Developmental validation of an efficient differential separation method incorporating the i-sep DL spin column with high sperm DNA recovery for the processing of sexual assault samples.

The differential separation method is key to recovering a DNA profile of the sperm donor from sexual assault samples. However, low numbers of spermatozoa from the perpetrator are often swamped by the victim's epithelial cells or lost during the separation process, with the separation process labor-intensive, time-consuming, and operator-dependent. The self-sealing filter of the i-sep DL spin column allows direct lysis of the substrate throughout the differential separation process while preventing intact sperm cells from passing through, maximizing DNA recovery, and separation of non-sperm and sperm cells present. This study investigated the efficacy of a modified differential separation method, which incorporated the i-sep DL spin column in comparison with the conventional pellet-based differential separation method. Using semen dilution series and mock post-coital samples, the sensitivity, reproducibility, repeatability, and efficiency of sperm DNA recovery of the pellet-based differential to the i-sep method were evaluated side by side. The i-sep differential method was more sensitive in capturing sperm fraction DNA, with informative semen donor alleles detected from high dilutions of semen inputs where the pellet method has been unsuccessful. The i-sep differential method reduces manual handling, generating repeatable, and reproducible results between operators. Re-extraction of samples previously processed by the pellet or i-sep differential method showed that the pellet method failed to recover 15-88% of sperm fraction DNA, while the i-sep differential method was able to recover >99% in the initial extraction. The i-sep method is robust for processing sexual assault samples, overcoming the challenges of sperm DNA losses encountered by pellet-based methods.