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Overcoming Problems with Limiting DNA Samples in Forensics and Clinical Diagnostics Using Multiple Displacement Amplification

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Overcoming Problems with Limiting DNA Samples in Forensics and Clinical Diagnostics Using Multiple Displacement Amplification Overcoming problems with limiting DNA samples in forensics and clinical diagnostics using Multiple Displacement Amplification Firman Alamsyah Muharam Bachelor of Science / Bachelor of Arts, University of Queensland 2002 Cooperative Research Centre for Diagnostics School of Life Science, Faculty of Science Queensland University of Technology Brisbane, Australia A thesis submitted for the degree of Master of Applied Science (Research) at the Queensland University of Technology, Brisbane, Australia 2005 i ii ABSTRACT The availability of DNA samples that are of adequate quality and quantity is essential for any genetic analysis. The fields of forensic biology and clinical diagnostic pathology testing often suffer from limited samples that yield insufficient DNA material to allow extensive analysis. This study examined the utility of a recently introduced whole genome amplification method termed Multiple Displacement Amplification (MDA) for amplifying a variety of limited sample types that are commonly encountered in the fields of forensic biology and clinical diagnostics. The MDA reaction, which employs the highly processive bacteriophage φ29 DNA polymerase, was found to generate high molecular weight template DNA suitable for a variety of downstream applications from low copy number DNA samples down to the single genome level. MDA of single cells yielded sufficient DNA for up to 20,000,000 PCR assays, allowing further confirmatory testing on samples of limited quantities or the archiving of precious DNA material for future work. The amplification of degraded DNA material using MDA identified a requirement for samples of sufficient quality to allow successful synthesis of product DNA templates. Furthermore, the utility of MDA products in comparative genomic hybridisation (CGH) assays identified the presence of amplification bias. However, this bias was overcome by introducing a novel modification to the MDA protocol. Future directions for this work include investigations into the utility of MDA products in short tandem repeat (STR) assays for human identifications and application of the modified MDA protocol for testing of single cell samples for genetic abnormalities. iii KEYWORDS Whole genome amplification, multiple displacement amplification, φ29, phi29, DNA polymerase, single cell, low copy number, DNA, PCR, forensic biology, forensic science, clinical diagnostics, comparative genomic hybridisation. iv TABLE OF CONTENTS ABSTRACT ............................................................................................................................... iii KEYWORDS.............................................................................................................................. iv TABLE OF CONTENTS............................................................................................................ v LIST OF FIGURES................................................................................................................... vii LIST OF TABLES ...................................................................................................................viii LIST OF ABBREVIATIONS AND SYMBOLS..................................................................... ix LIST OF PRESENTATIONS AND PUBLICATIONS .......................................................... xi STATEMENT OF ORIGINALITY ......................................................................................... xii ACKNOWLEDGEMENTS.....................................................................................................xiii CHAPTER ONE: AN INTRODUCTION TO THE PROBLEMS OF LIMITING DNA SAMPLES AND THE AIMS OF THIS RESEARCH: WHOLE GENOME AMPLIFICATION IN FORENSICS AND CLINICAL DIAGNOSTICS ............................. 1 1.1. Introduction .......................................................................................................................... 1 1.2. The Problem of Limiting DNA Samples............................................................................ 2 1.2.1. The need for large amounts of DNA for high-throughput genetic studies ................. 2 1.2.2. The amount of DNA in single cells and examples of limiting samples....................... 4 1.2.2.1. Limiting samples in the field of forensic DNA testing ........................................... 5 1.2.2.2. Limiting samples in the field of molecular archaeology ..................................... 10 1.2.2.3. Limiting samples in the field of clinical diagnostics ........................................... 12 1.2.2.4. Overcoming the problem of limited samples........................................................ 15 1.3. Applying Whole Genome Amplification (WGA) Methods to Overcome Problems with Limiting Sample Quantities............................................................................. 16 1.3.1. PCR-based Whole Genome Amplification ................................................................. 18 1.3.1.1. Alu-PCR, Linker Adapter PCR (LA-PCR) and Tagged Random PCR (T-PCR)................................................................................................................................ 20 1.3.1.2. Degenerate oligonucleotide-primed PCR (DOP-PCR)....................................... 23 1.3.1.3. Primer Extension Preamplification (PEP)........................................................... 28 1.3.1.4. Summary on the characteristics of PCR-based WGA methods........................... 31 1.3.2. Non-PCR-based isothermal Whole Genome Amplification by strand- displacing Bst and φ29 DNA polymerases............................................................................ 33 1.3.2.1. Rolling Circle Amplification (RCA) by Large Fragment Bst DNA polymerase ........................................................................................................................... 34 1.3.2.2. Multiple Displacement Amplification (MDA) by φ29 DNA polymerase ............ 37 1.4. Research Rationale: Background to the Investigation..................................................... 46 1.5. Hypothesis and Research Aims......................................................................................... 48 1.5.1. Hypothesis .................................................................................................................... 48 1.5.2. Research Aim I ............................................................................................................. 49 1.5.3. Research Aim II............................................................................................................ 49 1.5.4. Research Aim III .......................................................................................................... 50 1.6. Research Design................................................................................................................. 50 1.7. Thesis Outline .................................................................................................................... 50 1.8. Conclusion.......................................................................................................................... 53 CHAPTER TWO: MATERIALS AND METHODS ............................................................. 54 2.1. Introduction ........................................................................................................................ 54 2.2. Sample Handling and Preparation of DNA Samples....................................................... 54 2.2.1. General sample handling ............................................................................................ 54 2.2.2. Dilution of human genomic DNA for sensitivity assays ............................................ 56 2.2.3. Collection of human fibroblasts for sensitivity assays .............................................. 56 2.2.4. Collection and preparation of simulated forensic specimens (buccal swabs, licked stamps, touched object samples and shed hairs) .......................................... 57 2.3. DNA Extraction and Quantification ................................................................................. 58 2.3.1. DNA extraction of population database bloodspots by 5% Chelex-100 .................. 56 2.3.2. DNA extraction of simulated low copy number forensic samples (buccal swabs, licked stamps, touched object samples) by a modified 5% Chelex protocol.......... 59 v 2.3.3. Extraction of DNA from shed hairs by a modified Buffer/Proteinase K digest protocol.........................................................................................................................59 2.3.4. Procedure for salt extraction of genomic DNA from whole blood for use in Comparative Genomic Hybridisation (CGH) .......................................................................60 2.3.5. Quantification of samples by quantitative real-time PCR (RT-QPCR) ....................61 2.3.6. Quantification of whole blood genomic DNA and MDA products by spectrophotometric 260nm/280nm absorbance ....................................................................62 2.4. PCR Amplification and Agarose Gel Electrophoresis .....................................................63 2.5. Multiple Displacement Amplification (MDA) .................................................................66 2.5.1. Multiple Displacement Amplification (MDA) using the GenomiPhi™ DNA Amplification Kit .....................................................................................................................66 2.5.2. A Modified MDA procedure using
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