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

Sexual assault sample processing, despite recent funding and research efforts, remains time-consuming, labourious, 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 were retained in sperm fractions for neat semen, neat vaginal, and semen-vaginal mixture eluates, respectively. Short tandem repeat (STR) analysis of mixture samples processed with this technique exhibited sperm fraction DNA profiles with mean male-to-female ratios of 1.74:1, which was a 3.01 ± 2.30-fold improvement in male-to-female ratios and led to the recovery of 5.90 ± 7.80 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 min. Future studies should investigate alternative "non-sperm" cell lysis methods to enhance lysis efficiency and minimize the potential for inhibition, as well as the optimization and automation of this technique.

Traditional sexual assault sample processing methods are time-consuming and inefficient.This modified differential lysis method produces lysates with sufficient DNA yield and quality.A combined technique using enzymatic and alkaline lysis can accomplish fractional separation.Lysis with prepGEM and NaOH absent purification is compatible with downstream processes.