Alternative direct‐to‐amplification sperm cell lysis techniques for sexual assault sample processing

Abstract

The prevalence of sexual assault cases and increasingly sensitive DNA analysis methods have resulted in sexual assault kit backlogs in the United States. Although traditional DNA extraction and purification utilizing detergents, proteinase K, and DTT have been the primary technique for lysing sperm cell fractions from these samples, it is labor‐intensive and inefficient regarding time and sperm DNA recovery – hindering the ability of forensic analysts to keep pace with evidence submissions. Thus, this study examined seven alternative sperm cell lysis techniques to develop a method that could efficiently lyse sperm and consistently generate high‐quality profiles while also reducing time, labor, and cost requirements. Microscopic examination of lysates indicated only Casework Direct and alkaline techniques could lyse all spermatozoa within samples, while quantification results demonstrated all methods performed comparably to the control method of forensicGEM™ Sperm (p > 0.06). Amplification with 0.25 ng DNA revealed that unpurified lysates from Casework Direct, alkaline, and NP‐40 techniques produced DNA profiles with acceptable mean STR peak heights and interlocus balance, both of which were similar to or better than the control. Overall, this study demonstrated the ability of Casework Direct, alkaline, and NP‐40 methods to efficiently lyse spermatozoa and provide high‐quality STR profiles despite the absence of a purification step. Ultimately, based on the data reported herein, alkaline lysis is the recommended alternative sperm lysis approach given its ability to generate high‐quality profiles, save time, and decrease the cost per reaction when compared to traditional sperm cell lysis methods.

Alternative direct‐to‐amplification cell lysis techniques for forensically relevant non‐sperm cells

“…Alkaline lysis (1 M NaOH) was used as the control for semen samples as it leads to complete lysis of sperm cells [14]. For this method, ten microliters of 1:10 semen was incubated in 7 μL of DPBS and 4 μL of 1 M NaOH (Thermo Fisher Scientific) at 75°C for five minutes in 0.2 mL PCR tube strips using the ProFlex™ 3 × 32‐well PCR System.…”

“…In addition, the fundamental structure of DNA is comparatively stable in an alkaline solution [19][20][21]. However, the alkaline approach can also lead to lysis of sperm cells if used at high concentrations (e.g., 1 M NaOH) [14,19,22]. Thus, to differentially lyse epithelial cells and not sperm cells, lower concentrations of NaOH would need to be utilized [14].…”

“…However, the alkaline approach can also lead to lysis of sperm cells if used at high concentrations (e.g., 1 M NaOH) [14,19,22]. Thus, to differentially lyse epithelial cells and not sperm cells, lower concentrations of NaOH would need to be utilized [14]. Radioimmunoprecipitation assay (RIPA) buffer and urea/thiourea lysis buffer are also commonly used for rapid and efficient cell lysis, as well as solubilization of proteins, from mammalian cells-releasing cytoplasmic, nuclear, and mitochondrial proteins [23][24][25][26][27][28].…”

“…In a recent study by Schellhammer et al, alternative direct‐to‐amplification sperm cell lysis methods were explored in order to provide a faster and cheaper alternative to the conventional sperm cell lysis technique [14]. Of the methods tested, the alkaline lysis method‑which uses 1 M sodium hydroxide (NaOH) for five minutes at 75°C‑was ultimately selected as the best alternative approach to accomplish sperm cell lysis [14].…”

“…In a recent study by Schellhammer et al, alternative direct‐to‐amplification sperm cell lysis methods were explored in order to provide a faster and cheaper alternative to the conventional sperm cell lysis technique [14]. Of the methods tested, the alkaline lysis method‑which uses 1 M sodium hydroxide (NaOH) for five minutes at 75°C‑was ultimately selected as the best alternative approach to accomplish sperm cell lysis [14]. However, identification of a similar direct‐to‐amplification lysis method for the non‐sperm fraction is also needed, as this is critical to achieve successful centrifugal separation of the cellular components.…”