Optimization of a novel temperature controlled differential extraction procedure for aged sample analysis

• Main Author: Roberts, Ashleigh
• Other Authors: Cotton, Robin
• Language: en_US
• Published: 2019
• Subjects: Biology
• Online Access: https://hdl.handle.net/2144/38690

There is a documented backlog of sexual assault samples in the United States. Of the many efforts made to reduce this backlog, efficient sample testing is among the most sought-after solutions. The Cotton lab at Boston University has developed a temperature controlled direct-lysis differential extraction protocol that has demonstrated efficient separation of spermatozoic and epithelial cell (e-cell) deoxyribonucleic acid (DNA) from fresh liquid samples. Here, efforts to adapt the protocol for use with dried and aged samples were examined. Varying ratios of epithelial cell to sperm cells from a semen stock were pipetted onto substrates and allowed to age at room temperature for various lengths of time, ranging from 0 to 20 weeks. Modifications were then made to the Cotton lab’s protocol in an effort to optimize DNA yield without compromising the quality of resultant profiles. These modifications focused on the traditional use of spin-baskets and performing lysis on the substrate itself, as opposed to the flow through of traditional spin-basket differential extraction. It was determined that ~95% of the spermatozoic DNA was retained by the substrate. Therefore, efforts were made to adapt the differential extraction protocol to be used directly on a substrate without the use of a spin-basket. It was determined that incubation of the substrate for one hour in water at room temperature prior to enzymatic extraction was beneficial to the percentage of sample recovery. The modified Cotton lab temperature controlled differential extraction (TCDE) protocol was used to extract the epithelial cell and spermatozoic DNA from mixed-sample substrates after aging. Quantitative polymerase chain reaction (qPCR) was performed and the percent DNA recovery was determined. Epithelial cell DNA recovery declined from approximately 85% to 50% as aging progressed. However, spermatozoic DNA recovery remained stable at approximately 80% on average. The resulting DNA profiles indicated that efficient separation of e-cells and sperm cells was achieved even after 20 weeks of aging. In instances where carry-over was detected, the carry-over signal was minimal, reaching a maximum of approximately 10% of the major contributor. Profiles were also analyzed to determine the degree of degradation of epithelial cell DNA and spermatozoic DNA over time. While some degree of degradation was observed after 20 weeks of aging, the probative quality of the profiles was preserved. A representative group of aged samples was successfully extracted using a modified Cotton lab TCDE protocol. Profiles generated from these samples yielded single-source profiles or mixed profiles with a readily distinguishable major contributor. The results from the adapted protocol are encouraging for future use in the analysis of sexual assault evidence and the reduction of the sexual assault DNA backlog.