Author: Renee Blackie
Blackie, Renee, 2017 The application and validation of direct PCR from various substrates for forensic practice, Flinders University, School of Biological Sciences
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Direct PCR is fast becoming a popular method in forensic science due to the advantages of saving time and money in the laboratory while increasing the probability of obtaining substantial results has a positive rippling effect. A laboratory is able to reduce the time spent on processing trace DNA samples, which can lead to investigators receiving important information in a timely manner that may not have been possible using standard methods. DNA extraction procedures are standard practice in the initial steps of DNA profiling when examining swabs, adhesive tapes and sections of fabric. Significant loss of DNA can occur during this process resulting in no DNA profile generated. Direct PCR circumvents the extraction process such that a DNA profile may be generated directly from the substrate. This saves time, increases the DNA yield, reduces tube changes, and minimises steps open to error or contamination in the laboratory.
To fully understand the benefits and limitations of direct PCR, several aspects of the method have been analysed in this thesis. The loss of DNA via extraction was investigated and determined for the most common extraction methods used in forensic science, as well as understanding the limit of detection for the commerical PCR human identification kits. From this, a statistical method to quantify the DNA template used in direct PCR amplifications based on the resulting magnitude of the allele peaks generated was developed. In this PhD study, trace evidence items investigated using direct PCR include: human hairs, canine hairs, fibres, human finger nails, and human teeth. Substrates have been analysed using both short tandem repeat (STR) and single nucleotide polymorphism (SNP) methods for identification. A high level of succesful profile generation has been found across all of the substrates. A successful profile produces five or more complete loci that can be up-loaded to the National Criminal Investigation DNA Database (NCIDD, Australia).
Current practice for many laboratories is that trace evidence items, such as the ones listed above, are generally not subjected to DNA testing as there is little chance of generating a meaningful DNA profile. All direct PCR methods, where applicable, have followed validated protocols as to maintain high standards and allow fast implementation within forensic laboratories. The only alteration to the PCR methods was an increase in the amount of DNA polymerase used to help overcome any potential inhibitors present on the substrate. This study highlights direct PCR sensitivity and the ability for trace DNA to be amplified without the need to increase cycle number or modify current protocols to obtain meaningful data. Based on the data presented in this thesis, a direct PCR approach is a viable option for the future of trace DNA recovery and analysis for forensic science purposes, vastly improving efficiency, sensitivity and the quality of results.
Keywords: forensic science, pcr, direct PCR, DNA, human identification, polymerase chain reaction
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
Completed: 2017
School: School of Biological Sciences
Supervisor: Adrian Linacre