Author: Belinda Martin
Martin, Belinda, 2022 Enhancement of trace DNA: Role of direct PCR in forensic practise, Flinders University, College of Science and Engineering
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DNA profiling success from trace genetic material, such as that deposited within a fingermark, is unpredictable and often low. In a forensic casework setting it may be highly advantageous to obtain genetic data from exhibitory samples to aid in investigations, prosecution, or acquittal. As touched items comprise a large portion of the samples submitted for analysis it is imperative to examine how improvements can be made to the production of DNA profile data.
Standard methods of DNA analyses involve sample collection, DNA extraction, PCR by means of a validated commercial STR kit, CE separation, and profile analysis. The portion of this workflow that can cause the largest reduction in profile information is the template DNA loss during an extraction. An alteration to this workflow, termed ‘direct to PCR’, involves the collected sample being incubated, but not extracted, with the lysate used in a subsequent PCR. This approach offers inhibitor dilution and no loss of template, however not all template is placed into a single PCR. In a further workflow alteration, termed ‘direct PCR’, the collected sample can be placed directly into a PCR with no pre-treatment. Direct PCR uses the entire sample in the PCR with maximum template provision, does not require extraction or incubation reagents, but does not facilitate the removal or dilution of any inhibitors present.
The initial part of this thesis looks at optimising direct PCR by analysing six commercially available kits using direct PCR to analyse touch DNA. The work provides informative data and assessments to operational and research-based laboratories of how each of these kits performed. This leads on to investigations into factors that affect touch DNA analysis in a number of ways. Examination of substrate types not previously studied was conducted using direct PCR to determine whether any substrates were more or less suitable for this workflow choice. To better understand how touch DNA acts for genetic amplification, cellular threshold requirements following swabbing and tapelifting for both ‘model’ cell types and corneocytes were examined through extraction-based and direct PCR workflows. The role of cell-free DNA on profiling success was also observed. This encouraged the assessment of other workflow possibilities for touch DNA analysis with rapid DNA technology being analysed for its suitability.
A crucial issue not previously addressed was whether replicate analysis of a sample could be performed using direct PCR. Here, the analysis of a novel tapelift method showed the ability to produce multiple concordant direct PCR profiles from a single sample. The method also allowed performing both a direct PCR amplification and extraction from a single sample. This provides a solution to a major limiting factor for the implementation of direct PCR to casework samples.
The final two chapters look at sub-optimal sample types or those submitted for examination following extreme conditions; improvised explosive device components post-detonation, small calibre fired bullet casings, and used matchsticks. These studies utilise the optimisation techniques developed through the previous chapters. The data presented within this work demonstrates that STR profiles can be obtained from sample types previously not possible.
Direct PCR is becoming a popular method for forensic science research for touch DNA analysis, due to advantages in cost and time reduction while increasing the probability of obtaining STR profiling data. However, it has not been taken up by operational forensic facilities. Prior to this thesis, the application framework of direct PCR for forensic analysis was not well defined. There was little consistency in STR kit choice, the nature of touch DNA template deposition, and a limited range of substrate types analysed. The outcomes of this work and original contribution to touch DNA analysis and direct PCR knowledge involve an extensive examination of STR amplification kit performance and substrate types, a brief consideration of PCR additive effects, the inclusion of cellular staining for experimental monitoring, determination of cellular template input requirements for PCR, and the examination of extreme conditions on touch DNA template viability.
Keywords: forensic science, direct PCR; human STR typing; DNA; trace analysis
Subject: Science, Technology and Enterprise thesis
Thesis type: Doctor of Philosophy
Completed: 2022
School: College of Science and Engineering
Supervisor: Adrian Linacre