Extraction Efficiency of DNA from Various Swab Types in Contrived Sexual Assault Samples
Sylvia Hamilton, Pamela Marshall (firstname.lastname@example.org) Forensic Science and Law Program, Duquesne University, Pittsburgh, PA 15282, USA
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Sexual Assault Kits (SAKs) are used in thorough examinations to collect biological evidence deposited from a perpetrator onto a victim (1). Bodily fluids are sources of DNA that, in high enough quantity, can generate a genetic profile to serve as evidence in a sexual assault investigation. The SAK collection process includes vaginal swabs that may collect residual seminal fluid. Many variables including the vaginal flora, pH and the time interval between incident to sample collection may impact the quality and quantity of DNA recovered from these evidentiary swabs (2). This can often lead to disproportionate ratios of male to female DNA, as female epithelial cells are shed onto the swab in large quantities during collection (3). Due to the low quantity of the perpetrator’s DNA, it is imperative that the separation of male and female fractions is effective. Currently, SAKs utilize the cotton swab as the primary collection device of biological fluid from a victim (4), but the entangled matrix of the cotton fibers often traps biological evidence which reduces the ability to recover DNA (5). This study tested a flocked nylon swab and a plastic cytobrush against the ubiquitously used cotton swab. The flocked nylon swab has perpendicular fibers that allow for rapid release of DNA and the plastic cytobrush lacks DNA ensnaring fibers. It was predicted that the flocked nylon swab and cytobrush would yield higher quantities of male DNA compared to the cotton swab, thereby increasing the likelihood of developing full DNA profiles from the perpetrator and the likelihood of convictions in sexual assault crimes. Furthermore, a recent study analyzed the cost-benefit analysis of the COPAN flocked nylon swab and describes several benefits of implementing these swabs into laboratory practices (6).
Figure 1. Collection Devices. Three types of collection devices were used in this study which include (A) Cotton Swab, (B) Flocked Nylon Swab, and (C) Cytobrush. Next to each device is a 10x magnification image showing the device matrix.
Materials and Methods
The DNA IQ™ System (Promega) was utilized according to the manufacturer’s recommendations on samples of neat seminal (BioIVT) and vaginal fluids (Lee BioSolutions) from single donors (7). The Quantifiler™ HP DNA Quantification Kit (Applied Biosystems) was then used to prepare the samples for qPCR on the QuantStudioTM 5 Real-Time PCR (Applied Biosystems) (8). The initial DNA quantification values were recorded and used to prepare dilutions.
The vaginal fluid (VF) was diluted by combining 173µl of VF and 627µl of PBS for a final concentration of 0.1ng/µl. The seminal fluid (SF) was diluted by combining 3µl of semen in 897µl of PBS in order to create a male to female ratio of 1:5. The contrived SA swabs were prepared utilizing a flocked nylon swab (COPAN), a cytobrush and a cotton swab in replicates of 5. Each swab was prepared by adding 50µl of VF, drying for 24 hours, adding 50µl SF and allowing to dry for another 24 hours.
The Differex™ System (Promega) was then used in place of a differential lysis to separate the male and female fractions according to the manufacturer’s recommendations (9). Quantifiler™ DUO Quantification was run according to standard protocol on the QuantStudioTM Real-Time PCR (Applied Biosystems) (10). Eight standards were run in duplicate to make a standard curve with an R2 value of 0.990.
The GlobalFiler™ PCR Amplification Kit was utilized to perform PCR amplification on the Veriti 96 Well Thermal Cycler (Applied Biosystems) according to Table 1 (11). Samples were then run on the SeqStudioTM Genetic Analyzer (Applied Biosystems) and analyzed using the GeneMarker® HID software.
Table 1. PCR Parameters.
Figure 2. Total human DNA quantity in the male fractions. Five replicates of cotton, flocked nylon and cytobrush swabs were processed using the Differex™ and DNA IQ™ systems (Promega). Total human DNA was then quantified by the Quantifiler™ DUO and QuantStudio Real-Time PCR (Applied Biosystems).
Figure 3. Male DNA quantity. Five replicates of cotton, flocked nylon and cytobrush swabs were processed using the Differex™ and DNA IQ™ systems (Promega). Male human DNA was then quantified by the Quantifiler™ DUO and QuantStudio Real-Time PCR (Applied Biosystems).
Discussion and Conclusion
This experiment was successful in the recovery of male DNA from three different contrived sexual assault swab types. The flocked nylon swabs recovered the highest male DNA quantity from the extracted male fraction with an average of 0.155± 0.11ng/µl. Cotton recovered the second highest male DNA quantity at 0.075±0.055ng/µl. The cytobrush recovered the lowest quantity with an average of 0.018±0.006ng/µl (Figure 3).
While the flocked nylon swab recovered the highest male quantity, it also had the highest standard deviation. This high variation may be due to replicate 3, in which the spin basket broke during centrifugation, causing a loss in sample within the swab tip. This error may be remediated by re-spinning the swab tip to recover the remaining solution.
The low quantity of DNA recovered from the cytobrush may be due to the brush’s inability to absorb all the fluids during the contrived sample preparation.
Due to the low initial concentrations, not all fractions were carried through to genetic analysis. The male fractions with the highest male DNA recovered from each swab type and their female fraction counterparts were amplified and analyzed. The flocked nylon swab female fraction generated the best genetic profile in which some loci presented four alleles, suggesting that both male and female DNA was present (results not shown).
An unfortunate limitation to this study was the small sample size. This limitation was due to the limited available source material. While these results are promising in suggesting that a flocked nylon swab type may retrieve higher quantities of DNA, the small sample size is a restriction on providing solid validation for this hypothesis.
The results of this study show that collection device could be a factor in DNA extraction efficiency in SAK samples. The research project will be continued by replacing the source material of purchased vaginal fluid to vaginal swabs collected from volunteer participants following Institutional Review Board (IRB) approval. The sample size will also be increased to improve statistical evaluation of the results. The ratios of male:female DNA examined will also be varied and tested at 1:1, 1:10 and 1:100. An alternative route is to also use real post-coital swabs rather than contrived SA samples.
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