Modified differential lysis for sexual assault samples using a combined enzymatic and alkaline approach

Abstract

Abstract Sexual assault sample processing, despite recent funding and research efforts, remains time-consuming, laborious, and inefficient. These limitations, combined with the prevalence of sexual assaults, have prompted the need to develop a cheaper, quicker, and more robust method for separating victim and perpetrator contributions within sexual assault evidence so that analysts can keep pace with submissions and cases can be resolved in a timely manner. Thus, this study examined the use of a combined enzymatic and alkaline approach for differential cell lysis—with the goal of developing a quick, cheap, and more efficient DNA isolation method. Quantification results for this assay revealed that 72.0 ± 18.3%, 15.8 ± 14.2%, and 29.5 ± 23.7% of total DNA was retained in sperm fractions for neat semen, neat vaginal, and semen-vaginal mixture eluates, respectively. STR analysis of mixture samples processed with this technique exhibited sperm fraction DNA profiles with mean M: F ratios of 1.74: 1, which was a 3.01 ± 2.3-fold improvement in M:F ratios and led to the recovery of 5.90 ± 7.8 unshared male contributor alleles in sperm fractions that were otherwise undetected in unseparated controls. Overall, this study presented a modified differential lysis approach using prepGEM™ and sodium hydroxide treatments that can accomplish cell elution and fractional lysis within 25 minutes. Future studies should investigate alternative non-sperm cell lysis methods to enhance lysis efficiency and minimize the potential for inhibition, as well as the automation of this technique.