Vintiner, SMelia, LSheeba, Elisha2018-08-012017https://hdl.handle.net/2292/37578Available to authenticated members of The University of Auckland.In the early days of RFLP DNA profiling, large amounts of intact DNA, up to 20 to 30 ng, was required to determine profiling results from a sample. The development of STR analysis overcame this issue to a large degree, as STR analysis is very well suited for the analysis of samples that contain smaller amounts of DNA, as well as highly degraded samples. As many forensic DNA samples often contain very low (trace) amounts of DNA, testing these samples often results in partial or incomplete profiles being generated or sometimes no profiling results are obtained. Standard polymerase chain reaction (PCR) conditions may not be sufficient for profiling results to be obtained from such samples. These low-level DNA amounts are generally known as low copy number (LCN) samples. Numerous studies have successfully demonstrated that it is possible to perform DNA analysis on samples containing less than 100 pg of template DNA. To maximise the DNA profiling results from LCN DNA samples, it is therefore necessary to increase the sensitivity of the profiling method being used. The most common way to do this is to increase the number of PCR cycles. Another way is to perform post PCR purification of the amplification product and run the purified and concentrated product at enhanced capillary electrophoresis injection conditions. The success of both methods have been well documented in the literature. Ultra-sensitive methods of DNA profiling, however, also has its drawbacks. Elevated levels of stochastic effects, such as allelic drop-out, allelic drop-in, heterozygote imbalance and increased stutter peaks can be observed. A reliable and robust interpretation strategy for results obtained from ultra-sensitive DNA profiling methods must take on board these profiling features. Before implementing any new technique into forensic casework, it is necessary that the laboratory conducts a validation study to not only test the robustness and reliability of the ultrasensitive DNA profiling method when used in their hands, but to also determine and define appropriate thresholds. limitations and guidelines to assist in interpreting the resultant profiles. This research was divided into two main parts. The main aims of this research were to 1. identify a new ultra-sensitive DNA profiling method to replace the current LCN test at the Institute of Environmental and Science and Research (ESR), SGM Plus at 34 PCR cycles, and 2. to conduct part of an internal validation study of the new method. In part 1, three different PCR amplification kits (Identifiler, Identifiler Plus and NGM Detect) were subjected to testing with both increased number of amplification cycles (from standard to 32 and 34 cycles) and post PCR purification, coupled with enhanced injection conditions. The performance of each method was assessed by investigating the results for the three main parameters: percentage profile, heterozygote balance and average peak heights. These results were also compared to the performance of 34 cycle SGM Plus using the same samples. From this comparison new ultra-sensitive DNA profiling method was selected. In part 2, an appropriate analytical threshold, stutter ratio guidelines and rates of allele drop-in and allele drop-out were determined. These findings form part of an internal validation study of ESR’s new LCN test. Finally, a small investigation on whether any benefits could be gained from reducing the PCR reaction volume by half was also conducted.Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher.Restricted Item. Available to authenticated members of The University of Auckland.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmhttps://creativecommons.org/licenses/by-nc-sa/3.0/nz/ThesisCopyright: The authorQ112934901