Abstract:
This project aimed to explore the possible alternatives of sampling devices and methods as well as the efficacy of direct amplification of casework samples. Forensic casework samples come in all conditions, from samples containing trace levels to dense amounts of DNA. This unknown factor in casework samples requires immense amounts of effort to fully introduce an appropriate sampling device which is able to collect the required amount to produce profiling results of high quality. Additionally, the direct PCR amplification method of casework samples, where it bypasses extraction and quantitation, have been of interest. The main body of this project was split into two sections. The first explores the efficiency of recovery and capture of DNA by testing a selection of alternative swabs and swab lubricants. These different swab-and-lubricant combinations were tested against the current method of using cotton-spun swabs with water or tape-lifts to determine if there is a better potential swab candidate and/or lubricant for casework samples. The list of swab candidates to be tested was initially narrowed down with the input from Auckland service centre scientists. Among the candidates were the fine-tip swabs used in medical examination kits, and a type of swab made with dissolvable swab fibres, to maximise release. The swab lubricant alternatives to water were 100% ethanol and 20% SDS (sodium dodecyl sulphate) solutions. Results indicated that even though the fine-tip swabs had the lowest DNA recovery, it yields better profiling results in terms of the numbers of alleles recovered compared to the cotton-spun swabs. The tape-lifts was more proficient at recovering samples of low levels of trace quantities than the rest. The second section looked at the direct amplification of various casework samples collected using swabs as this project's intention was to explore whether the method was possible and feasible at the Institute of Environmental and Science Research Limited (ESR). Sample types tested in this study included neat blood, swabs from drink cans or bottles, mixed saliva and mixed vaginal-semen and blood deposited on various fabrics and surfaces. The size of the swab portion was also investigated to determine the optimal amount required. These samples were tested and compared among the AmpFISTR® Identifiler®, Yfiler® Plus and AmpFISTR® Identifiler® Plus kits at different thermal cycling conditions. Lastly, samples were also amplified with and without the addition of TE buffer or water to the final PCR volume to observe any effects. The results showed that the Yfiler® Plus kit had an overall a more efficient allele recovery. The effects of the nature of the samples or substrates, such as fabric samples also impacted the amplification stage, disrupting the internal size standards used in capillary electrophoresis. It was apparent that swabbing stains from fabric bloodstains had an improved allele recovery as opposed to testing the fabric directly. A finding of interest was the decrease in overall peak heights when TE buffer was added to the PCR tube to make up the final volume. The portion size of a swab head that was able to recover alleles most consistently was approximately 1 x1 mm in size when testing blood samples. The possibility to repeat some of this work was not possible given the limited resources and time available.