title: The modified method of two-step differential extraction of sperm and vaginal epithelial cell DNA from vaginal fluid mixed with semen.
abstract: This investigation was undertaken as an efficient method for isolating sperm DNA from a mixed fluid sample which contains vaginal epithelial cells in a greater amount. The modified method of the two-step differential extraction procedure was found to be suitable for separating sperm DNA and vaginal epithelial cell DNA from the mixed stains. As the first step of digestion, vaginal epithelial cells in the mixed stains were lysed with Proteinase K and SDS, and sperm heads remaining in the lysed solution were collected by centrifugation. As the second step digestion, the sperm heads were lysed with the buffer containing Proteinase K, SDS and DTT as reducing agent. DNA fractions extracted from the two lysed solutions were enriched, one with sperm DNA and the other with vaginal epithelial cell DNA. MCT118(D1S80), ApoB VNTR and HLADQ alpha types of sperm DNA were detected and were confirmed by matching with corresponding male blood DNA. In the case of vaginal secretion mixed with semen of two males, the mixture of MCT118 types of the two males was detected in sperm DNA fraction.
authors.affiliation: National Research Institute of Police Science, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Yoshida
authors.forename: K
authors.initials: K
authors.lastname: Sekiguchi
authors.forename: N
authors.initials: N
authors.lastname: Mizuno
authors.forename: K
authors.initials: K
authors.lastname: Kasai
authors.forename: I
authors.initials: I
authors.lastname: Sakai
authors.forename: H
authors.initials: H
authors.lastname: Sato
authors.forename: S
authors.initials: S
authors.lastname: Seta
mesh_terms: D001483:Base Sequence; D001826:Body Fluids / Q000737:chemistry; D002469:Cell Separation / Q000379:methods; D004247:DNA / Q000302:isolation & purification*; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D005260:Female; D006801:Humans; D008297:Male; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012661:Semen / Q000737:chemistry; D012697:Serine Endopeptidases / Q000378:metabolism; D013094:Spermatozoa; D013347:Subcellular Fractions / Q000737:chemistry; D013997:Time Factors; D014621:Vagina / Q000166:cytology; D014626:Vaginal Smears
chemical_list: D017931:DNA Primers; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K