The use of a direct to PCR analysis method for disaster victim identification

Transcription

1 UNIVERSITY OF CANBERRA The use of a direct to PCR analysis method for disaster victim identification Clare Megan Berry Bachelor of Applied Science in Forensic Studies National Centre for Forensic Studies (NCFS) University of Canberra ACT 2601 A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Applied Science (Honours) at the University of Canberra November 2014

2 i Abstract The use of tissue preservative for the preservation of muscle samples has been shown to be an ideal method of preservation and is recommended by International Criminal Police Organisation (INTERPOL) in regards to mass disasters. It has been shown that preservatives such as salt saturated DMSO/EDTA (DMSO), TENT buffer and proprietary preservatives contain DNA in the preservative solution surrounding the tissue sample, showing that the need to handle tissue samples for DNA analysis including short tandem repeat (STR) profiling can be eliminated. Direct to PCR is a new analysis method that has shown promising results for numerous sample types. This analysis method involves the removal of DNA extraction and taking samples directly to PCR analysis including the production of STR profiles. This analysis method is ideal due to it being a faster method when compared to standard DNA analysis with an extraction step. This method is also more effective the production of STR profiles with trace DNA samples. The effectiveness of a direct to PCR analysis for a variety of sample types and the presence of DNA in preservative aliquots surrounding tissues samples shows the possibility for a direct to PCR analysis for the use with disaster victim identification (DVI). This method was found to be successful for use with DMSO, DNA Genotek solution and DNAgard preservatives for all days preserved with full STR profiles produced. These results were found with standard PCR assays without any modifications to the manufactures protocol.

3 iii Acknowledgments I would like to express my appreciation to my supervisors Dr Dennis McNevin and Dr Michelle Gahan. Thank you for all your help with laboratory training, writing up my thesis and the many questions throughout the year. Your guidance and expertise has been a big help in getting me through this year. I would like to thank Dr David Bruce and the Biology team at NSW Forensic and Analytical Science Service for all your help in genotyping my samples. I would like to extend my gratitude to Jaymi Kingston for all your help this year in the laboratory and out. Thank you so much for your support and advice it has been a big help in getting me though this year. I would also like to thank the University of Canberra and the National Centre for Forensic Studies for giving me the opportunity to undertake this research; it has been a goal since entering the University of Canberra that I can now cross off.

4 iv Table of Contents CHAPTER 1 INTRODUCTION Preservation of Forensic Samples Preservative Methods Salt saturated DMSO/EDTA Buffers Proprietary preservatives DNA Extraction Magnetic Bead Extraction Organic extraction Chelex extraction PCR Quantification STR genotyping PCR Inhibitors from preservatives EDTA Salt PCR inhibitors from sample Haemoglobin Hematin Melanin Humic acid Procedures to mitigate against inhibition Dilution Filtration MgCl Bovine Serum Albumin (BSA) Direct to PCR analysis Experimental Aims CHAPTER 2 - Method Preparation of solutions Sample Preparation Ethics Blood Saliva Muscle Tissue PCR... 18

5 v Target loci and primers Commercial PCR Preparations Standard PCR Setup Modified PCR setup PCR Cycling Conditions Gel Electrophoresis Quantification Genotyping PowerPlex Capillary Electrophoresis Statistical Analysis Normality Testing Kruskal-Wallis CHAPTER 3 RESULTS Optimisations studies Direct to PCR analysis on different kits Primer optimisation Effect on the addition of BSA Effect on altering MgCl 2 concentration Results from muscle samples Direct to PCR analysis Quantification Genotyping CHAPTER 4 - DISCUSSION Agarose gel electrophoresis Saliva and blood Muscle tissue Quantitation of DNA in tissue preservatives Capillary electrophoresis of PowerPlex 21 loci Conclusion Further direction REFERENCES APPENDIX I QUANTIFILER RESULTS APPENDIX II ELECTROPHEROGRAMS FOR DONOR TWO SAMPLES PRESERVED FOR 14 DAYS... 50

6 vi List of Tables Table 1.1 Summary of results from salt saturated DMSO for a variety of tissue types (Allen-Hall & McNevin, 2013)... 3 Table 1.2 Summary of results for different buffer preservation methods and a variety of tissue types (Allen-Hall and McNevin, 2013) Table 1.3 Summary of results from proprietary preservative kits (Allen-Hall & McNevin, 2013)... 7 Table 2.1 Preparation of solutions used for direct to PCR analysis and preservation of samples Table 2.2 FGA and Amelogenin primer Table 2.3: PCR component concentrations according to the manufactures protocols for each of the proprietary PCR preparations Table 2.4 PCR component concentrations with the addition of extra MgCl2 to the standard manufactures protocols for MyTaq DNA Polymerase kit at concentrations equal to that of EDTA present for each of the proprietary PCR preparations Table 2.5 PCR component concentrations with the addition of extra MgCl2 to the standard manufactures protocols for MyTaq Blood PCR Kit at concentrations equal to that of EDTA present for each of the proprietary PCR preparations Table 2.6 PCR temperature cycling conditions as per the manufactures protocols for each of the proprietary PCR preparations Table 2.7 Quantifiler standards according to the manufacture s protocol Table 2.8 PCR temperature cycling conditions as per the manufactures for Quantifiler Human assay Table 2.9 PCR cycling conditions for PowerPlex 21 system Table i Concentration of DNA (ng) determined from Quantifiler Human Assay after analysis from aliquots of preservative solution from surrounding tissue samples

7 vii List of Figures Figure 3.1 Agarose gel image of direct to PCR analysis using MyTaq Velocity kit (lanes 1-6) and MyTaq HS DNA Polymerase kit(lanes 8-13) kits for the FGA and Amelogenin loci Figure 3.2 Agarose gel image of direct to PCR analysis using MyTaq Blood kit for the FGA loci. 28 Figure 3.3 Agarose gel image of primer optimisation using MyTaq Blood PCR Kit for the FGA and Amelogenin loci Figure 3.4 Agarose gel image of primer optimisation using MyTaq DNA Polymerase kit for the FGA and Amelogenin loci Figure 3.5 Agarose gel image of primer optimisation using My Taq DNA Polymerase kit for the Amelogenin loci Figure 3.6 Gel image of direct to PCR analysis using MyTaq DNA Polymerase kit for FGA loci with BSA addition Figure 3.7 a. Partial gel image of direct to PCR analysis using MyTaq DNA Polymerase kit for FGA loci with BSA addition Figure 3.8 Partial gel images of direct to PCR analysis using MyTaq DNA Polymerase kit for FGA loci with extra MgCl Figure 3.9 Partial gel image of direct to PCR analysis using MyTaq DNA Polymerase kit for FGA loci with extra MgCl Figure 3.10 Gel image of tissue samples preserved by Allen-Hall & McNevin, 2012 for donor one analysed using MyTaq DNA polymerase for FGA loci Figure 3.11 Gel image of tissue samples preserved by Allen-Hall & McNevin, 2012 for donor one analysed using My Taq DNA polymerase for FGA and Amelogenin loci Figure 3.12 Gel image of tissue samples preserved by Allen-Hall & McNevin, 2012 for donor one analysed using My Taq DNA polymerase for Amelogenin loci Figure 3.13 Concentration of DNA (ng/µl) generated from aliquots of preservative solutions containing muscle sample Figure 3.14 Mean peak heights for the Amelogenin loci generated from aliquots of preservative solutions containing muscle sample Figure 3.15 Mean peak heights for the D21S11 loci generated from aliquots of preservative solutions containing muscle sample Figure 3.16 Mean peak heights for the Penta E loci generated from aliquots of preservative solutions containing muscle sample Figure 3.17 Mean peak heights for the Penta D loci generated from aliquots of preservative solutions containing muscle sample Figure 3.18 Reportable alleles generated from aliquots of preservative solutions containing muscle sample

8 viii Abbreviations bp Base pairs BSA Bovine serum albumin Ca 2+ DMSO DNA dntps EDTA EPGs FBI Calcium ion Dimethyl sulphoxide deoxyribonucleic acid nucleotide bases ethylenediaminetetraacetic acid Electropherograms Federal Bureau of Investigation GMID-X GeneMapper ID-X v1.4 INTERPOL International Criminal Police Organisation IPC Internal positive control KCl Potassium chloride LST Lysis storage and transport buffer Mg 2+ MgCl 2 NaCl NAME PCR Salt saturated DMSO STR Magnesium ion Magnesium chloride Sodium chloride National Association of Medical Examiners Polymerase chain reaction Salt saturated DMSO/EDTA Short tandem repeats