This study reports the development of a two-step protocol using pressure cycling technology (PCT) and alkaline lysis for differential extraction of DNA from mixtures of sperm and vaginal epithelial cells recovered from cotton swabs. In controlled experiments, in which equal quantities of sperm and female epithelial cells were added to cotton swabs, 5 min of pressure pulsing in the presence of 0.4 M NaOH resulted in 104 ± 6% recovery of female epithelial DNA present on the swab. Following the pressure treatment, exposing the swabs to a second 5-min alkaline treatment at 95 °C without pressure resulted in the selective recovery of 69 ± 6% of the sperm DNA. The recovery of the vaginal epithelia and sperm DNA was optimized by examining the effect of sodium hydroxide concentration, incubation temperature, and time. Following the alkaline lysis steps, the samples were neutralized with 2 M Tris (pH 7.5) and purified with phenol-chloroform-isoamyl alcohol to permit downstream analysis. The total processing time to remove both fractions from the swab was less than 20 min. Short tandem repeat (STR) analysis of these fractions obtained from PCT treatment and alkaline lysis generated clean profiles of female epithelial DNA and male sperm DNA for 1:1 mixtures of female and male cells and predominant male profiles for mixtures up to 5:1 female to male cells. By reducing the time and increasing the recovery of DNA from cotton swabs, this new method presents a novel and potentially useful procedure for forensic differential extractions.

A pressure‐based protocol for differential extraction has been optimized to provide a rapid and selective alternative to conventional differential extraction techniques with the advantage of an increased recovery of genomic DNA. The protocol involves treating cotton swabs containing mixtures of sperm and vaginal epithelial cells with two sets of pressure treatment using a Barocycler® NEP 2320 in alkaline conditions. This first step quantitatively and selectively removes female epithelial cells. The cotton swab is removed and further treated with alkali at 95°C for the removal of sperm cell DNA. The resultant solution provides a clean male profile at a 20:1 cell ratio. Furthermore, the inclusion of a pretreatment step involving immunomagnetic cell capture of the female cells, permits nearly complete isolation of male sperm cells at cell ratios of up to 200:1 female to male cells.

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