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Forensic Analysis of Expirated Blood

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Forensic Analysis of Expirated Blood Forensic analysis of expirated blood ANDREA DONALDSON A Thesis submitted for the degree of Master of Science in Biochemistry at the University of Otago, Dunedin, New Zealand January 2010 For my nephew Brayden, who taught me how precious life is and to always follow your heart. ii Abstract In forensic investigations the distinction between expirated bloodstains (blood from the mouth, nose or lungs) and impact spatter (blood from gunshots, explosives, blunt force trauma and/or machinery accidents) is often important but difficult to determine due to their high degree of size similarity, which may result in the patterns being incorrectly classified. Expirated bloodstains on an accused person’s clothing occur when assisting an injured person, a finding which would tend to exonerate that individual. Impact spatter stains on clothing tend to occur due to the proximity of the person to the bloodshedding event, implying guilt. Therefore this project determined the characteristics inherent in each bloodstain type by using high speed digital video analysis and developed a test using PCR analysis to distinguish between the two types of bloodstain patterns to allow for proper bloodstain classification. The current study developed a test involving PCR analysis using DNA from human- specific oral microbes as a biomarker for the presence of saliva and hence oral expirated bloodstains. This PCR method is very specific to human oral Streptococci, with no PCR product being made from human DNA or DNA from soil or other microbes that were tested. It is also very sensitive, detecting as little as 60 fg of target DNA. The PCR was not inhibited by the presence of blood and could detect target DNA in expirated blood for at least 92 days after deposit on cardboard or cotton fabric. In a blind trial, the PCR method was able to distinguish three mock forensic samples that contained expirated blood from four that did not. These data show that bacteria present in the oral cavity can be detected in bloodstains that contain saliva, and therefore can potentially be used as a marker in forensic work to distinguish mouth- expirated bloodstains from other types of bloodstains. High speed digital video analysis was used to examine the physical characteristics, droplet sizes and distance travelled of expirated bloodstain patterns generated by breathing, spitting and coughing. This analysis showed that some physical characteristics (beaded stains) are unique to expirated bloodstains and can be used to determine that the stain is expirated in origin. However, the video images also showed that these physical characteristics are not always present and that the type of expelling mechanism i.e. coughing, spitting or breathing, the distance the bloodstains travel, and iii the type of surface the bloodstains are deposited on all influence the presence of these physical characteristics. iv Acknowledgements I have received a huge amount of support and assistance throughout this project, for which I will be forever grateful, so thank you everyone. I wish to gratefully acknowledge all the willing participants who provided saliva, nasal and sweat for this Research project without whom, I would not have been able to carry out the project. Professor John Tagg, Megan Inglis, and the rest of the Tagg Lab who provided Streptococcus strains, references and advice on their growth and manipulation. Dr Dan Power and Dr Jacqui Horswell for all their suggestions and assistance with developing the project, and for Jacqui who proof read aspects of this thesis. Rachel Parkinson who provided the soil microbial DNA samples, Sally-Ann Harbison and her forensic biology team in Mt Albert ESR who analysed DNA samples for me, taught me about performing and analysing human DNA profiles and all the other ESR staff in Porirua and Christchurch who freely gave their time, knowledge, and assistance to help with this research. Additionally, thank you for the financial support in awarding me an ESR Postgraduate Scholarship. Dr Mik Black for all the help in showing me how to carryout and understand statistical analysis. To all the staff at the University of Otago Maori centre, thank you for all the support and tuition over the last five years, to enable me to achieve my goals. To my Lab 216, thank you for all the assistance, support, laughter, and listening to the complaints when things were not going right. I would like to especially acknowledge Lois Martin for all her help in teaching me how to perform DNA analysis and cloning techniques, I am very grateful, Thank you. v To my three incredible supervisors, Professor Iain Lamont, Dr Michael Taylor, and Dr Stephen Cordiner, for always having my best interest at heart, going above and beyond the call of duty at times, (coughing blood, thanks Steve) pushing me to work harder and strive higher, but most of all, for being three of the most inspirational men I know, both with regards to work and family. Thank you all, very, very much. To my friends Darnell Kennedy, Toni Sinclair, Sharleen Rae-Whitcombe, Dora Leahy, and the rest of the 4th year 2008 class, thank you for all the fun times, the laughter, the tears, and the constant companionship and support. To my wonderful husband Pete, Thank you for your love and support, for shifting across the country, to allow me to fulfil my dream of becoming a forensic scientist, and finally to my family, especially my mum, thank you for the love and support, always being there when I need you, and for believing in me always. vi Table of Contents Abstract ......................................................................................................................... iii Acknowledgements ......................................................................................................... v Table of Contents ..........................................................................................................vii List of Tables .................................................................................................................. xi List of Figures ...............................................................................................................xii Abbreviations ............................................................................................................... xiv Chapter one Introduction .............................................................................................. 1 1.1 Preface .................................................................................................................... 1 1.2 Bloodstain Pattern Analysis ................................................................................... 2 1.3 Bloodstain Pattern Categorisation ........................................................................ 2 1.4 Impact Spatter ....................................................................................................... 3 1.5 Limitations of the Bloodstain Pattern Classification. ............................................. 3 1.6 Expirated Bloodstain Patterns .............................................................................. 6 1.7 Current methods to determine if bloodstains are expirated ................................... 8 1.8 PCR and DNA analysis in Forensics. ................................................................ 9 1.8.1 Limitations of PCR ........................................................................................ 10 1.8.2 DNA extraction methods ............................................................................... 11 1.9 Oral Microbes .................................................................................................. 12 1.10 Diversity of the oral microflora in humans ....................................................... 14 1.11 Streptococci genomes and genes. ...................................................................... 15 1.12 Oral Microbial DNA analyses. .......................................................................... 16 1.13 High speed digital video analysis ................................................................. 17 1.14 Aims of this study ............................................................................................. 17 Chapter Two Methods and Materials......................................................................... 20 2.1 Chemicals and materials ....................................................................................... 20 2.2 Oligonucleotide Primers ....................................................................................... 20 2.3 Bacterial strains and plasmids .............................................................................. 21 2.3.1 Bacterial strains ............................................................................................. 21 2.3.2 Plasmids ......................................................................................................... 22 2.4 Media .................................................................................................................... 22 2.4.1 Liquid Media ................................................................................................. 23 2.4.2 Solid Media .............................................................................................. 23 2.4.3 Media Supplements ....................................................................................... 24 2.5 Growth and Maintenance of Bacteria ................................................................... 24 2.6 Isolation of bacterial
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