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Identification of Tissue Specific Differential Methylation in Human Body Fluids and Its Potential Application in Forensics

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Identification of Tissue Specific Differential Methylation in Human Body Fluids and Its Potential Application in Forensics Identification of Tissue Specific Differential Methylation in Human Body Fluids and its Potential Application in Forensics Farzeen Kader 209503949 School of Life Sciences, Faculty of Science and Agriculture, University of KwaZulu-Natal School of Life Sciences University of KwaZulu-Natal Westville Private Bag X 54001 Durban 4000 South Africa Tel: 031 260 3192 / 2029 Email: [email protected] Master of Science - Genetics Supervisor: Dr Meenu Ghai (UKZN - Westville Campus) IDENTIFICATION OF TISSUE SPECIFIC DIFFERENTIAL METHYLATION IN HUMAN BODY FLUIDS AND ITS POTENTIAL APPLICATION IN FORENSICS BY FARZEEN KADER Submitted in fulfillment of the Academic Requirements for the degree of Master of Science (MSc) in the Discipline of Genetics, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal (Westville Campus), Durban As the candidate’s supervisor, I have approved this dissertation for submission. Supervisor: Dr. Meenu Ghai Signed: Name: Date: PREFACE The experimental work described in this dissertation was carried out in the School of Life Sciences, Discipline of Genetics, University of KwaZulu-Natal (Westville Campus), Durban, South Africa from January 2014 to December 2015, under the supervision of Dr. M. Ghai and the co-supervision of Dr. O. T. Zishiri and Prof. A. O. Olaniran. These studies represent original work performed by the author and have not otherwise been submitted in any form for any degree or diploma to any tertiary institution. Where use has been made of work of others, it is duly acknowledged in the text. Supervisor: Dr. M. Ghai Signature: ________________ Co-supervisor: Dr. O. T. Zishiri Signature: ________________ Co-supervisor: Prof. A. O. Olaniran Signature: ________________ COLLEGE OF AGRICULTURE, ENGINEERING AND SCIENCE DECLARATION 1 – PLAGIARISM I, ………………………………………………………………………………….. declare that 1. The research reported in this dissertation, except where otherwise indicated, is my original research. 2. This dissertation has not been submitted for any degree or examination at any other university. 3. This dissertation does not contain other person’s data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This dissertation does not contain other person’s writing, unless specifically acknowledged as being sourced from other researchers. Where other research sources have been quoted, then: a) Their words have been rewritten but the general information attributed to them has been referenced; b) Where their exact words have been used, then their writing has been placed in italics inside quotation marks and referenced. 5. This dissertation does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections. Signed: ……………………………………. COLLEGE OF AGRICULTURE, ENGINEERING AND SCIENCE DECLARATION 2 – PUBLICATIONS DETAILS OF CONTRIBUTION TO PUBLICATIONS that form part and/or include research presented in this dissertation (including publications in preparation, submitted in press and published). Publication 1 Title: DNA Methylation and Application in Forensic Sciences. Journal: Forensic Science International. Citation: Kader, F. and M. Ghai. 2015. DNA Methylation and Application in Forensic Sciences. Forensic Science International. 249: 245-265. Publication in Preparation Title: Development of new tDMR-based markers for identification of blood and vaginal fluid for forensic applications. Authors: Kader, F., M. Ghai, D. Evans and F. Abader. Signed: ……………………………………. TABLE OF CONTENTS CONTENTS PAGE ACKNOWLEDGEMENTS i ABSTRACT ii LIST OF FIGURES iv LIST OF TABLES vii CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1 1.1 Introduction 2 1.2 Literature Review 5 1.2.1 Epigenetics 5 1.3 DNA Methylation 7 1.3.1 Localisation of DNA Methylation in the Human Genome 8 1.3.2 Tissue-Specific Differential DNA Methylation 9 1.4 Environmental Influences and Differential DNA Methylation 10 1.4.1 Age 10 1.4.2 Nutrition and Diets 11 1.4.3 Life Experiences 12 1.5 Mapping of Genome-wide DNA Methylation 13 1.5.1 Sodium Bisulfite Treatment of DNA Templates 14 1.5.1.1 Infinium HumanMethylation BeadChip Arrays 14 1.5.1.2 MALDI-TOF MS 15 1.5.1.3 Pyrosequencing 16 1.5.2 Methylation-Sensitive Restriction Enzyme PCR 16 1.5.2.1 Capillary Electrophoresis 19 1.5.2.2 Restriction Landmark Genomic Scanning 20 1.5.3 Affinity Purification of Methylated DNA 21 1.6 Role of Epigenetic Modifications in Gene Expression and Development 22 1.6.1 Differential DNA Methylation and Gene Expression 23 1.6.1.1 Imprinting 25 1.6.1.2 Regulation of ncRNAs 27 1.6.1.3 X Chromosome Inactivation 29 1.6.1.4 Control of Alternate Promoters 30 1.7 Tissue-Specific Differentially Methylated Regions (tDMRs) and Control 32 of Gene Expression 1.8 Tissue-Specific Differentially Methylated Regions (tDMRs) and its 37 Application in Forensic Science 1.8.1 Verification of DNA Samples 37 1.8.2 Identification of Forensically Relevant Body Fluids/Tissues by 38 Analysis of tDMRs 1.8.3 Sex Determination in Forensics 43 1.8.4 Ancestry Informative Markers of Fluid/Tissue Donor 44 1.9 The Current Status of Forensic Fluid and Tissue Identification 50 1.9.1 Conventional Methods Employed to Identify Forensically 51 Relevant Body Fluids 1.9.1.1 Saliva 51 1.9.1.2 Blood 52 1.9.1.3 Semen 52 1.9.1.4 Vaginal Fluid 52 1.9.2 The Use of Biological Markers 53 1.9.2.1 Protein-based Biomarkers 53 1.9.2.2 RNA-based Biomarkers 54 1.9.2.3 Microbial Biomarkers 56 1.10 Scope of the Present Study 58 1.10.1 Hypotheses tested 59 1.10.2 Aims 59 1.10.3 Objectives 59 1.10.4 Experimental Design 60 CHAPTER TWO: IDENTIFICATION OF TISSUE-SPECIFIC DIFFERENTIAL DNA METHYLATION IN 61 HUMAN BODY FLUIDS ABSTRACT 62 2.1 Introduction 63 2.2 Materials and Methods 68 2.2.1 Data Acquisition 68 2.2.2 Methylation Data Acquisition 69 2.2.3 Data Integration and Exclusion of Differentially Expressed 70 Genes Present in More than One Surrogate Tissue 2.2.4 Elimination of Genes with No Evidence of Protein Synthesis 70 2.2.5 Genes and Genome Data 71 2.2.6 Mapping CpG Island and Methylation Information 71 2.2.7 Final Selection of Candidate Tissue-Specific Differentially 71 Methylated Regions 2.2.8 Primer Design for PCR 72 2.2.9 Sample Collection 72 2.2.10 DNA Extraction and Quantification 72 2.2.11 Restriction Enzyme Treatment 73 2.2.12 MSRE-PCR 73 2.3 Results 74 2.3.1 Tissue-Specific Gene Expression Data 74 2.3.2 Exclusion of Differentially Expressed Genes Present in More than 75 One Surrogate Tissue 2.3.3 Elimination of Genes with Unconfirmed Protein Products 77 2.3.4 Methylation Data 81 2.3.5 Mapping CpG Island and Methylation Information 85 2.3.6 Final Selection of CGIs for Primer Design 88 2.3.7 Optimisation of the HhaI Restriction Reaction 90 2.3.8 Body Fluid Identification using MSRE-PCR 91 2.3.8.1 HPCAL1 Gene-based Primer Set 91 2.3.8.2 PTPRS Gene-based Primer Set 92 2.3.8.3 ZNF282 Gene-based Primer Set 93 2.3.8.4 PMEPA1 Gene-based Primer Set 94 2.4 Discussion 95 CHAPTER THREE: METHYLATION PROFILING OF SALIVA FROM THE DIVERSE SOUTH AFRICAN POPULATION 100 USING tDMR-BASED BIOMARKERS ABSTRACT 101 3.1 Introduction 102 3.2 Materials and Methods 108 3.2.1 Sample collection 108 3.2.2 DNA Extraction and Quantification 108 3.2.3 Selection of tDMR Markers and Primer Design for PCR 109 3.2.4 HhaI Enzyme Restriction Reaction 110 3.2.5 Multiplex MSRE-PCR 111 3.2.6 Statistical Analyses 113 3.3 Results 114 3.3.1 Individual DNA Methylation Profiling using MSRE-PCR 114 3.3.2 Calculation and Comparison of Methylation Levels of Selected 116 tDMRs between Four Ethnic Groups 3.3.3 Statistical Analyses 119 3.3.3.1 USP49 tDMR-based Marker 119 3.3.3.2 DACT1 tDMR-based Marker 121 3.3.3.3 L81528 tDMR-based Marker 123 3.3.3.4 PFN3 tDMR-based Marker 125 3.4 Discussion 127 CHAPTER FOUR: GENERAL DISCUSSION AND CONCLUSION 131 4.1 Research in Perspective 132 4.2 Conclusion 138 REFERENCES 139 APPENDIX A 179 APPENDIX B 204 ACKNOWLEDGEMENTS First and most importantly, I wish to express my profound gratitude to God Almighty for His grace, guidance and granting me the strength to complete this study. My sincere thanks go to my supervisor, Dr Meenu Ghai. Thank you for your patience, mentorship, assistance and guidance during the course of this study. I appreciate your time to critically review the dissertation and most of all, thank you for constantly motivating me to grow and do better. I am immensely grateful for all that you have done for me. I wish to extend my gratitude to Dr Oliver Zishiri, for his constant enthusiasm and motivation. Thank you for constantly reminding me that in the end, it will be worth all the hurdles and challenges. I acknowledge Professor Ademola Olufolahan Olaniran, for his support and advice throughout this study. I am very thankful not only for Bioinformatic assistance from Dr Dyfed Lloyd Evans, but also for his advice and improvement of the study. Thank you to Dr Shailesh V. Joshi for statistical analysis and assistance. I am grateful to the National Research Foundation for financial support. Thanks to all the volunteers who participated in the present study. I am immeasurably grateful to my parents Osman and Thohida, and my brother Faizaan. Thank you for your patience and blessings throughout the study.
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