Optimizing cell elution conditions for a novel enzymatic DNA extraction technique for spermatozoa on cotton swabs
Taveira, Caitlyn Nicole
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Fundamentals of forensic deoxyribonucleic acid (DNA) typing for sexual assault samples require the successful application of a differential extraction. Gynecological swabs containing vaginal epithelial cells and sperm cells are commonly encountered in forensic casework. A priority in sexual assault casework is the identity of the male contributor and in order to identify the male contributor, separation of the vaginal epithelial cells and sperm cells must be achieved. Two considerations when separating the different cell-types from a substrate are cellular elution and purity of the DNA fractions. Maximizing DNA yield is directly proportional to the number of cells eluted off of the swab and the extraction method utilized. The trypsin-ZyGEM extraction method has shown results of increased DNA recovery on liquid mixture samples compared with the standard differential extraction, which uses proteinase K and dithiothreitol (DTT). The trypsin-ZyGEM differential extraction protocol calls for the use of proteases EA1, incorporated in the forensicGEM extraction kit, and trypsin, a serine proteinase that has been discovered to effectively digest the DNA bound protamines in sperm cells. The trypsin-ZyGEM protocol follows a similar preferential lysis procedure to the standard differential extraction; however, everything is incorporated into one tube, therefore, minimizing loss of DNA from transfer techniques in the trypsin-ZyGEM protocol. Initially, epithelial cells are lysed using the forensicGEM enzyme and removed from solution after centrifugation. Samples are subsequently treated with trypsin, digesting sperm cells. The resultant solution contains the sperm cell DNA and other cell components. The standard differential extraction commonly uses a Qiagen silica membrane column to purify DNA away from cellular proteins and other contaminants, ensuring successful downstream DNA testing with the polymerase chain reaction (PCR). However, treatment with the trypsin-ZyGEM method is followed by a second incubation with forensicGEM after sperm cells are lysed with trypsin. Extracted samples can be quantified and followed through to DNA typing. Expanding previous studies and optimizing conditions of the dual enzyme approach were explored in this study for semen samples on cotton swabs. When comparing DNA yields of extracted samples, tris-ethylenediaminetetraacetic acid (TE) buffer recovered more cells from the cotton swabs than phosphate buffered saline (PBS) buffer and Buffer ATL, used in the Qiagen QIAamp® DNA Investigator Kit. Relative to DNA recovery of the standard Qiagen differential protocol, the trypsin-ZyGEM method on the cotton swab samples appeared subpar and could be attributed to ineffective cellular elution. Additionally, carryover DNA into the non-sperm cell fraction was exhibited. Procedures with Accumax, a cell detachment solution, were implemented in an attempt to elute more cells. The resulting DNA yields were significantly lower in the presence of Accumax. Contrastingly, incorporating trypsin directly into the elution TE buffer exhibited significant increases in DNA recovery. The direct lysis of sperm cells on the swabs, rather than the two-phase method of eluting the cells from the swab and subsequently extracting the DNA, proved to be much more effective. The swab remains from the elution and subsequent trypsin-ZyGEM method were re-extracted using the direct lysis method. Whilst comparing DNA yields, it was discovered that approximately 90% of the cellular DNA was retained on the swab after the two-phase extraction method was performed. Experiments utilizing direct lysis with the trypsin-ZyGEM method, the standard Qiagen differential protocol, and the trypsin lysis followed by Qiagen extraction were performed on swab samples comprised of different semen concentrations resulting DNA yields and short tandem repeat (STR) DNA profiles were compared. Results obtained indicated that the trypsin-ZyGEM method provided substantially larger DNA recovery yields and provided full STR profiles to a target mass of 0.0625 ng and average peak height ratios above 60%. Successful implementation of this extraction procedure will require further studies with the addition of epithelial cells on the swab samples to mimic vaginal swab mixture samples. These samples will help to determine purity of the DNA fractions and effects of epithelial cells on the trypsin-ZyGEM protocol.