Increased yield of DNA from dual enzyme differential extraction
Fisher, Matthew B.
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Forensic analysis of deoxyribonucleic acid (DNA) represents an important facet of criminal investigations. DNA extraction, the first step in sample processing where DNA is released from biological samples and isolated, is crucial for subsequent analysis. Minimizing loss of DNA during extraction as well as ensuring complete lysis of DNA-containing cells are two factors that must be considered when choosing an extraction method. Extraction procedures that minimize sample transfers, specifically single-tube extractions, are ideal for minimizing DNA loss. A DNA extraction kit made by ZyGEM (Hamilton, New Zealand) incorporates the use of a recently characterized proteinase; EA-1 achieves a single-tube extraction that requires no further purification. However, research published on this method reported that the extraction was unable to release DNA from sperm cells. Sexual assault cases routinely require DNA to be extracted from sperm cells, thus the ZyGEM method would be unsuitable for treatment of such samples. It could, however, possibly be used as part of a differential extraction procedure for samples containing a mixture of epithelial cells and sperm cells. The initial step in a differential extraction is the preferential lysis of any non-sperm cells. The DNA from these cells is separated and removed from the intact sperm, which are then lysed using more robust methods. The current research discusses the process of differential extraction and the investigation of an alternative method for sperm cell lysis. Development of this method was aided by studying the physical structure of sperm cells. Sperm DNA is packaged by small proteins, the protamines that replace histones during spermiogenesis. These proteins are comprised primarily of the amino acids Arginine and Lysine. The serine proteinase Trypsin, which cleaves peptides at Arginine and Lysine, was investigated as an alternative enzyme for sperm lysis in a differential extraction procedure. A sperm cell extraction method with Trypsin was developed, using the forensicGEM extraction to purify the sample following lysis with Trypsin. This method was compared to an extraction of sperm with Qiagen QIAamp® DNA Investigator kit. The results show that the new method using Trypsin and ZyGEM yields between 4 and 12 times more DNA than the Qiagen method. When low template samples were extracted and amplified, the new method was able to generate a full profile while samples extracted with Qiagen lost over 80% of the profile due to low yield in the extraction. Analysis of Peak Height (PH) showed the new method had slightly lower peak heights compared to Qiagen when similar amounts of DNA were amplified, however Peak Height Ratio (PHR) was not reduced in the new method. The results indicate that a new extraction method using Trypsin and ZyGEM provides greater amounts of DNA from extractions of sperm cells and that the method should be further developed into a differential extraction protocol to aid in mixture sample processing.