A novel differential extraction technique utilizing multiple enzymes: developing separation of non-sperm and sperm fractions
Martinez, Rachael Elizabeth
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Processing sexual assault samples is a difficult time consuming task for the forensic analyst. Samples tend to be a mixture of the victim’s epithelial cells and the male suspect’s sperm cells that need to be separated prior to extraction of Deoxyribonucleic Acid (DNA). Without separation of the two cell types, the DNA extract would result in an uninterpretable mixture. In 1985, Peter Gill and colleagues outlined a procedure known as preferential lysis that would aid in the separation of female and male cells from sexual assault samples. The basis of this procedure, commonly referred to as a differential extraction, utilizes differences in the packaging of DNA between the two cell types to preferentially lyse the female epithelial cells and leave the sperm intact. By pelleting the sperm and removing the supernatant (termed the Non-Sperm Fraction) the Sperm Fraction can now be extracted without the contaminating epithelial cells. This procedure has been widely implemented in forensic laboratories and is still being used today over 30 years later. However, there are certain conditions under which this procedure does not perform sufficiently including excess of female epithelial cells and low amounts of sperm. Unfortunately, both of these conditions are common among sexual assault samples. The procedure also is quite long, and with the backlog of sexual assault samples continually growing in the United States, there is a need for a new procedure that is faster and performs optimally under the previously mentioned conditions. This research explores the use of two enzymes, EA1 (marketed by Zygem Corporation as ForensicGEM Saliva™) and Trypsin to separate the cells. Due to the inability of Zygem to cleave the disulfide bonds present in the sperm DNA packaging proteins, treatment of mixed samples with Zygem will lyse the epithelial cells and leave the sperm intact. The incubation time of Zygem is much faster than that of the Gill method and can be performed in one tube, minimizing the chances of DNA loss and contamination during transfers. Treatment of the pelleted Zygem extract with Trypsin effectively and rapidly lyses the sperm cells. Combining these methods into a differential extraction protocol has the potential to be a rapid, easily implemented procedure. Results from the Zygem-Trypsin differential extraction method showed incomplete separation of the two fractions due to the incomplete lysis of the epithelial cells by Zygem. The resultant profiles did show a major male contribution with a minor female component, however these results are not sufficient enough for real casework. While further research and development of the protocol are necessary, the Zygem-Trypsin differential extractions performed here show the potential for a rapid, easy differential extraction procedure that could be easily implemented in any laboratory.