomenclature Issues …………………...…….. 147 viii Analysis of Forensic Casework Samples Using Y-STR Multiplexes ………………………….………………... 158 Case Number 2002-0513 ……………….………...…... 159 Case Number 2002-0800 ……………………………... 165 Conclusion ……………………………………………………. 170 Future Research ………………………………………………. 171 APPENDICES ………………………………………………………………………... 172 BIBLIOGRAPHY …………………………………………………………….………. 246 ix LIST OF FIGURES Figure: Page: 1-1 Short Tandem Repeat (STR) Marker ……………………………….....… 4 1-2 Polymerase Chain Reaction (PCR) …………………………………........ 7 1-3 Multiplex