Fig. 1.

Overview of the modified differential lysis technique using a combined enzymatic and alkaline approach. Cells are first eluted from a swab cutting (1). Enzymatic lysis of non-sperm cells is conducted using prepGEM™ () and heat () (2), then intact sperm cells are pelleted via centrifugation (3). The entire supernatant is removed as the non-sperm fraction (4), leaving behind the pellet as the sperm fraction (5). Both non-sperm and sperm fractions are then subjected to alkaline solution at 75°C to lyse all remaining cells (figure created in part using BioRender.com).

Fig. 2. Percentage of total DNA retained in sperm and non-sperm fractions after treatment of semen, vaginal, and mixture eluates with the prepGEM/alkaline assay (n = 10). This technique was able to retain 72.0% ± 18.3% and 29.5% ± 23.7% of total DNA in sperm fractions from semen samples and mixture samples, respectively.

Fig. 3. Percentage of total DNA retained in sperm fractions from single-source semen and vaginal eluates relative to the total DNA yield after treatment with the prepGEM/alkaline assay (n = 10). Linear trendlines indicated no correlation between cellular input (i.e., total DNA yield) and the percentage of DNA retained in sperm fractions for each sample type, signifying that prepGEM can lyse a wide range of cellular input.

Fig. 4. Representative blue channel STR electropherograms for mixtures processed using the prepGEM/alkaline differential lysis technique. Unseparated mixture controls exhibited a mean M:F ratio of 1:1.9 (A), while non-sperm fractions had a mean M:F ratio of 1:5.5 (B). Sperm enrichment was demonstrated in sperm fractions, with STR profiles experiencing a 7.97-fold M:F improvement over unseparated controls and a mean M:F of 1.7:1 (C). Asterisks denote unshared female alleles, while unshared male alleles are represented by ∇. Other colour channels demonstrated similar results.