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Journal of Forensic and Legal Medicine 47 (2017) 9e15

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Journal of Forensic and Legal Medicine

journal homepage: www.elsevier .com/locate/ jflm

Increased recovery of touch DNA evidence using FTA paper compared to conventional collection methods

Irina A. Kirgiz a, Cassandra Calloway a, b, *

a Forensic Science Graduate Program, University of California, Davis, 1909 Galileo Court, Suite B., Davis, CA 95618, USA b Children’s Hospital Oakland Research Institute, Oakland, CA, USA

a r t i c l e i n f o

Article history: Received 17 August 2016 Received in revised form 8 December 2016 Accepted 30 January 2017 Available online 31 January 2017

Keywords: Forensic science DNA typing Double swabbing Water-soluble tape sampling FTA paper scraping Touch DNA

* Corresponding author. Forensic Science Graduate fornia, Davis, 1909 Galileo Court, Suite B., Davis, CA 9

E-mail addresses: i_ilyenko@yahoo.com (I.A. (C. Calloway).

http://dx.doi.org/10.1016/j.jflm.2017.01.007 1752-928X/© 2017 Elsevier Ltd and Faculty of Forens

a b s t r a c t

Tape lifting and FTA paper scraping methods were directly compared to traditional double swabbing for collecting touch DNA from car steering wheels (n ¼ 70 cars). Touch DNA was collected from the left or right side of each steering wheel (randomized) using two sterile cotton swabs, while the other side was sampled using water-soluble tape or FTA paper cards. DNA was extracted and quantified in duplicate using qPCR. Quantifiable amounts of DNA were detected for 100% of the samples (n ¼ 140) collected independent of the method. However, the DNA collection yield was dependent on the collection method. A statistically significant difference in DNA yield was observed between FTA scraping and double swabbing methods (p ¼ 0.0051), with FTA paper collecting a two-fold higher amount. Statistical analysis showed no significant difference in DNA yields between the double swabbing and tape lifting techniques (p ¼ 0.21).

Based on the DNA concentration required for 1 ng input, 47% of the samples collected using FTA paper would be expected to yield a short tandem repeat (STR) profile compared to 30% and 23% using double swabbing or tape, respectively. Further, 55% and 77% of the samples collected using double swabbing or tape, respectively, did not yield a high enough DNA concentration for the 0.5 ng of DNA input recom- mended for conventional STR kits and would be expected to result in a partial or no profile compared to 35% of the samples collected using FTA paper. STR analysis was conducted for a subset of the higher concentrated samples to confirm that the DNA collected from the steering wheel was from the driver. 32 samples were selected with DNA amounts of at least 1 ng total DNA (100 pg/ml when concentrated if required). A mixed STR profile was observed for 26 samples (88%) and the last driver was the major DNA contributor for 29 samples (94%). For one sample, the last driver was the minor DNA contributor. A full STR profile of the last driver was observed for 21 samples (69%) and a partial profile was observed for nine samples (25%); STR analysis failed for two samples collected using tape (6%).

In conclusion, we show that the FTA paper scraping method has the potential to collect higher DNA yields from touch DNA evidence deposited on non-porous surfaces often encountered in criminal cases compared to conventional methods.

© 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

1. Introduction

Each year approximately 800 thousand vehicles are stolen in the United States1 and an estimated 4.9 million vehicles are stolen worldwide.2 Many of the stolen vehicles are used to transport illegal substances or are involved in other types of crimes.1 In many

Program, University of Cali- 5618, USA. Kirgiz), scalloway@chori.org

ic and Legal Medicine. All rights re

cases, touch DNA collected from the steering wheel is the only evidence that can link a perpetrator to the crime (e.g. most recent driver to the carjacking).3

Forensic analysis of touch DNA evidence was first described in 1997 by Van Oorschot and Jones (DNA fingerprints from finger- prints).4 Since then, the topic of touch DNA has become of partic- ular interest to researchers in the field of Forensic Science. Now this type of evidence is frequently used in criminal cases worldwide and the number of cases solved solely by virtue of touch DNA has grown dramatically.5 As of today, a number of research teams have tried to standardize the touch DNA collection protocol.3 Despite their effort,

served.

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there is a need for further improvements, since collection methods optimized for analysis of high copy number DNA evidence are not always effective for low copy DNA specimens.3 Touch DNA often yields only a partial profile that is frequently attributed to a low shedding status of a donor.6 However, low DNA yields can also be explained by ineffective collection methods that leave behind a portion of the deposited DNA.7 Not all techniques that are used for blood, sperm and saliva DNA collection are effective for collecting touch DNA. DNA transferred to an object is usually present in smaller quantities at the crime scene when compared to the bodily fluids DNA.3 Touch DNA is also invisible to the naked eye and often a forensic scientist can only approximate its location on the surface of an object.3

The most widely used method for collecting touch DNA evi- dence from surfaces uses cotton swabs and a double swabbing technique.6,8e10 Tape lifting using water soluble tape is an alter- native method that is used to collect touch DNA evidence.11,12 A scrapingmethod usingWhatman® FTA® cards13 is a novel approach which was used in one case study to collect touch DNA from the surface of a steering wheel.14 Limited research has been published on the comparison of different touch DNA collection methods.7,12,15

The goal of our research was to directly compare double swab- bing to tape lifting and FTA paper scraping methods for collecting touch DNA deposited on steering wheels of vehicles under cir- cumstances that closely resemble a real life situation. The steering wheel is an ideal surface for touch DNA collection as it is relatively smooth and prone to a long contact with the driver’s hands. In order to replicate a real life situation, we performed a blinded study with little or no information about the drivers, their shedding status, hygiene routine, and driving schedule. In order to maximize DNA recovery, DNAwas collected over the entire area of each half of the steering wheel.

2. Materials and methods

2.1. DNA collection

This study compared three different DNA collection techniques of touch DNA from steering wheels: double swabbing, tape lifting and FTA paper scraping. A total of 70 cars were selected to partic- ipate in this study. All of the selected cars were in fully operational conditions; all cars were driven for 2e60 min on the day of the touch DNA collection. The steering wheels of the cars were selected for hard plastic, relative cleanliness and absence of wheel covers.

The study was approved by the Institutional Review Board (IRB) at the University of California, Davis. Car owners were asked to provide a reference cheek swab and answer a ques- tionnaire. 35 cars were selected to compare the double swabbing method to tape lifting method, and another 35 cars were selected for double swabbing vs. FTA paper scraping method comparison. They were analyzed in the following way: one half of a steering wheel was sampled using a double swabbing technique, while the other side of a steering wheel was sampled using either a tape lifting technique (Fig. 1A) or the FTA paper scraping tech- nique (Fig. 1B). We randomly alternated sides in order to mini- mize the possibility that the difference in DNA yield was due to a difference in shedding status between the left and right hands or bias in driving hand.

The study was divided into two parts for a direct comparison to the double swabbing technique: double swabbing vs. tape lifting, and double swabbing vs. FTA paper scraping. Fisherbrand® small 6 inch single headed sterile cotton-tipped swabs (Cat No. 23-400- 115) were used for double swabbing procedure. The standard double swabbing technique8 was used with the following modifi- cation. Both swabs were moistened with three drops of de-ionized

water before the sample collection.16 Due to a relatively large sur- face area of a steering wheel the moisture left by the first swab dried out before we were able to apply the second swab.12 For the FTA paper scraping method, four drops of de-ionized water were applied to a 3.2 cm by 3.9 cm cut portion of a WhatmanWB120205 FTA Classic Card before the sample collection. For tape collection, a 6 cm piece of 3 M(™) Water-Soluble Wave Solder Tape 5414 used to collect DNA from one side of the steering wheel.

2.2. DNA extraction and analysis

Swabs and FTA cards were dried for at least 1 h in a drying box. DNA was eluted from the cotton swabs following the procedure described in “DNA Purification from Buccal Swabs”. The Whatman FTA card was cut into small pieces and placed into two extraction tubes. The DNA was eluted from the FTA paper following manu- facturer’s procedure for “Isolation of Total DNA from FTA and Guthrie Cards” (Qiagen, Hilden, Germany). Water Soluble Tape was placed in a beaker of water heated to 60 �C and agitated for 1 min. Once the DNA was eluted from the collection device, the standard DNA extraction procedure using the QIAGEN® QIAamp DNA Kit was used to extract DNA from all samples collected.17 The extraction procedure was standardized to minimize any variability that may be introduced due to differences in the extraction procedure. Dur- ing the last step of each extraction process 50 ml of nuclease-free water was used to elute DNA.

Real-Time qPCR analysis using the Promega® PlexorHY Human Quantitation kit (Promega, Madison, WI) was performed on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) in- strument following the manufacturer’s procedure to determine the amount of DNA present in each sample.18 Each sample was quan- tified two times to increase the accuracy of the estimated DNA amount; if a CT difference greater than 0.5 was observed between the replicates then the quantitative analysis was repeated.

Thirty-two samples were selected from both studies for Short Tandem Repeats (STR) analysis; eight pairs were selected from the double swabbing vs. tape lifting study and eight pairs were selected from the double swabbing vs. FTA paper scraping study. Samples were selected with DNA amounts of at least 1 ng total DNA. Only three DNA samples contained less than 100 pg/ml; these samples were concentrated using heat to allow 1 ng DNA input. STR analysis using the AmpFLSTR Identifiler™ kit (Applied Biosystems, Foster City, CA) was performed to generate a driver’s profile from the 32 selected steering wheel samples and the 16 corresponding buccal swabs. The manufacturer’s recommended procedures were fol- lowed for amplification using a Gene Amp® PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA) at the recom- mended 28 cycles and analyzed using a ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). GeneMapper®ID software (Applied Biosystems, Foster City, CA) was used for STR data analysis. The analytical threshold was set to the recommended 50 RFUs and the stochastic threshold was set to 150 RFUs.19 Results from the STR analysis were analyzed for mixed profiles and used to assess the completeness of STR profiles. DNA collection methods were compared based on how well they recovered the STR profile of the most recent driver by counting the number of observed al- leles. The goal of the STR analysis was to assess the difference be- tween quantifiable and typable results among the three collection methods.

2.3. Statistical analysis of data

The data were log10 transformed in order to improve the normality of variables. AWilcoxon Signed Rank test was performed in order to assess the significance levels between the DNA yields

Fig. 1. Schematic of touch DNA collection techniques. This schematic illustrates the techniques (A) water-soluble tape lifting or double swabbing and (B) FTA paper scraping or double swabbing that were used to collect touch DNA from the surface of the steering wheels.

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from the touch DNA collection techniques. Statistical analysis was performed using JMP software (SAS Institute, North Carolina).

3. Results

Detectable quantities of touch DNAwere recovered from each of the 70 steering wheels sampled, independent of collection method and randomized for collecting from the right or the left side for a total of 140 samples (Fig. 2A and B, Supplemental Tables 1A and 1B). However, the DNA yield was dependent on the DNA collection method. The mean DNA yield was two-fold higher for the double swabbing method compared to tape lifting (16.1 vs. 7.37 ng). The standard deviation was also higher for the double swabbing method, 27.6 vs. 16.5. However, the median DNA yield was only slightly higher for the double swabbing method than for the tape lifting (2.46 vs. 2.05 ng). DNA yields from 50% of the samples (samples in the interval between the 25% and 75%) collected using the double swabbing technique fell within the range of 0.87 nge26.6 ng; 50% of tape lifting samples fell within the range of 1.22 nge4.88 ng (Fig. 2A). AWilcoxon Signed Rank test was used on the log10 transformed data to determine if the differences in the DNA yield between swabbing versus tape lifting were statistically significant and no significant difference in the yield was observed (p ¼ 0.21) (Fig. 3A).

For the double swabbing versus FTA paper scraping comparison, touch DNA was collected from 35 steering wheels using either method, randomized for collecting from the right or the left side for a total of 70 samples (Fig. 2B). Samples collected from one steering wheel, which yielded 884 ng using the double swabbing method (26 fold higher than the next highest sample) and 114 ng DNA using FTA paper (7 fold higher than the next highest sample), was considered an outlier and was removed from the dataset. The mean DNA yields were similar using the FTA scraping method compared to the double swabbing (5.33 vs. 5.22). However, the median value was two-fold higher for the FTA scraping method (4.78) compared to double swabbing (2.26). DNA yields from 50% of the double swabbing samples (samples in the interval between the 25% and 75%) fell within 0.62 nge4.36 ng range; and 50% of FTA scraping samples fell within 1.89 nge7.89 ng range (Fig. 2B). A statistically significant difference in DNA yield between the FTA scraping and the double swabbing methods was observed using a Wilcoxon Signed Rank test on the log10 transformed data (p ¼ 0.0051) with

the FTA paper scraping method yielding higher amounts of touch DNA (Fig. 3B).

3.1. STR results

Based on the DNA concentration yield, a majority of the samples collected using double swabbing (55%) or tape (60%) would be expected to result in partial or no STR profile using standard STR kits which recommend 0.5 ngs of DNA (�0.05 ng/mL) compared to 35% of the samples collected using FTA paper (Fig. 2). Also, almost 50% of the samples collected using FTA paper (18 out of 34) yielded DNA concentrations high enough to allow for 1 ng DNA input for STR analysis compared to 30% for double swabbing and 23% for tape lifting. A subset of these higher yield samples were selected for STR analysis based on the DNA amount (>1 ng) to confirm that the DNA collected from the steering wheel was from the driver.

For the double swabbing versus tape lifting comparison, the mean % STR allele recovery of the driver was higher for double swabbing (91%) when compared to tape lifting (59%) (Fig. 4A). For the double swabbing versus FTA paper scraping comparison, the mean % STR allele recovery of the DNA was slightly higher for double swabbing (99%) than for FTA paper scraping (91%) (Fig. 4B). A full STR profile was observed for 21 out of the 32 samples (66%); four of the 21 full profiles were from a single source. No profile was generated for two of the 32 samples and both samples were collected using tape. The STR failure of these two samples is not likely due to PCR inhibition since the same tape brand was used for this study as a studywhich showed that the tape did not inhibit PCR even with increased lengths of tape.11

Nine samples (28%) yielded only partial profiles and five of these samples generated searchable partial profiles (containing STR al- leles at � ten CODIS Core Loci). A mixed STR profile containing two or more contributors was observed for 26 samples or 88%. For all cars sampled, the primary driver was the last driver and for 29 samples out of 32, the driver was also the major DNA contributor. For one car sampled, the last driver was not the major contributor (Supplementary Tables 2A and 2B).

4. Discussion

The results of the study show that each of the three DNA collection methods was able to recover detectable quantities of

A.

Swab (N =35)

Tape (N =35)DNA Collection Methods

B.

Swab N =34

FTA N =34 DNA Collection Methods

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Fig. 2. The amount of touch DNA collected. DNA was collected from one side of a steering wheel using (A) the double swabbing or tape lifting method or (B) double swabbing or FTA paper scrapping method and quantified in duplicate using qPCR. The total DNA amounts (ng) recovered from each steering wheel were plotted on a log scale. 50% of the data is located within the rectangles; the whiskers cover 90% of the data. The median is indicated by the solid line.

I.A. Kirgiz, C. Calloway / Journal of Forensic and Legal Medicine 47 (2017) 9e1512

A.

B.

P=0.21

P=0.0051

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Fig. 3. Matched Pair Difference, Wilcoxon Signed Rank Test. The graph shows the difference of the log10 transformed amount of the touch DNA collected by (A) tape lifting method and double swabbing method; (B) FTA scraping method and by double swabbing method. Wilcoxon Signed Rank test was used to determine if there were significant differences in yield for paired samples collected from different sides of the same steering wheel (A) p ¼ 0.21; (B) p ¼ 0.0051. The dark solid line represents the overall mean difference across the 35 cars.

I.A. Kirgiz, C. Calloway / Journal of Forensic and Legal Medicine 47 (2017) 9e15 13

DNA. However, not all methods were equally effective in collecting touch DNA from the steering wheels. The FTA paper scraping method was shown to yield significantly more DNA from steering wheels compared to double swabbing while no significant differ- ence was observed between double swabbing and tape lifting. DNA yields obtained from the double swabbing collection method were most highly dispersed (10,000 fold difference from lowest to highest). This higher variability in DNA yield across steering wheels using the double swabbing method may result from less efficient collection of the DNA or loss of DNA during extraction from the cotton swab.

Touch DNA yields collected by tape lifting were not as widely dispersedwhen compared to double swabbing. One explanation for the smaller range could be that the tape becomes saturated with materials, therefore reducing its ability to collect touch DNA. Also, tape attachment properties (stickiness) decreased when used over

a large surface area, therefore it was important to start the tape lifting collection at the region with the highest expected DNA concentration. Since there is great variability for the hand position of the driver, it may not always be possible to correctly estimate areas where the steering wheel was primarily handled. Due to a large surface area, the tape was able to cover the entire steering wheel. During the collection process the tape needs to be handled with caution as it can stick to itself or to other surfaces. Further, when the water soluble tape was dissolved in water, it formed a viscous mass that had a hard time passing through the QIAamp silica membrane. Even though that obstacle was overcome by increasing the centrifugation time, the amount of tape that could be processed for a particular sample was limited.

The FTA paper scraping method yielded significantly more DNA collected from the surface of a steering wheel when compared to double swabbing. One possible explanation for the higher DNA

Fig. 4. % STR Allele Recovery of Last Driver’s Profile. The figure compares the percent of STR peaks recovered from the last driver’s profile for eight cars. Touch DNA collection methods are compared based on how well they recovered the last driver’s profile. (A) Recovery mean: double swabbing 91%; tape lifting 59%. Paired T-test: p ¼ 0.10. (B) Recovery mean: double swabbing 99%; FTA paper scraping 91%. Paired T-test: p ¼ 0.29. The results for both studies are not statistically significant.

I.A. Kirgiz, C. Calloway / Journal of Forensic and Legal Medicine 47 (2017) 9e1514

yields could be that the chemical composition of the FTA paper allows for greater preservation and release of the DNA20 in com- parison to the cotton swab which can trap DNA in the fibers.9 Also, FTA paper has a greater surface area when compared to swabs and could therefore cover a larger area, potentially resulting in higher collection yields.3 The cotton swab has a relatively small surface area compared to FTA paper and tape, and therefore, the collection process was longer for double swabbing in comparison. The FTA paper did not dry as fast as swabs and still appeared wet at the end of the collection process. One limitation of the FTA paper was that in some cases scraping the wet paper across a rough surface resulted in the loss of some of the paper fibers. In the future, less water could be applied to the paper. Alternatively, the filter paper could be replaced by a sturdier matrix to minimize tearing and potential DNA sample loss.

Our results demonstrate that in some cases there is enough touch DNA on the steeringwheel of vehicles to yield a complete STR

profile of the last driver. We observed that steering wheels retain DNA of the recent driver even if vehicles were driven for a short period of time (as short as 2min, data not shown). However, as may be expected, a majority of the samples collected from steering wheels resulted inmixed STR profiles. Based on DNA concentration, DNA collected from steering wheels using FTA paper was more likely to result in a more complete STR profile compared to swab- bing or tape lifting as ~50% of the samples yielded concentrations high enough to allow for 1 ng input using FTA paper compared to 20e30%.

Touch DNA collected from a steering wheel is a powerful tool for solving crimes. The FTA paper scraping method has the potential to be used to recover touch DNA from the steering wheels of vehicles and similar objects for increased DNA yields. This novel method could be particular useful in situations when the exact location of touch DNA is unknown or small quantities of touch DNA are dispersed over a large surface. Additional studies are needed to

I.A. Kirgiz, C. Calloway / Journal of Forensic and Legal Medicine 47 (2017) 9e15 15

further evaluate the use of FTA paper as alternative touch DNA collection method or use in combination with other standard methods such as double swabbing.

Conflict of interest

The authors Irina Kirgiz and Cassandra Calloway have no financial or other conflict of interest to report.

Acknowledgements

This researchwas supported and funded by a research fund from the University of California, Davis Forensic Science Graduate Pro- gram. We would like to thank Cecilia VonBeroldingen and Robert Rice for their critical review of the study proposal. We would also like to thank Cecilia VonBeroldingen and George Sensabaugh for their critical review of the manuscript.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jflm.2017.01.007.

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