Using DNA Typing Methods to Identify Dental Specimens Exposed to Different Environmental Conditions

Home , DNA profiling, Forensic dentistry

Using DNA typing methods to identify dental specimens exposed to different environmental conditions

Nilufer Akman Dogan1,*, Yunus Emre Akman2, Gulten Rayimoglu3, Emel Hulya Yukseloglu3

______Abstract: The term identification explained the recognition and definition of a person living or dead and to reveal the features that distinguish it from other people. This study presents the findings on the validity and feasibility of DNA extraction analysis on tooth specimens which subjected to different environmental conditions (submerged in sea water, buried underground for some time) and teeth which are burned/subjected to extreme thermal injury. The motivators behind these is to aid in the STR profiling; identifying individuals to which these specimens belong, and propose a standard for forensics methods for recovered field tissue samples; i.e. teeth. A total sample pool of 88 teeth, irrespective of the level of caries, were processed for DNA isolation and PCR. In the sample preparing phase, the teeth samples were crushed in a standardized method, to be rendered as fine powder. The processed samples underwent DNA isolation through use of a commercial kit. Isolates were amplified with AmpFISTR primers and PCR application method was used to identify the unique marker sequences through amplification of the fragments. According to amplified target locus,16 STR profile, were calculated the rate of success. Further analysis was made possible by means of statistical methods. The proprietary program used was Matlab Version 7.6.0.324 for each of the sample to ensure consistency and reduce bias in the analysis, calculation and comparative stages in the results. Relevant literature is discussed further to outline the advantages and potential pitfalls of our methods. Key Words: STR typing, forensic DNA, dental DNA, dental identification.

dentification is the main study and research when exposed to the elements of sea water or when Iarea of forensic science because of deal with buried underground. The duration from the subject’s human body and human body identification. Many death and the exposure to the elements is crucial. From biological materials like bone tissue, hair follicles, the a forensic science point of view, which increasingly biopsy specimens, saliva, blood and other bodily tissues, is dependent on DNA profiling, the elements are have been and are used as the identification of people unfavourable and hostile to the DNA molecule. DNA in order laboratory tests successfully for DNA isolation. is fragile and rapidly denatures over time. The samples DNA can be observed in almost all tissues of the human which forensic scientists depend on become extremely body [1]. difficult, or even impossible to interpret when soft tissue The problem in analyzing samples from deceased is the sample source. subjects is that in soft tissue decays rapidly, especially We postulate the potential advantage of using a

1) Gebze Technical University, Department of Molecular Biology and Genetics, Kocaeli, Turkey * Corresponding author: Gebze Technical University, Department of Molecular Biology and Genetics, Kocaeli, Turkey, Tel: +90 262 605 25 01, Fax: + 00 90 262 605 25 05, E-mail: [email protected] 2) Baltalimani Bone Diseases Research and Training Hospital,Department of Orthopedics and Traumatology, Istanbul, Turkey 3) University of İstanbul, Institute of Forensic Science, Istanbul, Turkey

99 Dogan N.A. et al. Using DNA typing methods to identify dental specimens exposed to different environmental conditions hard biological tissue, like teeth or bone, as these can obtained burned bone samples belonging to iron serve as a more robust source of DNA. Such tissues are age which are obtained by archeological work in more resilient in comparison to soft tissues of the body. Bedd Branwen and Anglesey regions [8]. In their Indeed, the preservation of structural and experimental study, Tsuchimochi et al. demonstrated morphological integrity macroscopically may also that it was possible to extract DNA from the pulp of the translate to a maintenance of the crucial DNA which may teeth that were exposed to 300 Cº of heat. They reported be retrieved with careful processing methods [2]. Dental that following extraction they were successfull to type tissue (i.e. teeth) are far more resistant to moisture, high- DNA [9]. temperature, bacterial and fungal breakdown. Enamel, which provides the durable matrix covering of the tooth Materials and methods is the strongest material in the human body. It is very unusual for teeth to be absent in the deceased, but are Samples and Preparation exceptions. Edentulous individuals, those who have full Our subjects were comprised of males and dentures, or even rarer, if the deceased was submerged females (18 to 65 years), who donated the 88 dental into chemicals for dissolving body tissues, including specimens for the study. The teeth acquired from the teeth (thereby hiding an evidence of homicide). donors were dried and stored in plastic containers. Fortunately, these rarities do not occur at a significant The intra-oral buccal swabs collected were stored in a enough frequency to undermine resources dedicated to refrigerator at a constant temperature of +4 centigrade dental DNA extraction methods and research [3]. degrees. Although the resilience of dental specimens, We stratified and separated the test groups into when compared with soft tissue, is more robust to four arms. In the first group of the dental samples were conditions and decay over time, in an ideal case time maintained fresh without any intervention. The second delay should be kept as short as possible for best results. group were subjected to a thermal stress or burn injury. As well as DNA analysis for identifying the deceased is In the third group, the teeth samples were submerged promising and increasingly utilized, dental structure in sea water, for a duration ranging from 2 to 8 months. and records can further offset the disadvantage of tissue Finally, in the fourth group, we buried the dental samples breakdown or failure to extract DNA for analysis. Alas, underground for a time ranging 2 to 36 months. Teeth there is no controversy or doubt about the crucial role of which are burnt and submerged in sea water are shown dental tissue in forensic science [3]. in Figure 1 for sample. Anatomically, teeth are encased in a complex All four of the stratified groups of samples arrangement of epithelial tissue, connecting tissue, were further processed for preparation. The teeth in muscle and bone (alveoli or tooth sockets*. The pulp each of the groups were submerged in 10% bleach for chamber (containing nerves and blood vessels), dentin 10 minutes, and agitated every two minutes to ensure and the covering enamel which is firmly anchored by the complete exposure of the samples to the bleach solution. periodontal ligament which are comprised of organized This was followed by rinsing with distilled water and fibers; are all rich source of DNA. More specifically removal of excess fluid with fluid paper towels. this DNA-rich fibrous tissue is abundant in the unique Teeth showing areas of caries were carefully identifying 'short tandem repeat' (or STR) sequences. removed by filing and further cleaned. STR fragments are excellent and reliable constituents After these steps, the teeth samples were of DNA and, like DNA, are unique to each individual. incubated in distilled water, set at 56°C, for an hour. The Moreover, teeth and bone have been shown to maintain samples were then grated and pulverized to render the these DNA moieties long after decomposition of the samples as fine granules which were then deemed ready deceased [4, 5]. for the DNA isolation and profiling. The equipment Fingerprints have been used, and still continue we used in the fragmentation of the samples was the to be used as sources of identification. However, thermal proprietary TissueLYSER II (QIAGEN®). injury (fires, chemical burns) and decaying rapidly distort their architecture and make them unusable in DNA isolation the identification of post mortem subjects. Instead of DNA kits (Qiagen Microkit) were utilized finger prints, small amount of soft tissue samples, even in the DNA isolation. Teeth were standardized in an distorted, may be sufficient for a succesfull identification appropriate manner and adjusted as follows: 1 g of using current biomolecular techniques [6]. processed pulverized tooth powder per tooth sample. A Burned human remains have been a challenge to lysis buffer, containing the enzyme protein kinase K was the extraction of usable DNA specimens. Sajantila et al. added to the sample and incubated at 56°C overnight. sampled 10 burned corpses and successfully extracted After the overnight period, the sample mixtures were DNA from 26 dental samples [7]. Brown et al. have centrifuged at 14,000 rpm for a minute. We extracted the successfully extracted DNA from the archeologically supernatant for the next steps in the trial.

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Figure 1. Teeth which are burnt and submerged in sea water. accuracy evaluation of the DNA samples were made possible by the use of the Matlab version 7.6.0.324 program. The formula shown in Figure 2 was plugged into the software and applied to each of the samples; i.e. fresh; burnt; seawater exposed; and buried samples that make up the four dental sample groups. The analyzed raw Figure 2. Program calculations and formula legend - Matlab data demonstrates our findings in Table 1 and Table 2. Version 7.6.0.324. D: dental sample, i.e. teeth (measured); p: Number of samples; n: Number of loci; m:

Number of alleles per single locus; Dij: j. allele in the i. Locus of the dental sample. Discussion

DNA amplification, purification and We have evaluated 88 dental (tooth) samples in electrophoresis a study. The methods and results section clearly explain AmpFISTR primer was reacted with the the variables in these groups. Environmental stressors supernatant in preparation of the PCR amplification were simulated through burning; submersion in salt (sea) process. The primer target, the 16 STR locus, when water; and were buried underground, in a common soil isolated, was amplified with PCR. A supernatant volume matrix over various time periods. of 5 µL was sufficient for further analysis. The desired PCR We invested in reliable propriety DNA analysis kits products were further processed with the proprietary and software to be probed for the 16 STR loci. The tech- Amicon Ultra-4 Centrifuge Filter Device. nical method employed was through electroferogramic ABI 3130 Genetic Analysis Device, required processing and subsequent electrophoresis profiling. a further 12.5 µL aliquot volume of Hi-Di formamide The tangible findings of our study are expressed (Applied Biosystems); 0.5 µL GenScan 500 LITZ Size as percentage-concordance in reference to the buccal Standard (Applied Biosystems); and 1 µL of ultra refined samples. The 'fresh' dental samples showed concordance PCR product were all mixed together and then run of 79.4%; in 'burned' samples the concordance rate was through the ABI PRISM 3130 (Applied Biosystems) 73.6%; 63.9% for the samples submerged in seawater; device. The samples were mapped out by electrophoresis and, finally 46.7% in those buried in the soil. and electroferogram by GeneMapper ID v. 3.2.1 program In our wider research of the relevant literature, to home-in and identify the 16 STR locus through our a similar study design was adopted by Corte-Real et analyses. al. [10]. In their experimental design, they stratified the tooth samples into four groups. The simulated Results environmental stressors were submersion in sea water; burning; burying in soil; and finally teeth samples kept in In this section the findings, the results and out room temperature. The key difference in their study, was interpretation and analyses of our the data we generated in the method of DNA analysis. Instead of targeting the are presented. Table 1 details the profiles of each of the unique STR segments of DNA we used in this report, they four groups of STR profiles results which we have studied. extracted mtDNA and nuclear DNA, yielding arguably The detailed tabulated data contains the detailed more reliable results [10]. raw data in each of the experimental samples and STR A more recent study by Tilatta et al. [11], locus detection and amplification. The success rate and compared two different approaches of DNA extraction

101 Dogan N.A. et al. Using DNA typing methods to identify dental specimens exposed to different environmental conditions

Table 1. Results. The samples that supplied a complete profile were indicated with ‘‘Yes’’ ,whereas, ‘‘inc’’ means incomplete profile and ‘‘No’’ was indicated when no results were found from genetic analysis. For each sample, the numbers in parenthesis state the loci number completed and the total number analyzed. Air-dried samples Burned samples submerged in the sea water samples Buried samples Sample (21 Samples) (24 samples) (21 samples) (22 samples) 1 Yes(16/16) Inc.(13/16) Yes(16/16) No(0/16) 2 Inc.(13/16) Inc.(10/16) Inc.(1/16) Yes(16/16) 3 Inc.(6/16) Inc.(11/16) Yes(16/16) Inc.(1/16) 4 No(0/16) Inc.(11/16) Inc.(7/16) - 5 Yes(16/16) Inc.(8/16) Inc.(4/16) Inc.(7/16) 6 Yes(16/16) Inc.(13/16) No(0/16) - 7 Inc.(15/16) No(0/16) Inc.(8/16) No(0/16) 8 Inc.(7/16) Inc.(11/16) - Yes(16/16) 9 Yes(16/16) Inc.(12/16) Yes(16/16) Inc.(4/16) 10 Yes(16/16) Inc.(8/16) Inc.(7/16) No(0/16) 11 Yes(16/16) Inc.(13/16) Inc.(12/16) Yes(16/16) 12 Yes(16/16) Inc.(14/16) No(0/16) Yes(16/16) 13 - Yes(16/16) No(0/16) Yes(16/16) 14 Inc.(11/16) Yes(16/16) Yes(16/16) No(0/16) 15 Inc.(15/16) Inc.(15/16) Yes(16/16) Yes(16/16) 16 Inc.(15/16) No(0/16) Yes(16/16) Inc.(6/16) 17 Inc.(9/16) Inc.(7/16) Yes(16/16) No(0/16) 18 Yes(16/16) Yes(16/16) - Yes(16/16) 19 No(0/16) Yes(16/16) Yes(16/16) Inc.(9/16) 20 Yes(16/16) Yes(16/16) - Inc.(8/16) 21 Yes(16716) Inc.(6/16) Yes(16716) 22 Yes(16/16) No(0/16) Yes(16/16) No(0/16) 23 - Yes(16/16) Yes(16/16) Yes(16/16) 24 - Inc.(12/16) Yes(16/16) No(0/16)

Table 2. The number of the samples indicating the complete, incomplete and absent STR profiles and the rate of success calculated by Matlab Version 7.6.0.324 programme. The number of theThe number of theThe number of the Samples samples with complete samples with incomplete samples with absent STR Rate of success STR profile STR profile profile I (air dryed samples) (11)/(21) (8)/(21) (2)/(21) 79.4 II (burnt samples) (6)/(24) (15)/(24) (4)/(24) 73.8 III (submerged in sea water) (11)/(21) (6)/(21) (4)/(21) 63.9 IV (buried underground) (8)/(22) (7)/(22) (7)/(22) 46.7 from dental samples. The first method (same as our of the study. Out of the 32 specimens in the second arm method of initial processing) crushed and rendered of the study, they reported a 75% success rate in DNA the dental specimens into a powder form prior to DNA extraction reliable and fit for analysis [11]. extraction. The second group of teeth samples were Alakoc and Aka used the "orthograde entrance" processed in an arguably more labour intensive fashion. method in 72 archeologically acquired dental specimens. The experimenters utilized in vitro endodontic tooth Fifty eight of these dental specimens analyzed in this boring techniques, to reach the pulp chamber in order to method yielded DNA amplification products. Moreover, extract the DNA samples directly. Results from the first given the historical and archeological significance of these (powdered specimen) group contained 32 specimens. samples, they were also able to avoid irreversible damage Success rate of DNA extraction reported was 9%; or to the teeth, a further advantage for the advocates of this successful in 3 of the specimens. In the same group while technique [12]. In addition to the work that, there is a 24 of the tooth specimens (75%) did not yield any feasible further study worthy of mention [13], as it may give an or usable DNA profiling. This is in stark contrast to the impression that rendering the tooth samples in powder endodontic pulp-chamber DNA extraction method arm form may have an impact on the success of our study.

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Endodontic methods which are utilised to drill through to In conclusion, dental records and molecular the pulp, with subsequent isolation of the DNA has yielded DNA extraction techniques are valuable, credible, and more interpretive, meaningful results. From this it can be robust samples source for DNA identification. The said that the yield of DNA extracted from the samples have forensic utility of the methods that we have studied and dictated the direction of our study and findings. reviewed indicate that there is much space for refinement Our research demonstrates clearly that though and improvement in DNA extraction of samples. The the design of our experiment, DNA acquired for main challenges posed are environmental and DNA the purposes of STR profiling is the poorest in the extraction methods tailored to burned, immersed, buried, underground samples, followed by sea water exposure etc. samples may must be considered. It is estimated that and burnt teeth. Although the increasing efficacy is only the selection of the most appropriate samples, as the encouraging, it is debatable how reliable dental analysis heavily decayed dentition or those which have fillings in through the pulverizing/powder rendering method. site, can adversely effect the process of DNA extraction, is Finally, although we were as meticulous as possible the key point for a successful identification process. (clearing caries, decontamination), it is almost impossible to completely eradicate foreign DNA. (amalgam or 'Fuji Conflict of interest. The authors declare that white'), contain heavy metals and chemicals which would they have no conflict of interest concerning this article. almost certainly effect DNA. Further research could Acknowledgement. This study is supported by improve this promising methodology and we feel that Istanbul University Scientific Research Fund with the this is a paper which will be heavily referenced in the field Reference Number 11183. of forensic science.

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