Autosomal DNA-STR Profiling of Directly Captured Spermatozoa from Post-Coital (3-10 Days) Cervico-Vaginal Samples

Some victims of sexual assault provide vaginal samples more than 36-48 hours after the incident. In these cases, the ability to obtain a standard DNA (autosomal STR, aSTR) profile of the semen donor from the living victim diminishes rapidly as the post-coital interval is extended and normally it can't be obtained less than 3 days after the incident. The proposed methods should, for the first time, permit the recovery of single source CODIS-eligible autosomal STR profiles from the semen donor in extended interval (4-10 days) post coital cervicovaginal swabs. We hypothesize that a viable approach to be able to obtain aSTR profiles from extended interval post coital samples would be to carry out direct aSTR typing on selectively enriched, purified and pooled rare individual sperm cells obtained by direct physical recovery from the sample using simplified micromanipulation or digital cell sorting. Over the two-year project period, we propose to test and evaluate two methods, namely simplified manual micromanipulation and digital cell sorting (DEPArrayTM).

Using micromanipulation, sperm cells will be collected manually using a tungsten needle and water soluble adhesive, permitting direct transfer into collection tubes for analysis. Using digital cell sorting, cell-specific fluorescent labeling of epithelial and sperm cell populations permit the identification of sperm in admixed samples. Through the use of dielectrophoretic cages, sperm can be moved through a microfluidic cartridge into a chamber for collection.

Method optimization and performance evaluation will be carried out with vaginal secretions/semen mixtures using anonymous semen (n =15) and vaginal secretions (n=10) donors. This will determine the number of pooled sperm required to obtain a probative aSTR profile. Subsequently, optimized methods will be used to recover and perform aSTR profiling on appropriate numbers of pooled single sperm from bona fide post coital cervicovaginal swabs from 10 volunteer couples, self-collected 3-10 days after individual acts of sexual intercourse. For each time point, a pre-coital swab will be collected on day zero after at least 1 O days of abstinence to ensure no prior presence of male DNA. The DNA typing results will be compared to reference genotypes and the quality of obtained STR profiles will be evaluated (i.e. allele/locus drop-out, allele drop-in, and variation in profile recovery as the time since intercourse increases). Biological and technical replicates will be used to ensure reliability and validity of results. The project findings will be disseminated via publication in peer reviewed forensic journals and presentations at national/international forensics meetings.

This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14).

CA/NCF