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Validating Touch DNA collection techniques using cotton swabs
Salem K Alketbi a,b and W. Goodwin a
aUniversity of Central Lancashire
bGeneral Department of Forensic Science and Criminology in Dubai Police
05-07 MARCH 2024
DUBAI, UNITED ARAB EMIRATES
Touch or trace DNA analysis has become a fundamental aspect of forensic laboratory work and an indispensable tool for
investigators since its initial report in 1997 [1]. This technique has revolutionized DNA collection, allowing for the retrieval
of genetic material from a wide array of surfaces, including tools, knives, clothing, and firearms [2-4]. Typically, trace
samples like touch DNA are collected using cotton swabs, which are moistened and applied with pressure and rotation to
the target area for DNA collection. However, this method may not always yield optimal results, as a moist cotton swab
may only capture a portion of the available sample, leaving biological material behind [2]. Selecting the appropriate
collection technique can enhance trace DNA recovery, such as using the correct amount of reagent to moisten the swab
or employing a double swab technique (wet and dry) [5-6]. Post-collection, some cotton swabs are immediately extracted
or frozen while still moist. However, certain laboratories opt to air dry the swabs at room temperature or use swab drying
cabinets before extraction or freezing. However, these practices may not be suitable for collecting touch DNA, as allowing
the swab to dry before extraction often results in DNA loss [3,7]. Therefore, this paper presents two experiments. The first
experiment aims to validate three commonly used recovery techniques with cotton swabs: the single swab technique (half
wet and half dry), the single swab technique with the use of a plastic spray bottle to moisten the swab (developed in the
Dubai Police forensic DNA lab), and the double swab technique (wet and dry). The second experiment aims to validate
different conditions to explore the impact of immediate extraction, drying, and freezing of touch DNA collected using
moistened cotton swabs.
INTRODUCTION
MATERIALS AND METHODS
- EXPERIMENTAL SET UP AND DEPOSITION: A participant identified as a high shedder was instructed to wash their hands with
antibacterial soap and remain inactive for 5 minutes. Subsequently, the participant applied eccrine sweat from behind their ears to their
fingers to ensure sufficient DNA loading. After an additional 5 minutes, the participant touched a glass surface with their index, middle,
and ring fingers of both hands, exerting medium pressure on a5x7cm area for 1 minute each time. This process was repeated 48
times. Before each use, the surfaces underwent sterilization using 2% virkon and exposure to ultraviolet radiation (UV) for 15 minutes.
-EXPERIMENT ONE:A total of 24 samples were promptly collected using Copan 150C Cotton swabs moistened with distilled water,
a commonly employed agent in forensic labs [5]. Three distinct techniques were employed to moisten the cotton swabs prior to sample
collection, each utilizing 100 μl of distilled water: (a) Half of the cotton swab head was moistened using a pipette (n=8) –(b) The cotton
swab head was moistened using a spray bottle (n=8), with each spray delivering approximately 50 μL of water –(c) The cotton swab
head was moistened using a pipette, followed by a dry swab (double swab technique) (n=8). Subsequently, the swab heads were
promptly extracted using the QIAamp® DNA Investigator Kit (Qiagen) following the manufacturer's instructions, with a final elution
volume of 50 μL.
-DNA QUANTIFICATION AND ANALYSIS: The extracted samples underwent quantification using the Quantifiler® Human DNA
Quantification Kit, QuantStudio 5 Real-Time PCR (qPCR), and HID Real-Time PCR analysis software v1.3 (Thermo Fisher Scientific),
following the prescribed protocols from the manufacturers. Statistical analysis was conducted using RStudio, employing factorial
analysis of variance (ANOVA). Notably, the blank samples obtained from sterilized surfaces, as well as the negative controls for both
the collection and extraction methods, all tested negative for DNA when subjected to quantification. REFERENCES
ACKNOWLEDGEMENTS: This study was approved by the General Department of Forensic Science and Criminology in Dubai Police
and Ethical approval was granted by the School of Forensic and Applied Sciences, and the University of Central Lancashire’s Research
Ethics Committee (ref. no. STEMH 912). Many thanks to COPAN DIAGNOSTICS INC. for supporting this experiment with free swabs,
and to ThermoFisher Scientific™ for discounts on their products.
1- Van Oorschot, R.A.H. and Jones, M.K. (1997) 'DNA fingerprints from fingerprints', Nature, 387, p. 767.
2- Alketbi, S.K. (2018) 'The affecting factors of Touch DNA', Journal of Forensic Research, 9, p. 424.
3- Alketbi, S.K. (2023) 'Analysis of Touch DNA', Doctoral thesis, University of Central Lancashire. Available at: https://clok.uclan.ac.uk/46154/
4- Polley, D. et al. (2006) 'Investigation of DNA recovery from firearms and cartridge cases', J Canadian Soc Forensic Sci, 39,pp.217-228.
5- Alketbi, S.K. and Goodwin, W. (2019) 'The effect of surface type, collection, and extraction methods on Touch DNA', Forensic Science International. Genetics Supplement Series, 7(1), pp.704-706.
6- Pang, B.C.M. and Cheung, B.K.K. (2007) 'Double swab technique for collecting touched evidence', Legal Med, 9, pp.181-184.
7- Van Oorschot, R.A.H. et al. (2003) 'Improving collection methods can improve the ability to obtain typings from trace amounts of DNA from touched objects', The XIX International Congress of
Genetics, Melbourne, Proceedings.
RESULTS
DISCUSSION AND CONCLUSION
The utilization of cotton swabs for trace DNA collection is widespread, albeit associated with DNA wastage due to retention within the swab, influenced by the extraction
method employed [3,7]. Therefore, adopting an appropriate collection technique is imperative to enhance DNA recovery efficiency from the cotton swab. The double swab
technique, involving both wet and dry swabs, has demonstrated superior performance compared to the single swab technique, albeit its efficacy may vary depending on the
size of the sampled area. Nonetheless, it's important to note that DNA extraction may pose challenges with the double swab technique [3]. Moreover, the plastic spray bottle
method surpasses the pipette in moistening the swab by evenly distributing distilled water, minimizing the risk of contamination compared to the use of a pipette. While drying
cotton swabs before freezing may prove beneficial for long-term storage of certain biological materials like body fluids, this approach is not conducive to Touch DNA
samples. Allowing swabs to dry before DNA extraction results in diminished DNA recovery compared to immediate use of moist swabs [2]. Freezing the swab while still moist
after collection, rather than drying it before extraction, yields DNA recovery rates comparable to immediate extraction. Therefore, it is advisable to promptly freeze cotton
swabs after Touch DNA collection to optimize DNA recovery.
In Experiment One, a notable distinction was observed among the three recovery techniques employed for retrieving touch DNA with a cotton swab (p< 0.001). The
application of the spray bottle technique (b) or the implementation of the double swab technique (c) demonstrated greater efficacy in touch DNA collection. Conversely, the
single swab technique utilizing a pipette to moisten the cotton swab (a) exhibited limited effectiveness, resulting in trace DNA being left uncollected on the surface (mean: a –
0.05, b –0.09, c –0.07,all in ng/μL) (Figure 1). In Experiment Two, a similar trend was observed, with a significant disparity noted in the quantity of DNA collected among
the tested conditions prior to extraction (p< 0.001). Each of the three conditions exhibited discernible differences. Immediate extraction (a) or direct freezing of cotton swabs
post-collection (b) proved more conducive to preserving the collected touch DNA, while drying the samples before freezing (c) was associated with potential loss of some of
the trace DNA collected (means: a –0.09, b –0.08, c –0.05,all in ng/μL) (Figure 2).
Figure 1 illustrates the outcomes of Experiment One, showcasing the average
DNA recovered (n=24) through each technique: (a) single swab, (b) spray
bottle, and (c) double swab. The statistical analysis revealed a significant
difference (p< 0.001) among the techniques, with mean DNA quantities
recorded as follows: (a) 0.05 ng/μL, (b) 0.09 ng/μL, and (c) 0.07 ng/μL.
Figure 2 presents the findings of Experiment Two, displaying the average DNA
recovered (n=24) under each condition: (a) Immediate extraction of swabs, (b)
Swabs were only frozen before extraction, and (c) Swabs were dried and
frozen before extraction. Statistical analysis indicated a significant difference (p
< 0.001) among the conditions, with mean DNA quantities recorded as follows:
(a) 0.09 ng/μL, (b) 0.08 ng/μL, and (c) 0.05 ng/μL.
-EXPERIMENT TWO: A total of 24 samples were promptly collected using Copan 150C Cotton swabs, and a plastic spray bottle
technique, pioneered by the Dubai Police forensic DNA lab, was employed to moisten the swabs with 100 μl of distilled water.
Subsequently, three different conditions were applied to the collected swabs: (a) Immediate extraction after collection (n=8) - (b)
Freezing at -20°C for one week (n=8) - (c) Drying for 24 hours at room temperature followed by freezing at -20°C for six days (n=8).
The swab heads were then extracted using the QIAamp® DNA Investigator Kit (Qiagen) in accordance with the manufacturer's
instructions, with the final extracted sample elution volume being 50 μL.
