Comparison of downstream data quality of two differential extraction techniques through the examination of peak heights and peak height ratios
Sutherby, Danielle Marie
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Analysis of sexual assault evidence in a forensic laboratory setting requires accurate analysis of deoxyribonucleic acid (DNA) utilizing forensic DNA typing. This process can be complicated since most sexual assault samples are comprised of DNA from a female contributor as well as one or more male contributors. Generally female epithelial cells (e-cells) are in excess, making it more difficult to determine the short tandem repeat (STR) profile of the male contributor from the significantly fewer sperm cells present in the mixture. This complexity requires that the two contributing sources of DNA be separated in order to obtain a probative single source profile. Separation of the two fractions is accomplished by differential extraction. The most common protocol for a differential extraction involves preferentially lysing epithelial cells while leaving the sperm cells intact, separating the released female DNA from the sperm cells and finally lysing the sperm cells to obtain the sperm fraction. Research has shown that a Trypsin-ZyGEM extraction method produces higher yields of DNA compared to the standard differential extraction method. A previous study examined whether substrate type had an effect on the efficiency of the Trypsin-ZyGEM extraction. It was found that the Trypsin-ZyGEM protocol worked well at extracting DNA from aqueous semen, dried semen in a microfuge tube and semen dried on white cotton. However, results were inconclusive on the method’s ability to extract DNA from semen dried on denim fabric due to inhibition of the quantitative polymerase chain reaction (qPCR) used to quantify the samples. In this study, a subset of the denim samples was amplified and processed through capillary electrophoresis to verify that the extraction protocol had successfully recovered DNA from this substrate type and that the DNA could be amplified to create a STR profile. Full profiles were obtained for all three of the denim samples that were amplified to a target mass of 1 nanogram (ng). One full profile and two partial profiles were obtained for the same samples at a target mass of 0.25ng. Allelic dropout for the partial profiles varied with one profile having 3 alleles dropout while the other had 11 dropout. Peak heights and peak height ratios were more variable compared to the other substrate types but were still within an acceptable range for probative profiles. The results are promising and suggest that the Trypsin-ZyGEM protocol could possibly replace the current differential extraction technique. Further research is required to understand if this extraction protocol would be efficient on these substrate types with mixture samples of semen and e-cells. This research also builds upon the exploration of the effectiveness of the Trypsin-ZyGEM extraction on dried and aqueous semen samples compared to a modified Qiagen differential extraction. Samples that had been previously quantified were amplified and separated by size using capillary electrophoresis. Peak heights and peak height ratios (PHR) from the STR profiles were examined to assess profile quality of samples extracted using the Trypsin-ZyGEM protocol and the Qiagen protocol. Peak heights and peak height ratios (PHR) were found to be consistent between the two methods.