The separation of sperm from female epithelial cells has been a topic of interest in forensic DNA (deoxyribonucleic acid) analysis since the origin of the field. One of the most needed applications of DNA analysis in the identification of the perpetrator of a sexual assault, as often there is little to no other evidence for identification. The largest hurdle to forensic DNA analysis in these cases is that vaginal or oral swabs from sexual assaults will have a mixture of the victim’s epithelial cells and the perpetrator’s sperm cells. It is well known that the analysis of complex mixtures can be difficult to impossible, especially when there is an added concern of low template DNA. Separating these cell types in the mixture evidence is the best way to avoid the need to deduce these difficult mixtures. Sperm and Epithelial Cells are morphologically different both in cell shape and DNA packaging. Nuclear DNA in epithelial cells are more loosely packaged around histones in a structure called a nucleosome. Sperm DNA is tightly packaged around protamines rather than histones. These DNA packaging differences can be utilized to preferentially lyse sperm and epithelial cells in order to separate them. Traditionally this is done by lysing epithelial cells with sodium dodecyl sulfate (SDS) and proteinase K (PK), separating this epithelial DNA from the sperm by centrifugations and finally lysis of the sperm using dithiothreitol (DTT) which reduces the disulfide bonds in the sperm DNA packaging. This method was developed by Peter Gill in 1985 and is still used by forensic laboratories to date. This differential extraction is very labor intensive and time consuming. This dual-enzyme differential extraction can be performed in roughly one hour, which is highly advantageous with the large amount of backlogged sexual assault cases that forensic laboratories have. This work was undertaken to improve the separation of epithelial DNA from sperm cells in the dual-enzyme differential extraction. Here we found that the DNA carryover into the sperm fraction was due to a combination of an inability to completely separate the non-sperm fraction liquid from the sperm pellet and the decreased efficiency of ZyGEM to fully lyse epithelial cells in clumps. The solution to this problem includes the addition of a wash of the sperm pellet after initial separation of the fractions. This wash step decreased the concentration of epithelial DNA to the point that its detection may only occur with very low concentrations of sperm DNA. / 2017-11-03T00:00:00Z