Assessing DNA recovery from chewing gum

Abstract

The purpose of this study was to evaluate which DNA extraction method yields the highest quantity of DNA from chewing gum. In this study, several popular extraction methods were tested, including Chelex-100, phenol-chloroform-isoamyl alcohol (PCIA), DNA IQ, PrepFiler, and QIAamp Investigator, and the quantity of DNA recovered from chewing gum was determined using real-time polymerase chain reaction with Quantifiler. Chewed gum control samples were submitted by anonymous healthy adult donors, and discarded environmental chewing gum samples simulating forensic evidence were collected from outside public areas (e.g., campus bus stops, streets, and sidewalks). As expected, results indicate that all methods tested yielded sufficient amplifiable human DNA from chewing gum using the wet-swab method. The QIAamp performed best when DNA was extracted from whole pieces of control gum (142.7 ng on average), and the DNA IQ method performed best on the environmental whole gum samples (29.0 ng on average). On average, the QIAamp kit also recovered the most DNA from saliva swabs. The PCIA method demonstrated the highest yield with wet swabs of the environmental gum (26.4 ng of DNA on average). However, this method should be avoided with whole gum samples (no DNA yield) due to the action of the organic reagents in dissolving and softening the gum and inhibiting DNA recovery during the extraction.

Keywords

Forensic DNA chewing gum DNA IQ PrepFiler Chelex phenol-chloroform QIAamp Quantifiler

Introduction

DNA can be recovered from forensic evidence and reference samples containing body fluids and hair roots—and even fingerprints that contain a much lower quantity of cellular material.¹ DNA-containing forensic evidence can be instrumental in helping to identify and convict or exonerate a suspect in a case.¹ After probative evidence containing biological fluids or materials is recovered from a crime scene, presumptive tests are used to identify the body fluids tentatively that are present. Next, for probative samples, DNA is extracted using one of several published or commercial methods, and copied and quantified using polymerase chain reaction (PCR). If sufficient DNA is present, it is processed further using DNA typing methods. In cases where DNA is present in low quantities, also termed “low template DNA,” it is essential to recover the maximal quantity of DNA from the evidence. The goal of this study was to determine the DNA extraction method that recovers the highest quantity of DNA from chewing gum.

Chewing gum as forensic evidence is a low occurrence, but it can be a valuable source of DNA in criminal investigations.^1–3 Bond et al.² reported that chewing gum comprised 3.5% of casework samples from January to December 2006. In almost 50% of those samples, sufficient DNA was recovered for DNA typing and subsequent entry into the UK National DNA Database.² Matching the recovered chewing gum DNA profile to a suspect is dependent upon the location of recovery: within or outside of the immediate crime scene and/or in a communal area, as well as if the suspect’s DNA profile is included in the database.² Other studies on chewing gum have shown that the condition of the gum has little effect on the ability to obtain a DNA profile.⁴ Thacker et al.⁴ were able to obtain full STR DNA typing profiles from chewing gum following incubation at room temperature, in a humidity chamber, or after 30 hours of exposure to sunlight. However, exposure of gum to UV light caused longer alleles to drop out.⁴ Of the extraction methods used in that study, Chelex-100® (Sigma-Aldrich, St. Louis, MO) was found to be the most effective for recovering DNA from degraded samples.⁴

From our informal discussions with criminalists, various methods are used to extract DNA from chewing gum in forensic labs, including phenol-chloroform-isoamyl alcohol (PCIA), Chelex-100, QIAamp®, PrepFiler®, and DNA IQ™. In this paper, we report the quantities of DNA recovered from control and environmental chewing gum samples using those five methods.

Methods

DNA extraction

DNA from cellular material on chewing gum was extracted using the inorganic Chelex-100,⁵ organic PCIA,⁶ QIAamp DNA Investigator Kit (Qiagen, Valencia, CA),^7–9 DNA IQ System (Promega, Madison, WI), and PrepFiler (Life Technologies, Carlsbad, CA)¹⁰ methods. The commercial kits were used as directed by the manufacturers; published protocols were used for the remaining methods. All DNA extractions were performed in a DNA-free PCR workstation using sterile instruments, and the extracted DNA was stored at –20℃. Negative control samples were also processed using the DNA extraction protocols.

Samples

Different pieces of gum constituted the replicates, and each extraction method was used on three to five separate pieces of gum, either chewed by a volunteer (control) or collected in the environment. Saliva and control chewing gum samples were donated by adult human volunteers, and environmental discarded chewing gum samples were collected as approved by Towson University’s Institutional Review Board (IRB, 13-0X22). Prior to use with the chewing gum samples, the DNA extraction methods were validated using swabs (DNA-free PurSwab; Puritan Medical Products, Guilford, ME) with 50 µL of saliva and swabs with no sample. Control chewing gum (Stride spearmint sugar-free gum) samples were collected from volunteers after they had chewed the gum for 30 minutes at least 30 minutes after eating or drinking. Abandoned “environmental” chewing gum samples were collected on the Towson University campus on the morning of December 6, 2012. The chewing gum samples were collected in separate, clean, plastic sandwich bags by inverting the bag and collecting the gum without sealing. Supplementary Table 1 details the samples (n = 117) tested using each method. Five replicates were performed using all of the extraction methods for saliva swabs, wet-swabbed control gum, wet-swabbed environmental gum, and whole environmental gum. Three replicates were performed for all of the whole control gum samples, except the organic method for which there were five replicates. Each replicate consisted of a whole piece of gum. No single piece of gum was processed using more than one method.

DNA quantitation

The quantity of amplifiable DNA was determined via a TaqMan assay using the Quantifiler® kit (Applied Biosystems, Carlsbad, CA) on an ABI Prism 7000 real-time PCR instrument. A standard curve was created from a dilution series using DNA provided in the kit and used to calculate the DNA concentration in each of the extracted samples. The internal positive control (IPC) was used as an indicator of PCR inhibition.

After initial DNA quantitation following the elution step from the above methods, all of the chewing gum samples were concentrated by ethanol precipitation in order to attempt to re-quantify any samples that were previously below the detection limits. Specifically, 3 M of sodium acetate, pH 5.2, was added in an equivalent of one tenth of the elution volume, mixed by inverting, and two volumes of ice-cold 100% ethanol were added and mixed. The solution was stored at –20℃ overnight. After subsequent centrifugation, the supernatant was decanted, the pellet was washed with 1 mL of 70% ethanol, re-precipitated, and the alcohol was decanted. The pellet was re-suspended in 10 µL TE buffer and re-quantitated.

Statistics

The parametric analysis of variance test and non-parametric Kruskal–Wallis (H) test were employed in statistical tests to determine if there were significant differences in the yields using the different DNA extraction methods. The significance level is noted in Supplementary Table 1.

Results

The quality of the reagents and chemicals was validated using swabs aliquoted with saliva. DNA was successfully extracted from the saliva swabs using all of the methods and yielded amplifiable human DNA as quantified with Quantifiler (Supplementary Table 1 and Figure 1(a)). DNA was not detected from the sample-free swabs. The highest average DNA quantity was recovered using the QIAamp DNA Investigator Kit (α = 0.05; p < .005; Figure 1(a) and Supplementary Table 1).

Figure 1.

DNA yield from saliva swabs and control and environmental chewing gum samples using swabs or whole gum samples. (a) Saliva swabs; (b) wet-swabbed control (gray) and environmental (black) gum; and (c) whole control (gray) and environmental (black) gum samples.

The Quantifiler real-time PCR quantitation results indicate that all of the DNA extraction methods we tested yielded detectable amplifiable human DNA from at least four of the five replicates for the wet-swabbed control chewing gum (Supplementary Table 1 and Figure 1(b)); the DNA IQ method performed the best (Figure 1(b)). A zero quantitation value indicates that the sample DNA is below the detection limit of Quantifiler. For the wet-swabbed environmental gum samples, the PCIA method performed the best (Figure 1(b)). DNA recovery was very high (26.4 ng on average) for swabs of the gum extracted using the PCIA method. For the whole control gum samples, the highest DNA quantity (142.7 ng on average) was observed using the QIAamp method (Supplementary Table 1 and Figure 1(c)). For the whole environmental gum samples, the highest DNA quantity (29.0 ng on average) was observed using the DNA IQ method (Supplementary Table 1 and Figure 1(c)). No DNA was recovered using the PCIA method for whole gum samples (Supplementary Table 1 and Figure 1(c)). Samples from all extraction methods were subjected to ethanol precipitation. While ethanol precipitation did improve the DNA concentration of some of the samples, loss was observed in other samples (data not shown).

Significant differences (α = 0.05) between the method means were observed using an ANOVA test for the saliva swabs and control gum samples (swabbed and whole). A Kruskal–Wallis test revealed that there were significant differences (p < .10) among the DNA extraction methods for all sample types, except the swabbed control and environmental gum samples (Supplementary Table 1).

As all of the methods that we evaluated were successful in recovering DNA from chewing gum using the wet-swab method and as all methods, except PCIA, were successful in recovering DNA from the whole gum samples, a cost and time analysis was performed to determine the most cost- and time-effective method (Table 1). The Chelex-100 method was the most inexpensive of the methods tested, but the overnight or long incubation draws out the overall time required for DNA typing. Shorter incubation times have been shown to reduce the overall time, but often reduce DNA yields (unpublished results). The QIAamp and PCIA methods yielded high quantities of DNA but were two of the three most expensive methods. The DNA IQ method performed well and has an intermediate cost.

Table 1.

Comparison of lab preparation cost (US$) and time per sample for the methods evaluated.

Method	Cost (US$)	Time (min)
PCIA	4.03 (+filter device)	90 (+overnight)
PrepFiler	4.64	240
QIAamp	4.32	40
DNA IQ	2.92	90
Chelex-100	0.10	20 (+overnight)

Discussion

The commercially available QIAamp DNA Investigator, DNA IQ, and PrepFiler kits were chosen because they are widely used in forensic labs that have validated and implemented these kits for casework. These kits are automatable, have relatively short extraction times, and require no overnight incubations. The PCIA organic method is the oldest and “gold standard” method for obtaining high molecular weight, double-stranded DNA, and it typically yields the highest quantity of DNA using standard samples. The Chelex-100 method is an inexpensive, single-tube method that has performed well with degraded chewing gum samples.⁶ DNA quantification using Quantifiler is a well-accepted method, contains an IPC that can be used to indicate PCR inhibition, and is much more sensitive and species specific compared with older methods such as UV-Vis spectroscopy.

All of the methods (except PCIA applied to whole gum) recovered at least 1 ng of DNA, the suggested input for STR DNA typing kits, using either the swab or whole chewing gum sampling methods (Figure 1). However, with the added chemical complexity of gum base in chewing gum, the organic components in the PCIA method caused the gum to dissolve partially and to become sticky and interfere with the extraction chemistry, thus inhibiting recovery using the whole gum samples. The IPC was acceptable for all samples except the whole gum samples, indicating that the PCIA interaction is inhibitory. No detectable DNA was recovered using the PCIA method with whole gum samples (Figure 1).

The amplifiability of DNA extracted from environmental gum may vary widely based upon where the sample was collected (i.e., grass, soil, or sidewalk) due to the presence of PCR inhibitors and exposure to sunlight.⁴ Due to random variations in the quantity of cellular material on the substrate, the quantity of DNA recovered varied widely. A zero value was recorded for some sample replicates quantified with Quantifiler, indicating that the quantity of DNA in the samples was below the detection limit with the method. Other studies have demonstrated that DNA typing profiles may be obtained from samples that are below the detection limit of Quantifiler (although newer kits, including Plexor HY®, have been shown to have a lower detection limit).¹¹ Ideally, though, the quantity of DNA recovered from the evidence can be determined using the standard operating procedure so that the appropriate amount of sample can be inputted from DNA typing. Accordingly, the goal of this study was to determine which DNA extraction method yielded the highest quantity of DNA.

The use of spin columns and separation devices such as those used in this study has been shown to yield purer DNA extracts in previous research. Of the extraction methods evaluated, the QIAamp method is preferred for extracting DNA from chewing gum samples. It extracted DNA successfully from all of the environmental gum samples evaluated (10 replicates in total), yielded the largest average DNA quantity for the whole control gum (142.7 ng of DNA on average), and also yielded among the highest DNA quantities from the whole environmental gum (21.8 ng on average) and swabbed environmental gum samples (7.2 ng on average). The QiaAMP column has been shown to remove PCR inhibitors, and this kit has previously reported to be an excellent method of purification.⁹

The QIAshredder/QIAamp DNA Mini Kit (Qiagen)⁹ and a BTA™ lysis buffer/PrepFiler Forensic DNA Extraction Kit (Applied Biosystems)¹² have also been used to extract DNA from chewing gum. The Qiagen MagAttract DNA Mini M48 Kit for use with BioRobot M48 includes methods for working with chewing gum. In another study using the QIAamp kit, the highest DNA yields from 50 µL of whole blood samples were recorded using the QIAamp DNA Investigator Kit compared with the ZR Genomic DNA and AccuPrep Genomic DNA kits.¹³ Phillips et al.¹⁴ observed the highest DNA recovery using the QIAamp Investigator manual method compared with the automated and Chelex-100 methods using buccal samples. Our results are congruent with that study.

The DNA IQ method also yielded amplifiable DNA from all of the replicates and the highest DNA yield (29.0 ng of DNA on average) from the whole environmental gum samples and swabs of the control gum samples (3.5 ng of DNA on average). The DNA IQ kit utilizes a stronger lysis buffer than other methods do. This may explain the better overall yields for the whole environmental gum using the DNA IQ kit.¹⁵

DNA was also successfully extracted from the control and whole environmental gum samples using the PrepFiler (14.8 ng of DNA on average from the whole control gum—the second highest quantity) and Chelex-100 methods (1.2 ng of DNA on average from the control gum swab—the second highest quantity). The results using the PrepFiler kit are in agreement with previous research.¹²

The inter-sample variability observed in this study was due to chance alone. The poorer results using the DNA IQ method with the swabbed environmental gum may have been due to older/poorer gum samples—although all samples were selected at random—and it is impossible to know the history of the environmental samples collected on campus.

For comparison, in a previous chewing gum study by our group using Trident Watermelon Twist with xylitol chewing gum, the PCIA method yielded the highest quantity of DNA, the DNA IQ method yielded the second highest DNA quantity, dialysis the third, and Chelex-100 the fourth using a real-time PCR assay.¹⁶ PrepFiler and QiaAMP Investigator were not evaluated in that study.

Although we expected that the outdoor environmental samples we collected (exposed to UV radiation from the sun and/or to humic acid in soil) would yield less amplifiable human DNA than the control,⁴ this was not observed in this study. The IPC amplification was acceptable for all samples except the whole gum samples with the PCIA extraction method. Interestingly, in agreement with another study comparing indoor and outdoor samples (cigarette butts), several of our environmental gum samples yielded more DNA than the control gum samples.¹⁷

Conclusion

Chewing gum is known to be a source of DNA used in forensic investigations. Herein, we have presented the DNA yields from chewing gum in a systematic study of five DNA extraction methods. The aim of this study was to determine the extraction method that would recover the highest quantity of amplifiable DNA from chewing gum. Except for the PCIA method with the whole gum, all of the methods recovered at least 1 ng of DNA using either the swab or whole chewing gum sampling methods, thereby recovering sufficient DNA for DNA typing. Though further controlled degradation and aging experiments will need to be performed to understand fully the effects of the environmental conditions on evidence such as chewing gum, our results indicate that of the extraction methods evaluated, the QIAamp method is preferred for its high yields and reproducibility. QIAamp resulted in the highest quantity of extracted DNA from the saliva swab and whole control gum samples, and yielded among the highest quantities of DNA from all of the other gum and swab samples. The highest DNA yields for the whole environmental gum samples and wet-swabbed control gum samples were observed using the DNA IQ method. The PCIA method, although it performed well with wet-swabbed samples, should not be employed with whole gum samples due to the action of the organics in dissolving and softening the gum and inhibiting DNA recovery during the extraction.

Footnotes

Declaration of conflicting interests

The authors have no conflicts of interest to declare.

Funding

This work was supported by a Forensic Sciences Foundation Acorn grant and TU new faculty start-up funding (to KME).

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