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

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.