The Examination of Alternative Methods for Sperm Cell Lysis and Purification of Sexual Assault Samples

Sammanfattning: Forensic sexual assault samples typically contain a mixture of a small quantity of sperm cells and a high quantity of epithelial cells. This relationship complicates the generation of a DNA profile, which prompts the need for differential lysis and extraction of the DNA. Differential extraction is a lengthy method that includes multiple tube transfers and requires a purification step. Amicon Ultra centrifugal filters are employed to purify samples; however, these filters are costly and susceptible to potential breaks in the supply chain. The objective of this project was to examine alternative methods utilizing “off-the-shelf” reagents for sperm cell lysis or sample purification, with the goal of streamlining the current differential extraction protocol. Three methods for sperm cell lysis were studied: alkaline lysis, lysis with the nonionic detergent NP-40, and lysis with a natural decondensation assay. Isopropanol precipitation was examined as an alternative purification method. Additionally, reducing the number of washing steps in the differential extraction protocol was investigated. The results showed that optimized alkaline lysis (25 µL NaOH (0.2 M), 25 µL Tris-HCl (0.2 M) and TE buffer) was comparable to the differential extraction protocol in terms of DNA recovery. Alkaline lysis was also comparable to the differential extraction when performed on mock sexual assault samples. Isopropanol precipitation likewise showed equal DNA yields, complete DNA profiles and peak heights compared to Amicon filtration. Lastly, it was shown that reducing the number of washing steps of the reference protocol produced comparable DNA profiles. In conclusion, the findings suggest that the differential extraction protocol can be modified by using alkaline lysis for sperm cell lysis or isopropanol for purification, as well as by performing only one washing step.