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Received: 29 July 2021    Revised: 18 October 2021    Accepted: 20 October 2021
DOI: 10.1111/1556-4029.14932
TECHNICAL NOTE
Criminalistics
Touch DNA recovery from vehicle surfaces using different
swabs
Alexandre Giovanelli PhD  | Rodrigo Grazinoli Garrido PhD | Alípio Rocha MSc |
Tatiana Hessab PhD
Departamento de Polícia Técnico-­
Científica do Rio de Janeiro, Instituto de
Pesquisa e Perícia em Genética Forense,
Rio de Janeiro, Brazil
Correspondence
Alexandre Giovanelli, Instituto de
Pesquisa e Perícia em Genética Forense,
Rua Marquês de Pombal 150, Centro RJ,
Rio de Janeiro, Brazil, 20230-­240.
Email: agiovanelli@gmail.com
Funding information
This study was supported by FAPERJ
(Fundação de Amparo à Pesquisa do
Estado do Rio de Janeiro –­Processo E-­
26/290.054/2018).
J Forensic Sci. 2021;00:1–5. wileyonlinelibrary.com/journal/jfo
Abstract
Several methods of DNA collection are used in places or objects related to crimes,
the most common being the use of swabs. However, it is known that the efficacy of
touch DNA recovery can be affected by collection devices and surfaces. The aim of
this work was to evaluate the efficiency of three different types of swab in recover-
ing touch DNA collected from different parts of a vehicle. The following swabs were
tested: PurFlock® swab (Puritan, USA), 4N6FLOQSwabs™ (Copan S.p.A., Italy), and
cotton swab (Labor Import). The experiments were carried out in the same vehicle,
using the gearshift knob, the parking brake lever, and the steering wheel as support
for the collection of touch DNA. Swabs showed significant differences in the amount
of DNA recovered (Hc = 53.52; p < 0.05) and in the rate of allele amplification (Hc =
24.3; p < 0.05). The results indicated a greater DNA recovery efficiency by PurFlock®
swab, followed by cotton, and then 4N6FLOQSwabs™. However, there was no signifi-
cant difference among the surfaces analyzed. PurFlock® swab was more efficient for
recovering donor alleles than the others (cotton and 4N6FLOQSwabs™), especially
for small DNA amounts. This swab was, therefore, suitable for collections in vehicles
involved in crime. Furthermore, this study highlights the need to assess different ma-
terials and methods of collection of biological samples, considering collection, extrac-
tion, and amplification.
KEYWORDS
4N6FLOQSwabs™, cotton swab, PurFlock® swab, touch DNA, trace DNA, vehicle surface
Highlights
• Three types of swabs were tested for the collection of touch DNA in vehicles.
• The type of swab used in touch DNA samples was more important than the type of substrate
for the rate of allele recovery.
• PurFlock® swabs (Puritan, USA) were able to retrieve complete STR profiles from vehicle
surfaces.
• Gearshift knobs and steering wheel are good surfaces for contact DNA collection.
© 2021 American Academy of Forensic Sciences     1
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2      GIOVANELLI et al.
1  |  I NTRO D U C TI O N
In recent years, techniques associated with genetic material found
at crime scenes have advanced a lot [1]. In 1997, there was the first
report on obtaining full genetic profiles from objects handled by sus-
pects [2]. Since then, several types of objects have been investigated
for DNA collection, including documents, ammunition, tools, cloth-
ing, gloves, glasses, watches, vehicles, doors, among others [1, 3–­5].
This form of trace evidence has been called touch DNA, contact
DNA, or trace DNA. Such sample types often have DNA concentra-
tions well below the threshold for genetic profile detection and/or
interpretation [6]. For this reason, at any stage of sample processing,
loss of DNA fractions or even potential contamination can preclude
the pursuit of a satisfactory genetic profile [6, 7]. A key factor for
success in obtaining material from touch DNA is the method used for
DNA recovery. Several DNA collection techniques are used in crime-­
related places or objects. The most common are swabs of various
types, adhesives, scraping, and tissue cutting [8–­10].
Of these, the first is the most common in several forensic inves-
tigation agencies in Brazil and worldwide, which have employed a
technique known as double swab [11]. Notably, touch DNA recov-
ery efficacy can be influenced by the type of swab used [12, 13].
Overall, synthetic fiber swabs (rayon, nylon, and polyester) are more
efficient in absorbing solutions and releasing cells attached to sur-
faces. However, a higher absorption is not inherently linked to higher
DNA recovery rates [14]. Substrates from which samples are taken
can also have a direct impact on the efficiency of certain swabs [15].
In Brazil, there is a growing demand for touch DNA collected
from vehicles involved in robberies and murders [16]. Since efficient
methods for collecting touch DNA from vehicles must be established
to support the police investigation, our objective was to evaluate the
efficacy of three swab types for touch DNA recovery.
2  |  M ATE R I A L A N D M E TH O DS
2.1  |  Sample preparation and DNA collection
This study compared three swab types: PurFlock® (Puritan, USA),
consisting of polyester fibers; 4N6FLOQSwabs™ (Copan S.p.A.,
Italy), consisting of flocked nylon fibers, and cotton swab (Labor
Import). The experiments were carried out in the same vehicle, using
the gearshift knob, the parking brake lever, and the steering wheel
as a substrate to collect contact DNA. The steering wheel was made
of rigid plastic with no cover.
Four male donors were examined. The donors signed a consent
form. DNA reference profiles of all participants were generated
from FTA card (Whatman®) blood samples to compare with those
obtained in the experiments. Five minutes before the experiments,
participants were asked to wash their hands, without soap, to remove
excess cellular and cell-­free DNA. For the donor to touch the collec-
tion surfaces, a sequence of moves was set to be made with the car
turned off. These comprised fully turning the steering wheel once
to the left and once to the right, sequential shifting to six gears and
reverse, and release followed by activation of parking brake. After
each movement sequence, contact DNA collection was immediately
performed on each of the touched surfaces. This was repeated three
times for each donor. From one donor to another, the collection sur-
faces (steering wheel, gearshift knob, and parking brake lever) were
sterilized with gauze soaked in 1% sodium hypochlorite, followed by
70% alcohol. After sterilization, one swab sample was collected to
control persistent alleles. Twelve samples were taken from each col-
lection surface and swab type, totaling 36 per swab and 108 in total.
The samples were collected by the same person.
PurFlock® swabs (Puritan, USA), 4N6FLOQSwabs™ (Copan
S.p.A., Italy) and cotton swab (Labor Import) were moistened with
30 μl TE solution before sampling. For each sample, a single swab
was used. Swab heads were broken off into safe-­lock tubes for or-
ganic extraction.
2.2  |  DNA analysis
Samples were incubated in lysis buffer [10  mM Tris-­HCl; 100  mM
NaCl; 10 mM EDTA (pH 8.0); SDS 2%] plus 24 μg proteinase K and
48 μmol DTT for 2 h at 56 °C. Organic extraction was performed with
phenol-­chloroform-­isoamyl alcohol (25: 24: 1, v/v/v). Precipitation
was performed by adding 10% of the recovered volume of NaCl
(5 M) and 2x Ethanol AS, with incubation at −20°C for 2 h. Finally,
the DNA was resuspended in TE−4 buffer [10 mM Tris-­HCl; 0.1mM
EDTA (pH8.0)] and kept at 4°C until the time of PCR assay.
Real-­time qPCR analysis was performed using the Investigator
Quantiplex Human™ kit (QIAgen) and 7500 Real-­Time PCR system
(Applied Biosystems). To this end, we followed the manufacturer's
protocol except for half-­reaction volumes being used.
The DNA samples were amplified by the PCR (Polymerase Chain
Reaction) method using the PowerPlex® Fusion 6C System for
human identification (Promega Corporation, USA), according to the
manufacturer's protocol.
PCR-­amplified products were subjected to capillary electropho-
resis on an ABI 3500 Genetic Analyser for Human Identification
(Thermo Fisher Scientific) and further analyzed by the GeneMapperTM
ID-­X software (v 4.0), according to the manufacturer's protocol. The
analytical threshold was set to 150 RFU. An individual peak was con-
sidered to be homozygous if the peak height was at least three times
greater than the next highest allele. In the case of mixtures, all alleles
with a peak size greater than half the size of the largest allele were
included, up to a maximum of four alleles.
2.3  |  Statistical analysis
First of all, the number of alleles in the reference profiles of each
donor was counted. Then, the same process was applied to the sam-
ples collected during the experiment. Finally, the ratio of recovered
alleles in relation to total alleles for each donor was presented as
GIOVANELLI et al. |
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TA B L E 1  Mean and standard deviation of the amount of DNA

percentage. The total amount of DNA recovered was determined for
recovered for each experimental treatment (type of swab and
each treatment. Mean and standard deviation (SD) were calculated collection surface)
for each evaluation, respectively.
Mean Standard
A Shapiro-­Wilk test was performed to assess the normality of
Type of swab Collection surfaces (ng/µl) deviation
DNA yields from each touch DNA collection procedure. It was fol-
PurFlock® swab gearshift knob 0.38 0.55
lowed by a Kruskal–­Wallis test to assess the impacts of surface types
and swab materials on the resulting profile. Post-­hoc testing was also steering wheel 1.55 2.93

conducted with a Bonferroni adjustment to the significance level to parking brake lever 0.56 0.72

assess where these differences originated. Cotton swab gearshift knob 0.04 0.04
Spearman's correlation test was performed to examine associa- steering wheel 0.04 0.05
tions between allele retrieval rate and DNA amount for each swab parking brake lever 0.03 0.05
type. Statistical analyses were performed using the PAST software 4N6FLOQSwabs™ gearshift knob 0.01 0.02
v. 4.0.3 (Hammer et. al., 2001) [17]. steering wheel 0.02 0.02
parking brake lever 0.01 0.02

3  |   R E S U LT S

Control DNA amplification collected after switching donors and

surface sterilization resulted exclusively in amounts lower than
0.08 ng/µl. About 97% of the samples collected with the PurFlock®
swab resulted in one or more alleles. These values were lower for
cotton swab (72%) and 4N6FLOQSwabs™ (69%). The cleaning pro-
cess performed before collections was unable to fully eliminate
material left over from the previous donor. Eight samples collected
with PurFlock® swab showed mixture profiles, consisting of alleles
from the previous donor and the experimental donor. One mixture
profile was obtained with a cotton swab and three mixture profiles
with 4N6FLOQSwabs™. However, in most mixture samples, previ-
ous donor alleles had low signal intensity, on average, lower than
400 RFU. F I G U R E 1  Box-­and-­whisker plots showing the relative
The amount of DNA recovered and the allele amplification distribution of allele recovery rates (number of donor alleles
retrieved in the sample in relation to the total reference profile
rate showed non-­normal distribution for all treatments (cotton,
alleles of that donor) for each treatment, wherein: PF—­PurFlock®,
PurFlock®, and 4N6FLOQSwabs™), according to the Shapiro-­Wilk CT—­cotton, 4N6—­4N6FLOQSwabs™, Gear—­gearshift knob,
test. In all cases, the W-­value was lower than the p-­value at a 5% Wheel—­steering wheel, and parking brake—­parking brake lever.
significance level. Error bars indicate the standard error of each treatment. Each box-­
The amount of DNA recovered from all samples was below and-­whisker plot shows percentiles between the 25th and 75th of
the observed profile proportions, whereas the middle line denotes
0.5 ng/µl, except in 11 of the 36  samples of PurFlock® swab
the median value. The range between the lowest and highest
(about 31%). PurFlock® swab had the highest mean amounts of
values is shown as a dotted line
DNA recovered for all surfaces compared with cotton swab and
4N6FLOQSwabs™ (Table 1).
The Kruskal–­Wallis test showed significant differences between
DNA recovered amounts in the different swabs (Hc = 53.52; p < showed significant differences for all treatment-­comparison pairs.
0.05). Bonferroni's post hoc test also showed significant differences Figure 1 shows the allele recovery rate (number of donor alleles in
between pairs of groups. The results indicated a greater DNA re- relation to the total number of alleles in a sample) for each treat-
covery efficiency by PurFlock® swab, followed by cotton, and then ment evaluated, considering swabs and surfaces. PurFlock® was
4N6FLOQSwabs™. However, there was no significant difference more effective than the others on all surfaces, followed by cotton
among the surfaces analyzed (Hc = 1.228; p > 0.05). Therefore, and 4N6FLOQSwabs™. Although samples collected from gearshift
the place of collection (gearshift knob, steering wheel, and parking knobs and steering wheels had higher allele retrieval rates, differ-
brake) had less influence than did swab type. ences were not significant (Hc = 0.808; p > 0.668).
Regarding allele amplification, even at a small recovered DNA Spearman's correlation was analyzed between the amount of
amount, several samples showed results. The Kruskal–­Wallis test DNA recovered from each swab and their respective allele amplifica-
showed a significant difference in amplification between the tion efficiency. There was a significant positive correlation between
swab types (Hc = 24.3; p = 5.3 × 10−6). Bonferroni's post hoc test percentages of amplified alleles and DNA amounts recovered from
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4      GIOVANELLI et al.
cotton swab (Spearman rs = 0.886; p = 7.1 X 10−13) and PurFlock®
swab (Spearman rs = 0.765; p = 8.57 × 10−8). Conversely, there was
no significant correlation between DNA amounts recovered from
4N6FLOQSwabs™ and percentages amplified alleles (Spearman rs =
−0.0642; p = 0.71).
There was a visible difference in the success of allele recovery
among the four male donors. Figure 2  shows the total number of
samples (nine for each treatment) wherein 50% or more of the donor
alleles were recovered. Some participants had consistently high or
low allele deposition. Donors 4 and 3 (D4 and D3) had the best
deposition rates, while donor 1 (D1) had the lowest rates, regardless
of the swab used.
On the other hand, PurFlock® swab had the best allele recovery
rates for all donors while 4N6FLOQSwabs™ had the worst recovery
rates per donor. Cotton swab worked well for some types of donors
(D4 and D3), but performed poorly for D2 and D1 donors.
4  |  D I S C U S S I O N
Our findings point to greater efficiency of the PurFlock® swab, both
for the amount of DNA recovered and amplified alleles, regardless
of the surface. It is worth noting that all surfaces were composed
of the same material (rigid plastic); however, the steering wheel dif-
fered by being larger in size and having small grooves. Of all swabs,
4N6FLOQSwabs™ had the worst performance.
Both collection devices [8, 12, 18] and surfaces [1, 10, 13, 18]
influence the success rate of DNA recovery. For example, one study
showed that polyester and foam swabs were more efficient in recov-
ering alleles on hard plastic (screwdriver handle) and wooden sur-
faces, while cotton swabs were more efficient on smooth surfaces
(aluminum can and glassware) [12]. On the other hand, some stud-
ies have shown greater efficiency of 4N6FLOQSwabs™, including
steering wheels [19], when compared with other collection devices.
However, swabs from the same material and the same manufacturer
F I G U R E 2  Total number of samples
(9 for each treatment) from which 50%
or more of the target donor alleles were
recovered for each treatment, wherein:
PF—­PurFlock® swab, CT—­cotton swab,
4N6FLOQSwabs™—­4N6 swab, and D1,
D2, D3, and D4 indicate each of the
sample donors
can have totally different efficiencies, such as the 4N6FLOQSwab
developed for oral collection, which showed better DNA recovery
efficiency than the similar 4N6FLOQSwab developed for crime
scene collection. [14]. In turn, cotton swabs (Bode SecurswabTM)
have shown much higher efficiency than nylon-­flocked swabs
(Copan 4N6) [15].
As extraction methods highly influence DNA recoverability
[8], collection devices can have their efficacy improved by the ex-
traction technique used [16, 18]. This may be related to either DNA
absorption or retention by each swab type, which varies consider-
ably [19]. In this study, we only used organic extraction, which had its
efficiency proved for low DNA-­content samples in a previous study
[20]. Therefore, further studies should be carried out with different
extraction methods so that the performance of different swabs on
the same surfaces (gearshift knob, steering wheel, and parking brake
lever) could be assessed.
Furthermore, this study showed large differences in terms of
shedders status. This corroborates findings in the literature, which
show that some people leave more genetic material than others
on objects. In this context, individual, gender, and age-­related dif-
ferences of shedders status have been reported [1, 8, 9, 21–­23].
Another study on touch DNA recovery from steering wheels also
showed that primary donor alleles persist longer than secondary
donor ones [24].
5  |  CO N C LU S I O N S
Our results show that even brief contact by a driver with parts of a
vehicle is capable of releasing enough DNA to amplify a complete
STR profile. However, it was observed that the success in DNA re-
covery was influenced by the type of swab used and by the shedders
status. In the experimental conditions tested, the PurFlock® swab
was more efficient for recovering donor alleles than the others (cot-
ton and 4N6FLOQSwabs™), especially for small DNA amounts. This
GIOVANELLI et al.
swab is therefore suitable for collecting contact DNA in vehicles in-
volved in crimes, with the gearshift knob and steering wheel being
the most suitable surfaces for DNA recovery. However, when com-
pared with gearshift knobs, one disadvantage of the steering wheels
is their large surface area to be sampled. This hinders identifying the
area touched by suspects since the way to hold the steering wheel
varies from person to person, which can interfere with DNA recov-
ery rates.
Furthermore, this study highlights the need to assess different
materials and methods of collection of biological samples [15], con-
sidering collection, extraction, and amplification jointly.
ORCID
Alexandre Giovanelli  https://orcid.org/0000-0002-7452-323X
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How to cite this article: Giovanelli A, Grazinoli Garrido R,
Rocha A, Hessab T. Touch DNA recovery from vehicle
surfaces using different swabs. J Forensic Sci. 2021;00:1–­5.
https://doi.org/10.1111/1556-­4029.14932