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

English. 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.

English. Sperm cells are usually the most interesting biological material to analyze in forensic sexual assault samples. However, today’s method for extracting DNA from sperm is both time-consuming and requires purification with expensive filters. It is therefore relevant to investigate alternative protocols that can help streamline the process. The term “sexual offense” it the Swedish legal umbrella term for crimes of a sexual nature, with major crimes including sexual assault. Typically, the biological material of highest interest in sexual assault samples is sperm cells, as DNA can be extracted and potentially linked to a suspect. However, due to the nature of most sexual assault samples, where sperm cells are present in lower quantities in comparison to other cell types (e.g. epithelial cells), successful extraction and analysis of sperm cell DNA is difficult. Because of this, a method called differential extraction is typically performed, where the sperm cells and other cells in the mixture are treated separately. Differential extraction is a lengthy protocol that requires multiple transfers of material between tubes, which can lead to loss of DNA and contamination. On top of this, it uses the strong reagent dithiothreitol (DTT) to successfully break open the sperm cells and release the DNA. DTT interferes with downstream analysis and DNA profiling, for which reason the samples must be purified. At the National Forensic Centre (NFC), purification of samples is done with centrifugal filters called Amicon filters. As there is only one supplier of these filters, the process is susceptible to disruptions in the supply chain, which occurred during the Covid-19 pandemic. Furthermore, Amicon filters are expensive. Therefore, it was relevant to examine alternative methods for sperm cell disruption (lysis) and sample purification that could simplify the current method, preferably while using easily accessible reagents. This project examined three alternative methods for sperm cell lysis, and one alternative method for purification of samples treated with DTT. Also, reducing the number of times the intact sperm cells are washed was assessed, as this is a time-consuming step in the current protocol, and requires a lot of pipetting. Alkaline lysis, a method where the sperm cells are lysed by being subjected to heat and a strong base, was seen to produce DNA yields equal to the current protocol, as well as complete DNA profiles. Likewise, precipitation with isopropanol was seen to purify samples as well as Amicon filtration. Lastly, reducing the number of washing steps resulted in equal DNA recovery in comparison with the protocol used today. Thus, incorporating these methods could simplify today’s differential extraction protocol significantly.