Optimization of the temperature controlled differential extraction for casework-type samples
Hoffman, Emily Elizabeth
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Differential extraction has proven to be a challenging and time-consuming process, often requiring up to six hours of a forensic analyst’s concentration. With the ever-increasing backlog of sexual assault evidence kits, the forensic community is using new ways to diminish this backlog, including more streamlined evidence processing and sample analysis. The goals for processing sexual assault samples include efficient recovery of sperm deoxyribose nucleic acid (DNA), simplified sample processing, and the development of a profile eligible for forensic analysis. Cost and time can also be limiting factors. The Cotton Research Lab at Boston University has developed a novel method of differential extraction that combines separation of epithelial and sperm cell fractions, nuclease treatment to reduce female DNA carryover and a direct-cell lysis protocol. With the exception of a single centrifugation step, the entire protocol is conducted using a thermalcycler in the DNA extraction laboratory. Thus, the process is a Temperature Controlled Differential Extraction (TCDE), and has been effectively adapted for use with liquid, dried, and aged samples. The purpose of this research is to explore methods which further adapt the protocol for best use with forensic casework samples, namely vaginal swabs. Sexual assault evidence collection kits may contain a variety of items, and commonly include cotton swabs for the collection of fluids from intimate sources. To simulate casework-type samples, swabs were prepared with liquid epithelial cell preparations and various semen dilutions (ranging from 1:1 to 1:1000). Amendments were made to the TCDE protocol for best DNA recovery from a swab, and buffer changes were made to enhance compatibility with polymerase chain reaction (PCR)-amplification kits widely utilized in forensic labs. Finally, post-coital swabs from female donors were analyzed using the TCDE protocol with modifications for forensic casework samples. Preliminary testing of casework-type swabs with protocol modifications showed high yields of DNA and successful separation of epithelial and spermatozoa fractions. The epithelial fraction, when yielding a mixed profile, demonstrated a clear major female contributor, and the spermatozoa fractions showed little to no female carryover, often exhibiting single source male profiles. The TCDE protocol with modifications for casework-type samples requires approximately 2 hours and 30 minutes of an analyst’s time, from the moment the swab is removed from its evidence packaging to an extraction ready for DNA quant and short tandem repeat (STR) amplification. The method provides increased DNA recovery, can be used with various amplification kits, and generate probative profiles and is time efficient. This robust and promising new method that has the potential to be automated and to contribute to the effort to reduce the backlog in the analysis of sexual assault evidence kits.