Identification of spermatozoa by tissue‐specific differential DNA methylation using bisulfite modification and pyrosequencing

The focus of this study is to evaluate the application of epigenetic markers as a forensic tool for the determination of semen present in sexual assault cases. A series of genetic loci were screened in order to identify certain epigenetic markers displaying differential methylation that can allow semen to be differentiated from blood, buccal cells, skin epidermis, and vaginal epithelial cells. Of the different loci tested, a panel of six markers, DACT 1, USP 49, DDX 4, Hs_ INSL 6_03, Hs_ZC3H12D_05, and B_ SPTB _03 were identified to contain tissue‐specific differential methylation. Samples ranging from 9–21 for each tissue type were collected and subjected to bisulfite modification. The bisulfite modified DNA was amplified by PCR , and analyzed by pyrosequencing to quantitate the level of methylation at each marker. All six markers successfully differentiated semen samples from the other four tissue types analyzed. Sperm DNA was hypomethylated in all but one marker, B_ SPTB _03, where this marker showed hypermethylation. Mean methylation percentages for semen samples were statistically significant from mean methylation percentages of the other four tissues studied ( p < 0.01). The results of this study demonstrate the applicability of epigenetic markers as a novel tool for determination of spermatozoa and to identify the tissue source of origin of a DNA sample.