Collection techniques of touch DNA deposited on human skin following a strangulation scenario

Trace DNA is a significant type of evidence for its ability to be collected from touched items or surfaces at crime scenes to link suspects to their crimes. In cases of violent crimes like assault, sexual offences, or even homicide, often touch DNA is collected from the victim’s skin. However, the collection of touch DNA from the victim’s skin can be complex because of the mixture of DNA present, as there is likely to be a small quantity of the offender’s DNA compared to the victim’s DNA. Validating different collection methods or techniques can improve touch DNA sampling; therefore, this study investigated three collection techniques involving cotton and nylon swabs to test their efficiency for the collection of touch DNA from the human neck. There was a significant difference between the three recovery techniques used to recover touch DNA with a cotton swab (CS) (p < 0.05) and nylon swab (NS) (p < 0.05), with more alleles observed when the neck skin was moistened with 100 μL of distilled water using a spray bottle before collection with both swabs.


Introduction
Touch or trace DNA is a signi cant type of evidence for its ability to be collected from any touched items or surfaces to link the suspects to their crimes. Commonly, it is collected from tools, weapons, clothes, or even human skin but is more challenging compared to other types of biological evidence because of the various factors that can in uence the amount of collected Touch DNA [1][2][3][4][5][6], such as surface type and collection method [7]. In cases of violent crimes like assault, sexual offences, or even homicide, often Touch DNA is collected from the victim's skin, however, there is a lack of research investigating Touch DNA collection methods from human skin [8][9]. In manual strangulation, there is intense physical contact between the offender and the victim, thus the offender's epithelial cells are deposited on the victim's neck but the collection of Touch DNA from the victim's skin can be complex because of the mixture of DNA present containing a small quantity of the offender's DNA compared to the victim's DNA.
Validating different collection methods or techniques can improve Touch DNA sampling [10][11][12][13][14][15][16], therefore, this study investigated three collection techniques involving cotton and nylon swabs to test their e ciency for the collection of Touch DNA from the human neck.

Experimental setup and deposition
Two male donors previously identi ed as high and low shedders were asked to wash their hands with antibacterial soap and refrain from any activity related to using their hands for 10 min. The neck skin of the receiver participants (male and female) was disinfected using alcohol wipes (70% isopropyl alcohol), then cleaned with distilled water and air-dried for 10 min. A donor was asked to hold the neck of the rst receiver (male vs. female) as described in Fig. 1. Previously, measurements were taken from three males and one female participating in the strangulation scenario of this experiment (Fig. 2) for accurate sampling. After deposition, the neck was marked in three equal sections (11 cm x 10 cm) using a temporary marker pen for the recovery of touch DNA via three collection techniques. The DNA deposition was repeated for the other two participants (male vs. male). Furthermore, three recovery techniques were used to collect the randomly deposited DNA form the marked three sections (11 cm x 10 cm) to avoid using the same recovery technique for the same area. This was done to have a more effective sampling average for each technique used, as DNA amounts may shed differently from the hand of the donor during the physical contact in the strangulation process. Importantly, the deposition and collection processes were conducted at room temperature to avoid any environmental factors related to low or high temperature that can in uence the skin such as sweating.

DNA recovery and extraction
After each deposition, Touch DNA was recovered immediately with a Copan cotton swab (150C) (CS) and Copan nylon ocked swab (4N6 FLOQSwabs®) (NS) using three collection techniques as described in Real-Time PCR (qPCR) and HID Real-Time PCR analysis software v1.3 (Thermo Fisher Scienti c) according to the manufacturer's instructions. Next, DNA ampli cation was performed using a GlobalFiler™ PCR ampli cation Kit on an ABI GeneAmp® 9700 PCR System (Life Technologies) for 30 cycles following the manufacturer's protocol. Finally, ampli ed products were size-separated and detected on an ABI 3500 Genetic Analyzer (Life Technologies) using 1 µl PCR product, 9.6 µl Hi-Di™ formamide, and 0.4 µl GeneScan™ 600 LIZ® Size Standard v2.0 (Thermo Fisher Scienti c). Factorial analysis of variance (ANOVA) was performed in RStudio and Microsoft Excel. The negative controls for the collection and extraction methods were DNA free when quanti ed and ampli ed. Control samples from the hands of the donors and the neck of the receivers were recovered after each sterilisation, which generated full single DNA pro les related to the participants without any sign of mixtures or contamination.

Results
There was a signi cant difference between the three recovery techniques used to collect Touch DNA with a cotton swab (CS) (p < 0.05) and a nylon swab (NS) (p < 0.05). For the CS, moistening the neck before collection (c) recovered more DNA than moistening the swab rst (a) or using a dry swab (b) (mean a -0.25, b -0.37, c -0.59 all in ng/µL; Fig. 3). By contrast, for the NS, using a dry swab (b) recovered more DNA than moistening the swab rst (a) or moistening the neck before collection (c) (mean a -0.62, b -1.02, c -0.54 all in ng/µL; Fig. 3), which is related to the neck skin being naturally moist and a nylon swab is more sensitive to moist surfaces than a cotton swab [6]. Nylon swabs (NS) recovered more DNA from the neck skin than cotton swabs (CS) and that could relate to the nature of the nylon bres. The NS is much rougher on the skin compared to the CS, therefore may collect more cellular material from the victim's skin. The Touch DNA collected from the victim's skin usually contains a mixture of pro les comprising alleles from the victim's DNA and the perpetrator's DNA, so the amount of DNA does not necessarily lead to more alleles being recovered. All the collected samples produced mixture pro les, but the number of alleles observed was not consistent (p < 0.05). The number of alleles observed was much more consistent when neck skin was moistened before collection (c) for both swabs (CS alleles recovery a-81%, b-87% and c-94% vs. NS alleles recovery a-87%, b-88% and c-96%; Fig. 4).

Discussion And Conclusion
Recovering trace DNA deposited on human skin is more challenging than collecting from touched items, because Touch DNA deposited by a human on a skin often produces a mixed DNA pro le. Therefore, collection methods can affect the quality of the DNA pro le collected. Based on a study by Kallupurackal et al.,[8], cotton and nylon swabs are equally effective on collecting Touch DNA from human skin, while SceneSafe Fast™ minitapes appear to be the least effective method for the scenario of Touch DNA collection from the skin of the victim. However, using the appropriate technique with cotton or nylon swabs can enhance their performance on collecting Touch DNA from human skin, or touched items [10][11].
In conclusion, Moistening the neck with 100 µl of distilled water using a spray bottle before sample collection with a dry cotton or nylon swab increased the alleles recovery rate from neck skin in the strangulation scenario in this study. It is worth noting that the quantity of solution used can affect the amount of DNA collected, hence it is recommended not to exceed 100 µL which is the amount commonly used to wet cotton swabs.

Declarations Con ict of interest
None. Figure 1 DNA deposition to mimic a strangulation scenario and collection of Touch DNA from neck skin using three collection techniques (A, B and C) with a cotton swab (CS) and a nylon swab (NS).  The mean DNA recovered (n= 48) from neck skin by the three techniques using a cotton swab (CS) and nylon swab (NS): (a) moistening the swab with 100 μl of distilled water using a spray bottle for CS and moistening the swab with 30 μl of distilled water using pipette for NS, (b) dry swab and (c) moistening the neck with 100 μl of distilled water using spray bottle before collection using dry swabs.

Figure 4
Number of alleles observed (n= 48) for each technique using a cotton swab (CS) and nylon swab (NS): (a) moistening the swab with 100 μl of distilled water using a spray bottle for CS and moistening the swab with 30 μl of distilled water using pipette for NS, (b) dry swab and (c) moistening the neck with 100 μl of distilled water using spray bottle before collection using dry swabs. Homozygous loci were counted as one allele, while heterozygous loci were counted as two alleles.