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The Development of Novel Str Miniplex Primer Sets for The THE DEVELOPMENT OF NOVEL STR MINIPLEX PRIMER SETS FOR THE ANALYSIS OF DEGRADED AND COMPROMISED DNA SAMPLES A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Denise T. Chung August 2004 This dissertation entitled THE DEVELOPMENT OF NOVEL STR MINIPLEX PRIMER SETS FOR THE ANALYSIS OF DEGRADED AND COMPROMISED DNA SAMPLES BY DENISE T. CHUNG has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by Bruce R. McCord Associate Professor of Chemistry Leslie A. Flemming Dean, College of Arts and Sciences CHUNG, DENISE T. Ph.D. August 2004. Chemistry and Biochemistry The Development of Novel STR Miniplex Primer Sets for the Analysis of Degraded and Compromised DNA Samples (217 pp.) Director of Dissertation: Bruce R. McCord New Miniplex primer sets have been designed where the target sequence is much closer to the repeat region and will therefore produce smaller amplicons to facilitate the analysis of degraded DNA. The effectiveness of these primer sets on degraded DNA samples were tested on enzymatically degraded DNA, environmentally insulted bone samples, and real case work samples and compared to amplifications with a commercial multiplex kit. For both types of degraded DNA, the Miniplex primer sets were capable of producing more complete profiles due to the smaller amplicon size. A concordance study of 532 DNA samples was performed to check for the existence of potential point mutations in the Miniplex primer binding region or insertion/deletion between commercial primers and Miniplex primers. Out of the 532 samples, there were 15 samples that showed discrepancies at one allele. DNA sequencing of these samples revealed the presence of new mutations and polymorphisms. Another challenge associated with forensic samples is the presence of PCR inhibitors. The presence of these compounds can interfere with the amplification process resulting in preferential amplification of one locus, allele drop out or no amplification at all. The effect on amplification efficiency of the Miniplex primer sets due to several known PCR inhibitors was investigated. The use of BSA and LMT agarose to relieve PCR inhibition of the Miniplex primer sets were also investigated. Lastly, validation studies in accordance with the TWGDAM guidelines were performed to demonstrate the robustness of the Miniplex primer sets in typing compromised forensic samples. The Miniplex primer sets have been optimized to work with 100 pg/25 µL of DNA template. Overall, these Miniplex primer sets have demonstrated improved signal with degraded DNA in comparison to commercial sets. These Miniplex sets will provide a very useful tool to pick up loci that fail to amplify with commercial kits due to severe DNA degradation, PCR inhibition, and low copy number of template. In addition, these primers can also be used to check for potential allele drop out due to primer binding site mutations, as the primer binding sites for these Miniplexes do not overlap with commercial kit primer locations. Approved: Bruce R. McCord Associate Professor of Chemistry Acknowledgments First of all, I would like to express my deepest gratitude to my research adviser, Dr. Bruce McCord for giving me the opportunity to pursue my interest in forensic DNA typing. I never thought I would have the chance to make this dream come true because where I grew up, this area of research is not popular and in many case, not even heard of. In the Philippines, lab equipment is close to obsolete, remote places do not even broadcast CSI (the show that brought forensics to mainstream consciousness), and forensic science seemed to be so foreign, distant and unattainable. But somehow God had a way of making things happen and led me to this place called Athens, Ohio where my dream became a reality. In this journey, I have met people who have made me become the better person I am now. To Dr. John Butler of the NIST, I thank you for laying out the blueprint of this project. To Yin Shen, I thank you for educating the totally clueless person I was when I first came here. To Jiri Drabek and Kerry Opel, I thank you for working on this project with me. Without the both of you, I would not have accomplished this much. To the members of my committee, Dr. Peter Harrington, Dr. Nancy Tatarek, and Dr. Susan Evans, I thank you for all the ideas and professional aid you have given me. To the National Institute of Justice, I thank you for funding the research project and saving me from becoming a lab rat for the undergraduates. To my friends in the department, I thank you for keeping me alive and sane during these solitary years of being in graduate school. To my parents, I thank you for always believing in me and supporting me. And to God, I thank you for watching over me every day and giving me the strength to continue. As I move to the end of this journey, I just look forward to a new dream that I soon would be a part of once again. 6 Table of Contents Abstract................................................................................................................................3 Acknowledgments................................................................................................................5 List of Tables .....................................................................................................................10 List of Figures....................................................................................................................12 Abbreviations.....................................................................................................................15 Chapter 1 Introduction to DNA Typing.............................................................................16 1.1 Brief History of DNA Typing..........................................................................16 1.2 DNA Polymorphisms......................................................................................17 1.3 Overview of Forensic DNA Typing Markers .................................................18 1.3.1 RFLP Analysis ..................................................................................18 1.3.2 PCR-Based Assays............................................................................19 1.4 Biology of DNA..............................................................................................21 1.4.1 Basic Structure of DNA.....................................................................22 1.4.2 Chromosomes, Genes, and DNA markers.........................................23 1.4.3 Nomenclature for DNA markers .......................................................24 Chapter 2 Introduction to Short Tandem Repeat Markers.................................................26 2.1 Short Tandem Repeat Markers (STRs)..........................................................26 2.1.1 Characteristics of STRs Used in Forensic DNA Typing..................27 2.1.2 Allelic Ladders..................................................................................28 2.1.3 The 13 CODIS STR loci ....................................................................29 2.1.4 Commercial STR Kits........................................................................31 2.1.5 Additional STR loci...........................................................................31 2.2 Issues with STR Markers..............................................................................33 2.2.1 Stutter Products ................................................................................33 2.2.2 Non-Template Addition.....................................................................34 2.2.3 Microvariants, Mutations, and Polymorphisms................................35 Chapter 3 DNA degradation ..............................................................................................36 3.1 Process of degradation ..................................................................................36 3.2 The Problem of Degraded DNA ....................................................................40 Chapter 4 Primer Design....................................................................................................47 4.1 Important Parameters to Consider When Designing Primers........................47 4.1.1 Primer Length ...................................................................................48 4.1.2 Melting Temperature (Tm)................................................................48 7 4.1.3 GC Content .......................................................................................49 4.1.4 Priming Efficiency ............................................................................49 4.1.5 Secondary Structures ........................................................................50 4.2 Multiplex STR Primer Design ......................................................................51 4.3 Miniplex Approach to the Problem of Degraded DNA................................52 4.4 Miniplex Primer Design................................................................................54 4.5 Multiplexing Miniplex Primers.....................................................................58 Chapter 5 Materials, Methods, and Technology................................................................60 5.1 DNA Extraction .............................................................................................60 5.1.1 DNA Extraction
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