The development and optimization of a direct lysis differential extraction method

• Main Author: Yakoo, Mike
• Language: en_US
• Published: 2018
• Subjects: Biology
• Online Access: https://hdl.handle.net/2144/26954

With the publication of the first article on the forensic application of deoxyribonucleic acid (DNA) analysis, the authors demonstrated the ability to process samples acquired from victims of a sexual assault—samples, typically vaginal swabs, that oftentimes contain a mixture of sperm and epithelial cells (E-cells). The authors proposed a method that preferentially lyses E-cells while leaving sperm DNA, present in an ultra-compact nucleoprotein complex specific to sperm, out of solution. This method, commonly referred to as the Gill method, has since been extensively used, relatively unmodified, in the processing of sexual assault samples. While DNA extraction and analysis of forensic samples has improved significantly throughout the years, both in terms of process efficiency and the quality of results, the Gill method remains a labor intensive process. Additionally, most DNA extraction methods require purification of nucleic acids in order to perform downstream analyses, which inherently results in DNA loss. Various methods have been developed that allow for the extraction of DNA without a subsequent purification step, often referred to as direct lysis methods. However, none have been shown to be viable options for use in sexual assault samples. ZyGEM (Hamilton, New Zealand) manufactures DNA extraction products containing the thermophilic EA1 protease, which has been shown to effectively digest DNA samples that are suitable for downstream processes, without the need for purification. We demonstrate the ability to utilize this protease, along with nuclease digestion of residual E-cell DNA (a process referred to as selective degradation), to produce a sperm sample with little to no E-cell DNA carryover, with only one centrifugation step. Subsequent sperm cell lysis is carried out with a second direct lysis product, Acrosolv (ZyGEM, Hamilton, New Zealand). The total processing time has been reduced to approximately two-and-a-half hours compared to over six hours with currently used extraction and purification methods.