Optimizing an elution buffer to improve the recovery of sperm cells from cotton swabs
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Citation
Abstract
Sexual assault samples often contain a complex, disproportionate mixture of male sperm cells, male epithelial cells, and female epithelial cells, and require the precise, independent extraction of each cell type. This differential extraction (DE) procedure involves the initial lysis of male and female epithelial cells to release both male and female deoxyribonucleic acid (DNA). Male sperm cells, immune to this enzymatic treatment, remain intact and can then be extracted separately. In theory, this produces a clean female epithelial fraction (EF) and male sperm fraction (SF). In practice, however, various complications can occur that jeopardize the acquisition of a single-source male and female DNA profile. Perhaps most threatening is the well-documented poor release of sperm cells into the SF due to strong adhesion of the cells to the sample swab. While the exact mechanism for how the sperm adhere to cotton is unknown, surface glycoproteins that comprise the sperm glycocalyx are believed to play a vital role. This research sought to develop an elution solution that improves sperm cell recovery from cotton swabs and that can be easily integrated into commonly practiced DE procedures. Cotton swabs stained with either 53 nanograms (ng) or 100 ng of male sperm DNA were incubated in one of several solutions for various times and temperatures. Solutions of sucrose, histidine, polyvinylpyrrolidone (PVP) and Tween 20, and bovine serum albumin (BSA) and Tween 20 were selected for this study based on their recovery success demonstrated in past research, as well as to investigate if these small, soluble molecules could displace sperm cells from the swab. Solutions containing proteinase K (PK), PK and sodium dodecyl sulfate (SDS), and forensicGEM™ Bacillus sp. Erebus antarctica 1 (EA1) were tested to explore the activity of these proteases on the sperm glycoproteins. The eluate and material fraction (MF) from each sample were extracted and quantitated to measure the amount of sperm DNA recovered from the SF, as well asthe amount of DNA left on the swab. Of all solutions tested in this study, samples incubated forensicGEM™ EA1 returned the highest average recovery yields with up to 80% of total DNA extracted from the SF. Though more research is needed to understand the exact enzymatic mechanism at play, these results suggest that forensicGEM™ EA1 exhibits activity on the sperm glycoproteins that facilitates greater release of sperm cells into solution. Before these solutions can be incorporated into current DE protocols, validation tests using mock sexual assault samples are necessary.
Description
2024