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. / 2022-03-31

Recovering deoxyribose nucleic acid (DNA) from items of evidence can provide critical information in criminal cases. Since the development of the polymerase chain reaction (PCR) and use of short tandem repeats (STR) to create unique profiles from an individual’s genome1, sampling items of evidence for the presence of DNA has become routine. Biological evidentiary specimens are commonly collected at crime scenes as well as sampled from collected items of interest by using a cotton swab which can then be easily stored and tested as needed. However, even with modern advances in technology and methods, large amounts of DNA can be either lost throughout processing or remain on the substrate used for collection of the sample, such as a cotton swab2. While many of the downstream processes of evidence evaluation have been vastly improved through the use of automated procedures, engineered buffers, and commercially available extraction kits, the front-end procedures are typically more technician dependent; it is an area in which opportunities to fine-tune techniques remain. The most recent change to generalized stain recovery occurred after Sweet et al. achieved an increased efficiency of recovery by using what they referred to as the “double swab technique”. The classic method of collection before this time used a single, wet cotton swab. Based on a need to increase the effective collection of DNA from saliva samples, the double swab method was developed. The classic method was modified by using a second, dry swab to collect remaining moisture deposited by the first, wet swab3. To continue the effort to maximize cellular and DNA recovery from cotton swabs the use of trehalose in the cotton swab wetting solution was explored. D-(+)-Trehalose dihydrate is a naturally occurring disaccharide composed of two alpha glucose molecules. An alpha, alpha-1, 1 bond connects the two molecules which lends high resistance to acid hydrolysis, giving the molecule unique properties. Specifically, these properties allow the compound to maintain stability even during exposure to high temperatures and in acidic conditions4,5. In nature, trehalose can be found in plants and small organisms where it is thought to act as a protectant against fluctuations in moisture and temperature. Synthesis and release of trehalose by lower life forms during stressed states shows protective properties to cellular integrity by inhibiting protein denaturation6. The objectives of this study are to investigate the use of trehalose as an additive in DNA collection processes. The experiments examine the ability of trehalose to increase efficiency of cellular release from cotton swabs during the elution step and compares trehalose to other common buffer additives, bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS), when utilized as a pre-treatment or moistening agent on the cotton swab. Two procedures were developed to test the ability of trehalose to increase efficiency of cellular and DNA release from cotton swabs. The first procedure tested trehalose at 0.2 molar (M) and 1 M concentrations as the incubating solution over1 hour and 18 hour time periods after which the cotton swab was eluted using a spin-x insert and centrifugation. Both eluate and cotton swab were then processed using ZyGEM direct lysis and quantified. Quantification results of the eluate and swabs incubated in trehalose solution were not significantly different from controls. However, it is apparent that a large portion of deposited DNA remained on the swabs even after elution and ZyGEM direct lysis. The second procedure tested trehalose against BSA and SDS as treatments to cotton swabs before DNA collection. A pre-treated group (solution was applied to the swab and dried overnight; DNA was deposited to the dried swab) and a moist group (solution was applied and DNA deposited immediately) were tested after deposition of a set volume of saliva cell suspension. Quantification and amplification results of SDS treated samples indicated significant differences of DNA recovery and average peak height of profiles compared to water and buffer controls. Trehalose samples did have some significant improvement in DNA yield; however, the addition of trehalose as a moistening agent for cotton swabs does not prove to be of forensic value.