5-validation-de ungria.pmd P.R.L. Sales et al. 25 SCIENCE DILIMAN (JANUARY-JUNE 2019) 31:1, 25-36 Val idation of Two Extraction Methods for Human DNA from Cigarette Butts Paul Ryan L. Sales Dorothy Emma C. Ferrer Gay vell ine C. Calacal Jazelyn M. Salvador Maria Corazon A. De Ungria* DNA Analysis Laboratory Natural Science Research Institute University of the Philippines Diliman ABSTRACT Cigarette butts found in crime scenes may be used to identify persons and link them to a crime through DNA prof iling of epithelial cells from saliva stains on these materials. Downstream analysis of cigarette butts p o s e s s o m e c h a l l e n g e s b e c a u s e t h e s e a r e o f t e n e x p o s e d t o c h e m i c a l c o n t a m i n a n t s a n d e n v i r o n m e n t a l c o n d i t i o n s w h i c h l e a d t o D N A degradation. In this study, s everal factors were tested to compare the amount and quality of DNA obtained from cigarette butts extracted using an organic procedure and the QIAamp® DNA Micro Kit (QIAGEN). Results show that exposure to an outside environment had a signif icant effect o n D N A y i e l d a n d a m p l i f i a b i l i t y f o r b o t h e x t r a c t i o n p r o c e d u r e s . Prolonged storage of cigarette butts of up to six months affected the amount of DNA that can be extracted using the QIAamp® DNA Micro Kit. However, complete DNA prof iles can be generated from cigarette butts stored for six months provided that these samples are stored indoors under controlled temperature conditions and with minimal exposure to contaminants. Keywords: Cigarette butts, DNA typing, DNA yield, forensic science _______________ *Corresponding Author ISSN 0115-7809 Print / ISSN 2012-0818 Online Validation of Two Extraction Methods for Human DNA from Cigarette Butts 26 INTRODUCTION DNA analysis of evidentiary materials is a powerful tool for linking an individual to a victim or a crime scene (Hochmeister et al. 1991; Walsh et al. 1992). Proper crime scene collection and handling are essential to preserve the integrity of the evidence. Saliva may be found on various surfaces found at the crime scene, such as cigarette butts (Hochmeister et al. 1991; Watanabe et al. 2003), leftover food (Sweet and Hilderbrandt 1999; Abaz et al. 2002), and bite marks on human skin (Pretty and Sweet 2001; Chávez-Briones et al. 2015). DNA from epithelial cells present in saliva may be used for DNA analysis. However, saliva stains on most surfaces, once dry, are invisible to the naked eye. In addition, DNA preparations can potentially contain co-extracted inhibitors that could affect downstream DNA analysis. In a report by Watanabe et al. (2003), certain kinds of dyes in cigarette butts inhibit PCR amplif ication. Thus, a key determining step in the success or failure of obtaining a DNA prof ile relies on the extraction method used to recover amplif iable DNA. With the availability of multiplex Short Tandem Repeat (STR) DNA marker systems targeting autosomal chromosomes (aSTRs) or gonosomal chromosomes (XSTRs and YSTRs), generating DNA prof iles with a high capacity to differentiate human sources was made possible (Hochmeister et al. 1991; Balogh et al. 2003). In a 2009 survey, about 28.3% of Filipinos aged 15 years and older, or about 17.3 million individuals smoked cigarettes in the Philippines (WHO 2009). Hence, cigarette butts are likely to be present in crime scenes that may contain DNA from (1) the perpetrator who smoked while waiting for the opportune moment to commit the crime; (2) the victim/s, including those whose location remains unknown; and (3) the witness who could provide vital information for the investigation. Many crimes in the Philippines are located outdoors where the average temperature is 28.3°C, and the humidity ranges from 71 - 85% (PAGASA 2016). When DNA contained in crime scene samples are not recovered soon after a crime, these outdoor conditions promote microbial growth which leads to the degradation of DNA. In the present study, we compared the utility of an organic procedure and a silica- based DNA extraction method for handling cigarette butt samples. We also tested the effect of storage time and conditions, and cigarette type, on the generation of human DNA prof iles from these samples. P.R.L. Sales et al. 27 MATERIALS AND METHODS Institutional Review Clearance to conduct the study was provided by the Natural Sciences Research Institute, University of the Philippines Diliman. Sample Collection Samples were collected from two volunteers who are regular smokers. Both volunteers were asked to smoke three types of cigarettes, namely (1) regular-type; (2) light or less nicotine; and (3) light with menthol, following their normal routines. For the 24-hour stored samples, three regular-type cigarette butts from each volunteer were collected and stored for 24 hours indoors at room temperature (25°C - 27°C). Volunteers were asked to smoke three cigarettes in one day and each cigarette was smoked at a different time of the day. Each cigarette butt was split into two slices with the cut parallel to the seam of the cigarette. Each half was placed in separate sterile tubes, suspended, and incubated in 12 mL phosphate buffered saline (PBS) solution for three hours with shaking. For every cigarette butt, one half was subjected to organic DNA extraction, while the other half was subjected to DNA extraction using the QIAamp® DNA Micro Kit. For the six-month storage samples, four cigarettes for each cigarette type (regular, lights, and lights-menthol) were collected from each volunteer. For each cigarette type, volunteers were asked to smoke four cigarettes per day with two cigarettes smoked in the morning and two cigarettes smoked in the afternoon. Volunteers were also asked to leave some time before smoking the second cigarette. Samples were stored at room temperature prior to extraction. Each cigarette butt was split into two slices with the cut parallel to the seam of the cigarette. One half was subjected to organic DNA extraction, while the other half was subjected to DNA extraction using the QIAamp® DNA Micro Kit. For the indoor storage set-up, four cigarettes for each cigarette type (regular, lights, and lights-menthol) were collected from each volunteer and stored for six months indoors. As for the outdoor storage set-up, four cigarettes for each cigarette type (regular, lights, and lights-menthol) were collected from each volunteer and left outdoors for six months. Volunteers smoked each cigarette in intervals. Validation of Two Extraction Methods for Human DNA from Cigarette Butts 28 Cigarettes left outdoors were deposited directly on soil and were exposed to external elements, such as direct sunlight and rain. Cigarettes were also exposed to any animal, insect, and microbial activity that may occur in the area the cigarettes were deposited on. DNA Extraction QIAamp® DNA Micro Kit extraction was carried out using the manufacturer’s protocol with some modif ications. Modif ications include the use of 640 μL of lysis solution to submerge the cigarette butt samples and incubating these in lysis solution for two hours. Organic extraction was performed using the protocol described by Budowle et al. (2000) with some modif ications. The modif ied protocol made use of a 1000-μL cell lysis solution containing 835 μL of TEN buffer (10 mM Tris, 10 mM EDTA , and 100 mM NaCl), 100 μL of 20% SDS (Invitrogen), 40 μL of 1 M DTT (Roche), and 25 μL of 20 mg/mL Proteinase K (Novagen). Samples were incubated with shaking at 56°C for ~24 hours in an Eppendorf Thermomixer® (Eppendorf ). An equal volume of 25:24:1 mix of phenol (Invitrogen), chloroform (Merck Millipore), isoamyl alcohol (J.T.Baker), and a MaXtract® Low Density (QIAGEN) phase-lock gel was used to separate organic and aqueous layers. DNA extracts were purif ied using a Microcon® 100 centrifugal f ilter (Merck Millipore) and was eluted using 40 μL of TE-4 buffer. DNA Ampl if ication DNA was quantif ied using the PowerQuant® System (Promega Corporation) (Ewing et al. 2016), and quantitation results were analyzed using the PowerQuant® Analysis Tool following manufacturer’s instructions. A 0.50-ng DNA template was amplif ied using half-volume reactions (12.5 μL) of the PowerPlex® 16 HS System (Promega Corporation) kit. Polymerase Chain Reaction (PCR) conditions were as follows: initial denaturation at 96°C for 2 minutes, followed by 10 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 70°C for 45 seconds. This was followed by 22 cycles of denaturation at 90°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 70°C for 45 seconds. PCR amplif ication was completed with a f inal extension at 60°C for 30 minutes and a f inal hold at 4°C. P.R.L. Sales et al. 29 Fragment Detection and Profile Analysis Detection of the amplif ied DNA fragments was carried out in the Applied Biosystems® 3500 Genetic Analyzer (Thermo Fisher Scientif ic). Generated prof iles were analyzed using the GeneMapper® ID-X Software version 1.2 (Thermo Fisher Scientif ic). An analytical threshold of 50 relative fluorescence units (RFU) was used to distinguish true peaks from background noise. Allele Recovery Computation Blood samples from volunteers were collected and were used to generate their reference DNA prof iles for comparison with the DNA prof iles generated from the cigarette butts. The number of alleles in a volunteer’s reference prof ile was considered as the volunteer’s “expected” number of alleles. The number of alleles in the DNA prof ile from the cigarette butt replicate was considered as the “observed” number of alleles for that sample replicate. Allele drop-ins detected in cigarette butt samples were not considered part of the “observed” alleles. To determine the percentage allele recovery for each sample replicate, the observed allele number was divided by the expected number of alleles, then multiplied by 1 0 0 . Statistical Analysis Data in the comparison of DNA yield from the two DNA extraction methods, storage environment, and storage time were analyzed using the unpaired Student’s t-test. Data from the comparison of DNA yield from different cigarette types were analyzed using one-way Analysis of Variance (ANOVA). All statistical analyses were performed using the GraphPad Prism 7 software (GraphPad Software, Inc.). Validation of Two Extraction Methods for Human DNA from Cigarette Butts 30 RESULTS AND DISCUSSION Comparison of DNA yield and allele recovery between the standard organic procedure and the QIAamp ® DNA Micro Kit method for DNA extraction For regular-type cigarettes extracted after a 24-hour storage period, there was no signif icant difference (p=0.078) between the DNA yields of the two extraction methods (mean org =719.97±303.16; mean QIA =1188.33±500.35) (Table 1). Complete DNA prof iles were generated from all DNA extracts of 24-hour stored samples. In samples stored indoors for six months prior to processing, difference in DNA yield (p=0.008) was signif icant (mean org =1013.55±565.03; mean QIA =139.45±229.31) (Figure 1a and Table 2). Allele recovery across extraction methods (p=0.073) was not signif icant. Table 1. DNA recovery from regular-type cigarette butt samples extracted using organic QIAamp® DNA Micro Kit extraction methods after a 24-hour indoor storage period. DNA Extraction Method DNA Yield (ng DNA/g cigarette butt) Mean ± SD Organic Extraction 719.97 ± 303.16 QIAamp® DNA Micro Kit 1188.33 ± 500.35 Figure 1. Comparisons of DNA yield across different factors. (a) Comparison of DNA yields between the organic and the QIAamp® DNA Micro Kit extraction methods from regular-type cigarette butt samples stored indoors for six months prior to extraction. Difference in DNA yields between the two extraction methods is signif icant (p=0.008). (b) Comparison of DNA yields from cigarette butt samples extracted using the organic method with respect to indoor storage times. Difference in DNA yield (p=0.877) between the two storage durations is not signif icant. (c) Comparison of DNA yields from cigarette butt samples extracted using the QIAamp® DNA Micro Kit with respect to indoor storage times. Difference in DNA yield between the two storage durations is signif icant (p=0.0001). P.R.L. Sales et al. 31 After six months of storage prior to processing, DNA extracts from the QIAamp® DNA Micro Kit have lower DNA yields compared to those obtained via the organic extraction procedure. This observation illustrates the limitation of a silica membrane- based DNA extraction method, such as the QIAamp® DNA Micro Kit. When the lysis solution is transferred to the QIAamp® MinElute Column, DNA binds to the silica particles in the membrane while other molecules remain in the lysis solution. Since the amount of silica particles of the QIAamp® MinElute Column is limited, the amount of DNA that may be recovered from the lysis solution is also limited. Competitive binding among human DNA, non-human DNA, and other substances occurring in the silica membrane contributes to the decrease in human DNA yield using silica membrane-based DNA extraction. Competitive binding occurs when negatively-charged non-human DNA or other molecules bind to the positively- charged silica surface instead of the target human DNA. Furthermore, human DNA bound to the silica surface could be displaced by molecules with a stronger aff inity to the silica surface. Mundorff and Davoren (2014) noted that PCR inhibitors and non-human DNA may also co-isolate with human DNA during extraction. Allele recovery data show that the amount of amplif iable DNA recovered during silica column extraction generated partial (>80% alleles recovered) to full prof iles. Effect of storage environment on DNA yield and allele recovery DNA yield in cigarette butt extracts exposed outdoors for six months was lower compared to samples stored indoors at room temperature. Samples extracted using the organic extraction method exhibited a signif icant difference in DNA yield with respect to storage environments (p=0.0001). Similarly, the difference in DNA yield between samples stored in different environments and extracted using the QIAamp® DNA Micro Kit was signif icant (p=0.001). Poor allele recovery was observed in samples exposed outdoors (0 – 17%), while better allele recovery was remarked in DNA Extraction Method DNA Yield (ng DNA/g cigarette butt) Mean ± SD Organic Extraction 1013.55 ± 565.03 QIAamp® DNA Micro Kit 139.45 ± 229.31 Table 2. DNA recovery from regular-type cigarette butt samples extracted using organic and QIAamp DNA Micro Kit extraction methods after a 6-month indoor storage period Validation of Two Extraction Methods for Human DNA from Cigarette Butts 32 samples stored indoors (27 – 100%). Poor allele recovery of samples left outdoors was supported by DNA quantitation results, wherein the PowerQuant® Analysis Tool flagged 33% of samples as degraded, while 54% had a zero quantitation value. This result is consistent with the study of Casey et al. (2013) who reported a lower count of nucleated cells from cigarette butts left outdoors compared to those stored indoors. Effect of storage time on DNA yield and allele recovery DNA extracts obtained using the QIAamp® DNA Micro Kit exhibited a signif icant difference in DNA yield with respect to storage times (p=0.0001). For these samples, higher DNA yield was observed after storage for 24-hours compared to samples stored for six months. No signif icant difference was observed in the DNA yields of samples extracted using the organic method and stored for 24 hours versus those stored for six months (p=0.877) (Figure 1b). By contrast, the DNA yields of 24-hour and 6-month samples extracted using the QIAamp® DNA Micro Kit were signif icantly different (p=0.0001) (Figure 1c). DNA left on cigarette butts is highly prone to oxidation from reactive oxygen species (ROS), such as nicotine present in cigarette smoke (Ginzkey et al. 2012). Exposure of dehydrated (Matsuo et al. 1995) and lyophilized (Molina and Anchordoquy 2008; Bonnet et al. 2010) DNA to atmospheric oxygen at room temperature can also lead to DNA oxidation. In the oxidized state of DNA, the DNA molecule becomes less negatively charged, which in turn makes the binding of DNA to silica weaker. Thus, the prolonged exposure to oxidative stresses during long-term storage can decrease the adsorption eff iciency of silica columns in DNA extraction. It is therefore recommended that evidentiary samples that may have been exposed to harsh environmental conditions, such as cigarette butts left in a crime scene, must be processed immediately to decrease DNA loss during storage. No signif icant difference in allele recovery with respect to storage durations was observed in both organic (p=0.408) and QIAamp® DNA Micro Kit (p=0.051) extraction methods. P.R.L. Sales et al. 33 Effect of cigarette type on DNA yield and allele recovery Smoking habits differ from person to person. Smokers would have preferences on the type of cigarette based on the amount of nicotine and presence of other ingredients (e.g. , menthol). Three cigarette types, namely regular, less nicotine (lights), and less nicotine with menthol (lights-menthol), were tested to determine the effect of cigarette type on the amount and quality of DNA recovered. For both extraction procedures, the results showed that cigarette type had no significant effect on DNA yield (p org = 0.465; p QIA = 0.748) nor on allele recovery (p org = 0.613; p QIA = 0.297) (Tables 3 and 4). CONCLUSIONS This study shows that the amount and quality of human DNA on cigarette butts deteriorate over time, and the extent of DNA loss is more pronounced outdoors under warm and humid conditions. A suitable extraction method for a certain type of cigarette butt sample would differ on a case-to-case basis. For samples collected Table 3. DNA yield and allele recovery from cigarette butt samples of three types of cigarettes extracted using the organic extraction method after a six-month indoor storage period. Regular 760.17 ± 528.53 99.58 ± 1.18 Light 1653.89 ± 2003.18 99.58 ± 1.18 Light with Menthol 1272.32 ± 1013.93 100.00 ± 0 % Allele Recovery Mean ± SD Cigarette Type DNA Yield (ng DNA/g cigarette butt) Mean ± SD Table 4. DNA yield and alele recovery from cigarette butt samples of three types of cigarettes extracted using the QIAamp® DNA Micro Kit after a six-month indoor storage period. Regular 104.59 ± 214.50 81.64 ± 26.17 Light 176.64 ± 245.51 88.33 ± 20.39 Light with Menthol 178.21 ± 141.56 97.32 ± 7.58 % Allele Recovery Mean ± SD Cigarette Type DNA Yield (ng DNA/g cigarette butt Mean ± SD Validation of Two Extraction Methods for Human DNA from Cigarette Butts 34 indoors and stored for a short period of time, it is more practical to use the QIAamp® DNA Micro Kit because the process is faster, makes use of less hazardous chemicals, and involves less tube transfers, which in turn decreases the risk of contamination during processing. If possible, extraction of DNA from cigarette butts that are submitted as evidence should be processed immediately to maximize DNA recovery. For samples collected outdoors and stored indoors for a long period of time, the use of the organic extraction method is advised, since it can yield more DNA, allowing increased number of analysis despite low copies or low quality of DNA. Storage of cigarette butt samples in laboratories with controlled temperature ranging from 22oC to 25oC and with reduced humidity slows but does not completely stop DNA degradation. This is particularly important in the Philippines where delays in the collection and submission of evidence to DNA laboratories result in the prolonged outdoor exposure of samples, including cigarette butt samples, in locations that are subject to warm and humid conditions. ACKNOWLEDGEMENTS The authors would like to acknowledge the Off ice of the Vice Chancellor for Research and Development (OVCRD) of the University of the Philippines Diliman for providing the Outright Research Grant (OVCRD Project No. 111106 PNSE) that funded this work. The authors would also like to thank the volunteers for their participation and Miriam Ruth M. Dalet, of the DNA Analysis Laboratory, for support. REFERENCES Abaz J, Walsh SJ, Curran JM, Moss DS, Cullen J, Bright J, Crowe GA, Cockerton SL, Power TE. 2002. Comparison of the variables affecting the recovery of DNA from common drinking containers. Forensic Science International. 126:233-240. Balogh MK, Burger J, Bender K, Schneider PM, Alt KW. 2003. STR genotyping and mtDNA sequencing of latent f ingerprint on paper. Forensic Science International. 137:188- 195. Bonnet J, Colotte M, Coudy D, Couallier V, Por tier J, Morin B, Tuffet S. 2010. Chain and conformation stability of solid-state DNA: Implications for room temperature storage. Nucleic Acids Research. 38(5):1513-1546. Budowle B, Smith J, Moretti T, DiZinno J. 2000. DNA typing protocols: Molecular biology and forensic analysis. Natick (MA): Eaton Publishing. p. 20-22, 228. Casey L, Engen S, Frank G. 2013. Quantitative analysis of the DNA distribution on P.R.L. Sales et al. 35 cigarette butt f ilter paper. Journal of Forensic Science. 58(2):470-473. Chávez-Briones ML, Hernández-Cortés R, Jaramillo-Rangel G, Ortega-Martínez M. 2015. Relevance of sampling and DNA extraction techniques for the analysis of salivary evidence from bite marks: A case repor t . Genetics Molecular Research. 14(3):10165- 10171. Ewing MM, Thompson JM, McLaren RS, Purpero VM, Thomas KJ, Dobrowski PA, DeGroot G A , Ro s m o s E L , S to r t s D R . 2 0 1 6 . H u m a n D N A q u a n t i f i c a t i o n a n d s a m p l e q u a l i t y a s s e s s m e n t : D e v e l o p m e n t a l v a l i d a t i o n o f t h e P o w e r Q u a n t . F o r e n s i c S c i e n c e International: Genetics. 23:166-177. Ginzkey C, Stueber T, Friehs G, Koehler C, Hackenberg S, Richter E, Hagen R, Kleinsasser NH. 2012. Analysis of nicotine-induced DNA damage in cells of the human respiratory tract. Toxicology Letters. 208:23-29. Hochmeister MN, Budowle B, Jung J, Borer UV, Comey CT, Dirnhofer R. 1991. PCR-based typing of DNA extracted from cigarette butts. International Journal of Legal Medicine. 104:229-233. Matsuo S, Tokoyumi S, Osaka M, Hamazaki S, Sugiyama T. 1995. Degradation of DNA in d r i e d t i s s u e s b y a t m o s p h e r i c o x y g e n . B i o c h e m i c a l a n d B i o p h y s i c a l Re s e a r c h Communications. 208(2):1021-1027. Molina MC, Anchordoquy TJ. 2008. Degradation of lyophilized lipid/DNA complexes during storage: The role of lipid and reactive oxygen species. Biochemica et Biophysica Acta. 1778:2119-2126. Mundorff A, Davoren JM. 2014. Examination of DNA yield rates for different skeletal elements at increasing post mortem intervals. Forensic Science International: Genetics. 8:55-63. Philippine Atmospheric, Geophysical and Astronomical Services Administration. Climate of the Philippines [Internet]. Manila: PAGASA; [cited 2016 October 17]. Available from http: //pagasa.dost .gov.ph/index.php/climate-of-the-philippines. Pretty IA, Sweet D. 2001. A look at forensic dentistry – Par t 1: The role of teeth in the determination of human identity. British Dental Journal. 190(7):359-366. Sweet D, Hilderbrandt D. 1999. Saliva from cheese bite yields DNA prof ile of burglar: A Case Repor t . International Journal of Legal Medicine. 112:201-203. Walsh DJ, Corey A, Cotton RW, Forman L, Herrin Jr GL, Word CJ, Garner DD. 1992. Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva. Journal of Forensic Science. 37(2):387-395. Watanabe Y, Takayama T, Hirata K, Yamada S, Nagai A , Nakamura I, Bnai Y, Ohya I. 2003. DNA typing from cigarette butts. Legal Medicine Journal. 5:S177-S179. World Health Organization [Internet]. 2009. Geneva, Switzerland. Philippines’ Global Adult Tobacco Survey; [cited 2017 May 29]. Available from https://www.who.int/tobacco/ surveillance/2009_gats_report_philippines.pdf?ua=1. Validation of Two Extraction Methods for Human DNA from Cigarette Butts 36 _____________ Paul Ryan L. Sales is a University Research Associate at the DNA Analysis Laboratory of the Natural Sciences Research Institute, UP Diliman. He earned his Bachelor of Science in Biology degree at the Institute of Biology, UP Diliman and is currently enrolled as a graduate student in the MS Biology Program at the Institute of Biology, UP Diliman. His research interests are in the use of DNA technology for forensic applications. Dorothy Emma C. Ferrer is a Chemist at the Forensic Division of the Investigation Off ice of the Commission on Human Rights of the Philippines. She earned her Master of Forensic Sciences in Crime Scene Investigation at the George Washington University and her Bachelor of Science in Chemistry degree at the Institute of Chemistry, UP Diliman. Gay vell ine C. Calacal is a senior scientist working in the f ield of forensic DNA typing/forensic genetics at the DNA Analysis Laboratory, Natural Sciences Research Institute, UP Diliman. She obtained her Master of Science degree in Microbiology from the Institute of Biology, UP Diliman and completed a Forensic Genetics and DNA Database Technology course from the Graduate School of Biomedical Sciences, University of North Texas Health Science Center at Fort Worth Texas. USA. She is credited as one of the pioneers in the development of forensic DNA typing research in the country, in the validation of analytical procedures for handling different types of biological samples for forensic applications, developing a system for the collection, handling and analysis of DNA evidence, human remains identif ication and generation of the Philippine reference population genetic databases. Jazelyn M. Salvador is currently a University Researcher I at the DNA Analysis Laboratory, Natural Sciences Research Institute, UP Diliman. She finished her Bachelor of Science in Biology degree from the UP Manila and her Master of Science in Microbiology degree at the UP Diliman. Her research interest includes molecular population genetics and forensic DNA applications. Dr. Maria Corazon A. De Ungria heads the DNA Analysis Laboratory of the Natural Sciences Research Institute, University of the Philippines Diliman. Her research thrusts include the development of molecular procedures and studying human genetic variations for forensic applications. She currently holds a University Researcher V position and is a Scientist 2 of the DOST- CSC Scientif ic Career system.