Isolation of Sperm DNA Through Protamine Capture

As submitted by the proposer: Sexual assault is a horrible crime imparting both physical and psychological damage on over 89,000 individuals annually in the U.S. alone. Prosecution of the perpetrators is enabled through identification of the individual with DNA STR profiling of sperm collected from the victim. Current protocols heavily rely on separation of sperm from epithelial cells prior to DNA analysis in a laborious process known as differential separation. This process is not well suited for automation and as such there currently exists a backlog of sexual assault evidence samples numbering over half a million. New approaches are needed to relieve this backlog and process samples in a quick and efficient manner. Our team has conceived a novel approach for processing sexual assault samples that eliminates differential extraction and utilizes a process known as chromatin immunoprecipitaiton (ChIP) to capture sperm specific DNA by virtue of a unique class of chromatin proteins only found in sperm called protamines. To date we have used two different antibodies to capture sperm DNA and have shown the process to be specific for sperm when lysates from epithelial cells are added in excess. The current workflow is based on existing ChIP protocols and we have identified numerous steps in the process that can be improved to allow adaptation of this approach for sexual assault samples. We will utilize anonymized sperm samples obtained through commercial fertility banks along with post-coital swabs obtained commercially from a separate supplier from consensual volunteers at specified time points to confirm our methods will be applicable for actual sexual assault samples. We will verify performance using in-house PCR validations as well as through generation of STR profiles analyzed by an external entity. Ultimately, this process may eliminate differential extraction and allow automation through direct lysis of sample material from the swab, and downstream processing through modification of existing sample processing systems such as those used for buccal swab processing. As our approach will purify sperm specific DNA through proteins unique to the sperm, our process may allow for samples to be processed that are currently considered unanalyzable due to lack of intact sperm- but likely containing sperm DNA. Data from the project will deposited in an appropriate archive and a summary report will be provided in addition to a peer-reviewed publication. ca/ncf