There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures. Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile. Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.

A two-step protocol has been devised as a rapid and selective alternative to conventional differential extraction techniques with an increased recovery of DNA. The protocol involves pressure cycling with the Barocycler® NEP 2320 from Pressure Biosciences. Inc. in alkaline conditions for epithelial cell lysis and removal. This step is followed by alkaline lysis at 95º C for extraction of sperm cell DNA. At 1:1 or 2:1 female to male cell ratios, high selectivity and complete separation can be achieved. But at higher ratios, male allelic dropout is observed. This protocol has been modified to generate a clean male profile at a 20:1 cell ratio through optimization of NaOH concentration and inclusion of an additional pressure cycling step. Validation studies have been performed to assess the efficiency of this method under various conditions. An additional immunomagnetic cell capture pretreatment allowed for nearly complete separation at cell ratios of up to 200:1.