Development of electrospun nanofiber mesh for the enhancement of DNA recovery from spermatozoa in sexual assault evidence

Abstract

One out of every six American women has been the victim of an attempted or completed rape in her lifetime as well as one out of every thirty-three American men. Following a sexual assault, a forensic examination is performed during which probative deoxyribonucleic acid (DNA) evidence can be collected and preserved using a sexual assault kit (SAK). Over 150,000 untested SAKs currently exist in crime laboratories across the United States. Aside from the staggering number of untested SAKs, sexual assaults continue to occur. The untested SAKs need to be tested as do the SAKs collected from the sexual assaults that continue to happen because each SAK that remains untested contains possible DNA evidence that is not aiding in the identification of the suspects of these crimes.Literature discusses both DNA analysis methods for testing SAKs and engineering products used in separation techniques. Current literature does not, however, discuss the application of engineering products used in separation techniques to the DNA analysis methods for testing SAKs. This lack of current literature provides the basis for and the necessity of this study. The intent of this study was to enhance the efficiency of the current DNA extraction technique for sexual assault samples in order to expedite the analysis of SAKs and to ultimately work toward the goal of ridding the United States of its existing backlog of untested SAKs, preventing a backlog from happening ever again, and aiding in the identification of the suspects of these crimes. This research aimed to converge the fields of Engineering and Forensic Science by implementing an engineering product, electrospun nanofiber meshes (ENMs), into the traditional sexual assault sample DNA extraction technique, the differential extraction, in an attempt to physically separate sperm cells from epithelial cells based on their inherent size differences. The engineering products that were implemented were the product of a process called electrospinning. Three pilot protocols were designed and performed. The first two pilot protocols attempted to physically separate sperm cells from epithelial cells: the first by using an ENM made of polycaprolactone as a filter, and the second by using a polycaprolactone film as a nanosieve. The third pilot protocol also attempted to physically separate sperm cells from epithelial cells but by the incorporation of an ENM into a modified DNA analysis process. Extraction, quantification, amplification, and genetic analysis were all performed on simulated sexual assault samples. The resulting DNA profiles were interpreted and evaluated. Although the DNA profiles obtained were mixed profiles rather than single source profiles, this research was significant based on three positive discoveries. ENMs retain sperm cells; melting the ENMs during incubation does not inhibit PCR or any other aspect of downstream processing; and the resulting protocol merges the typical differential extraction protocol with a solid-phase DNA extraction, which has been automated, thus presenting potential to automate the developed protocol. Future research should be conducted on each pilot protocol attempted in this study. This includes research to develop membranes that can withstand the heat of a laser, research to determine a way to adhere ENMs to the insides of microcentrifuge tubes, and research to design an automated electrospinning system capable of producing consistent, uniform ENMs. The pilot protocols used in this study prove that the potential to converge Engineering and Forensic Science via separation of sperm cells from epithelial cells based on their inherent size differences exists but that additional research is needed in order to successfully implement ENMs into forensic DNA analysis.