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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 26  |  Issue : 4  |  Page : 393-397

DNA detection in tooth exposed to different temperatures: An in vitro study


Department of Oral Medicine and Radiology, SVS Institute of Dental Sciences, Mahabubnagar, Telangana, India

Date of Submission21-Jul-2014
Date of Acceptance09-Apr-2015
Date of Web Publication22-Apr-2015

Correspondence Address:
Rama Raju Devaraju
Department of Oral Medicine and Radiology, SVS Institute of Dental Sciences, Yenugonda, Mahabubnagar - 509 002, Telangana
India
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Source of Support: This study received no external support and was funded by the authors, Conflict of Interest: None


DOI: 10.4103/0972-1363.155681

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   Abstract 

Introduction: Human identification is an important field of study and research in forensic science and aims at establishing human identity. Several biological materials such as bone, hair, a biopsy sample, saliva and blood have been employed in isolation of DNA for human identification. It is possible to obtain DNA from virtually all human body tissues with variations in the quantity and quality of the DNA extracted from each tissue. Aims and Objectives: A study was carried out in our department to detect the presence of DNA from burnt teeth samples at various temperatures and to highlight the importance of DNA obtained from tooth in identifying a deceased in fire accidents. Materials and Methods: The work included 13 extracted teeth of patients who were indicated for therapeutic extraction and those who were diagnosed clinically and radiographically with caries and periodontitis who were indicated for extraction. Out of the 13 extracted teeth, two were decayed (One had class I dental caries C 1 and the other was grossly decayed C 2 ), four were periodontally compromised teeth and the other seven were therapeutically extracted. The freshly extracted teeth were immediately subjected to varying temperatures, from 100°C to 800°C using a Delta burnout furnace for 15-20 minutes. They were cryogenically crushed using a mortar and pestle to make samples of the tooth, which were analysed for DNA. Results: When teeth were incinerated from 100°C-800°C, genomic DNA was obtained only between 100°C and 300°C whereas it was not obtained above this temperature. When the teeth were incinerated from 300°C to 800°C mtDNA was extracted from 300°C to 700°C, but no DNA was obtained above 700°C. Conclusion: Teeth are good sources for DNA, even in cases where the specimens are highly decomposed.

Keywords: DNA, identification, teeth


How to cite this article:
Devaraju RR, Gantala R, Ambati M, Vemula A, Kubbi JR, Gotoor SG. DNA detection in tooth exposed to different temperatures: An in vitro study. J Indian Acad Oral Med Radiol 2014;26:393-7

How to cite this URL:
Devaraju RR, Gantala R, Ambati M, Vemula A, Kubbi JR, Gotoor SG. DNA detection in tooth exposed to different temperatures: An in vitro study. J Indian Acad Oral Med Radiol [serial online] 2014 [cited 2019 Sep 17];26:393-7. Available from: http://www.jiaomr.in/text.asp?2014/26/4/393/155681


   Introduction Top


Disasters such as earthquakes, floods, wildfires, hurricanes, or tornadoes may be caused by nature. In addition, disasters may be human-made, caused by people through mishap or neglect, such as a work accident or an apartment fire, or by deliberate intention, as with terrorism. Mass disasters have been increasing in the recent years, resulting in increased fatality with difficulty in identification of the deceased that poses an important task to the forensics team.

Human identification is an important field of study and research in forensic science and aims at establishing human identity. [1] The discovery of DNA in 1953 by Watson and Crick [2] led to a revolution in nearly all fields of scientific study. Jeffrey et al. [3] in 1985 created radioactive molecular probes that could recognize highly variable regions of DNA which could determine the specific patterns in each individual called DNA fingerprints. The currently performed DNA fingerprinting are accepted as legal proofs in the courts, such as for investigation of paternity and human identification. [4] Several biological materials such as bone, hair, a biopsy sample, saliva, and blood have been employed in isolation of DNA for human identification. It is possible to obtain DNA from virtually all human body tissues with variations in the quantity and quality of the DNA extracted from each tissue. [5],[6]

Fingerprints have been historically utilized for recognition. Not much information can be recovered from body remnants when exposed to elements such as fire, flames, heat, and explosions. [7],[8] However, due to the relatively high degree of physical and chemical resistance of the dental structure to such elements, the teeth play an important role in identification and criminology. [9] The teeth, thus, are an important source of DNA which may help in identification of an individual in case of failure of conventional methods for dental identification. [10]

This study was carried in the department of Oral Medicine and Radiology to detect the temperatures at which DNA can be obtained from burnt teeth samples. As this study was a pilot study and aims at detection of DNA extracted from teeth incinerated at different temperatures, the sample size was small. This study was just a prototype and single objective driven. There are no available standard procedures to incinerate teeth and extract mitochondrial DNA (mtDNA) in the realm of forensic odontology. Moreover there is a significant lack of literature pertaining to recent studies demonstrating mtDNA extraction from burnt teeth.


   Materials and Methods Top


The work included the extracted teeth of patients who were indicated for therapeutic extraction (normal teeth) such as in patients undergoing orthodontic treatment, and those who were diagnosed clinically and radiographically with caries and periodontitis who were indicated for extraction. Patients who were not willing to give consent were excluded from the study. This study was approved by the institutional ethical committee and informed consent was obtained from all the patients who came for extraction. They were informed about the details of the procedure regarding tooth extraction and the study.

Thirteen extracted teeth were collected from the Department of Oral and Maxillofacial surgery. Out of the 13 extracted teeth, two were decayed (One had class I dental caries C 1 and the other was grossly decayed C 2 ), four were periodontally compromised teeth and the other seven were therapeutically extracted. The freshly extracted teeth were immediately subjected to varying temperatures using a Delta burnout furnace for 15-20 minutes [Figure 1]. Two decayed teeth and a therapeutically extracted tooth in this group were subjected to 100°C, whereas each of the four periodontally compromised teeth were subjected to 200°C, 300°C, 400°C, and 500°C, respectively. Each of the remaining therapeutically extracted teeth were subjected to 300°C, 400°C, 500°C, 600°C, 700°C, and 800°C, respectively. The incinerated teeth obtained were kept in a liquid nitrogen container for 48 hours at Palamur Bio Sciences, Mahaboobnagar, Telangana, India. They were cryogenically crushed using a mortar and pestle to make samples of the tooth [Figure 2]. Because of high chances of misidentification, the isolation of each tooth sample from different individuals was carried out separately from start to finish. Before every use the mortar and pestle were autoclaved and heat treated.
Figure 1: Image showing burnt teeth

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Figure 2: Crushed teeth sample

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The teeth samples were sent to Bio-Axis DNA Research Centre, Hyderabad, Telangana, India, for detection of DNA. The following method was used to detect DNA. Seven hundred microliters of lysis buffer [10 mM Tris, pH 8.0, 100 mM NaCl, 50 mM EDTA, pH 8.0, 0.5% SDS pH 8.0 and 20 μL proteinase K (20 mg/mL)] was added to 0.2 g of sample, vortexed, and incubated at 56°C, overnight. Further, 720 μL of extraction buffer (phenol:chloroform:isoamyl alcohol of 25:24:1 ratio) was added to the sample, vortexed, and then centrifuged for 2 minutes at 15000 rpm. The upper aqueous layer was transferred to a sterile microcentrifuge tube. This process of extraction buffer was repeated twice. Later, 720 μL of isobutanol was added to the aqueous layer, vortexed, and centrifuged (2 minutes) at 15000 rpm. After this stage, the lower aqueous layer was used and transferred to the reservoir column of a Centricon-100 concentrator (Millipore). Then, 1 mL of Tris-EDTA buffer for washing was added into the same reservoir column, followed by centrifugation at 3000 rpm for 20 min or until the sample had spun through. This washing step was repeated twice. Finally, the sample in the collection column which contained the mtDNA was transferred to a sterile microcentrifuge tube run on 1.2% agarose gel electrophoresis.

Agarose gel electrophoresis

  1. Forty mililiters of 1.2% agarose gel was prepared and kept in a microwave oven at power level 800 V for 2 minutes for proper dissolving and to get a clear transparent solution.
  2. The agarose solution was allowed to cool at room temperature and 5 μl of ethidium bromide was dissolved in it.
  3. The gel casting tray, chamber and combs were wiped and cleaned with 70% ethanol.
  4. The boundaries of the tray were sealed with 'cello tape' carefully.
  5. The agarose gel was poured into the tray, comb was placed properly and the gel was allowed to solidify for about 20-30 minutes.
  6. After solidification the comb and tape were removed carefully.
  7. The loading samples were prepared by mixing 10 μl of the extracted DNA and 5 μl of loading dye.
  8. The samples were loaded in the corresponding wells made by removing the comb.
  9. The gel was allowed to run for 45 minutes to 1 hour at 100 volts.



   Results Top


The present study showed that DNA was obtained from all the extracted teeth samples (decayed, periodontally compromised and therapeutically extracted teeth). When teeth were incinerated from 100°C to 800°C, genomic DNA was obtained only between 100°C and 300°C whereas it was not obtained above this temperature [Figure 3] and [Table 1]. The amount of DNA yielded from the samples C1, C2 and therapeutically extracted teeth that were exposed to 100°C was 55, 49, and 60 μg/ml, respectively, whereas the samples kept at 200°C and 300°C yielded 41.5 and 39.5 μg/ml of DNA, respectively. As genomic DNA could not be obtained above 300°C, the teeth subjected to higher temperatures were evaluated for mtDNA. When the teeth were incinerated from 300°C to 800°C mtDNA was extracted from 300°C to 700°C, but no DNA was obtained above 700°C [Figure 4]a-b and [Table 2].
Figure 3: The genomic DNA gel doc picture

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Figure 4: The mtDNA gel doc picture of teeth samples exposed to (a) 300-500°C and (b) 600-800°C

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Table 1: Amount of genomic DNA obtained at various temperatures

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Table 2: Amount of mtDNA obtained at various temperatures

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   Discussion Top


Teeth are a preferred source of DNA for several reasons. Due to their unique composition and location within the jaw bones they are largely protected from the environmental and physical conditions that act to accelerate the processes of post-mortem decomposition and DNA decay. [11],[12] Therefore, DNA extracted from teeth are often of high quality [13],[14] and is less prone to contamination than DNA extracted from bones. [15]

This study was carried out with the main objective of isolation of DNA from teeth after subjecting them to different temperatures. Out of the 13 samples taken for the study, DNA was extracted from 12 samples and only one tooth subjected to a temperature of 800°C had not yielded DNA. The Genomic or Nuclear DNA, which is found in the nucleus of each cell and represents the DNA source for most forensic applications, was obtained in five samples subjected to temperatures below 300°C, whereas it was not retrieved above 300°C. The same inference was given in a study done by Vemuri et al. [16]

This study also revealed that, more the tooth is carious less the amount of DNA is obtained, which is, however, sufficient for identification. In addition to genomic DNA, cells contain mtDNA, which can also be used for identification. While analysing samples containing no nuclear DNA, mtDNA analysis was performed in seven teeth by subjecting to temperatures of 300°C and above. We could able to retrieve mtDNA from six teeth when subjected to a temperature from 300 to 700°C, but no DNA was detected in the sample subjected to 800°C. This infers that as the temperature increases, the amount of DNA obtained decreases significantly, which is not sufficient for identification. The reason for detection of mtDNA in teeth exposed to even higher temperatures could be due the presence of mtDNA in more quantity and more robustness to decomposition, due to its cellular location when compared to the nuclear DNA. Thus, It is more likely to be preserved in highly degraded tissues [17] and is especially valuable in missing persons cases, [18],[19] wherein the DNA can be compared with even distant relatives. [20],[21]


   Conclusion Top


In cases of human identification, when there is little remaining material to perform an identification (e.g., in fires, explosions, decomposing bodies, or skeletonized bodies), forensic dentistry plays a very important role. Moreover, teeth are good sources for DNA, even in cases where the specimens are highly decomposed. Also, extraction of DNA from the teeth is cost effective when compared to DNA extraction from bone tissue.

 
   References Top

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Da Silva RH, Sales-Peres A, de Oliveira RN, de Oliveira FT, Sales-Peres SH. Use of DNA technology in forensic dentistry. J Appl Oral Sci 2007;15:156-61.  Back to cited text no. 1
    
2.
Watson JD, Crick FH. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 1953;171:737-8.  Back to cited text no. 2
    
3.
Jeffreys AJ, Wilson V, Thein SL. Hypervariable 'minisatellite' regions in human DNA. Nature 1985;314:67-73.  Back to cited text no. 3
    
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Pötsch L, Meyer U, Rothschild S, Schneider PM, Rittner C. Application of DNA techniques for identification using human dental pulp as a source of DNA. Int J Legal Med 1992;105:139-43.  Back to cited text no. 4
    
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Corach D, Sala A, Penacino G, Iannucci N, Bernardi P, Doretti M, et al. Additional approaches to DNA typing skeletal remains: The search for "missing" persons killed during the last dictatorship in Argentina. Electrophoresis 1997;18:1608-12.  Back to cited text no. 5
    
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Miyajima F, Daruge E, Daruge E Jr. The importance of dental science in human identification: A casework report. Arq Odontol 2001;37:133-42.  Back to cited text no. 8
    
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Morgan OW, Sribanditmongkol P, Perera C, Sulasmi Y, Van Alphen D, Sondorp E. Mass fatality management following the South Asian Tsunami disaster: Case studies in Thailand, Indonesia, and Sri Lanka. PLoS Med 2006;3:e195.  Back to cited text no. 9
    
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11.
Schwartz TR, Schwartz EA, Mieszerski L, McNally L, Kobilinsky L. Characterization of deoxyribonucleic acid (DNA) obtained from teeth subjected to various environmental conditions. J Forensic Sci 1991;36:979-90.  Back to cited text no. 11
    
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Alvarez García A, Muñoz I, Pestoni C, Lareu MV, Rodríguez-Calvo MS, Carracedo A. Effect of environmental factors on PCR-DNA analysis from dental pulp. Int J Legal Med 1996;109:125-9.  Back to cited text no. 12
    
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Alonso A, Andelinoviæ S, Martín P, Sutloviæ D, Erceg I, Huffine E, et al . DNA typing from skeletal remains: Evaluation of multiplex and megaplex STR systems on DNA isolated from bone and teeth samples. Croat Med J 2001;42:260-6.  Back to cited text no. 13
    
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Ricaut FX, Keyser-Tracqui C, Crubézy E, Ludes B. STR-genotyping from human medieval tooth and bone samples. Forensic Sci Int 2005;151:31-5.  Back to cited text no. 14
    
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Gilbert MT, Rudbeck L, Willerslev E, Hansen AJ, Smith C, Penkman KE, et al. Biochemical and physical correlates of DNA contamination in archeological human bones and teeth excavated at Matera, Italy. J Archaeol Sci 2005;32:785-93.  Back to cited text no. 15
    
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Vemuri S, Ramya R, Rajkumar K, Rajashree P. Influence of various environmental conditions on DNA isolation from dental pulp for sex determination using polymerase chain reaction. SRM J Res Dent Sci 2012;3:231-5.  Back to cited text no. 16
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Foran DR. Relative degradation of nuclear and mitochondrial DNA: An experimental approach. J Forensic Sci 2006;51:766-70.  Back to cited text no. 17
    
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Budowle B, Bieber FR, Eisenberg AJ. Forensic aspects of mass disasters: Strategic considerations for DNA-based human identification. Leg Med (Tokyo) 2005;7:230-43.  Back to cited text no. 18
    
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Alvarez-Cubero MJ, Saiz M, Martinez-Gonzalez LJ, Alvarez JC, Eisenberg AJ, Budowle B, et al. Genetic identification of missing persons: DNA analysis of human remains and compromised samples. Pathobiology 2012;79:228-38.  Back to cited text no. 19
    
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Budowle B, Allard MW, Wilson MR, Chakraborty R. Forensics and mitochondrial DNA: Applications, debates, and foundations. Annu Rev Genomics Hum Genet 2003;4:119-41.  Back to cited text no. 20
    
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Ginther C, Issel-Tarver L, King MC. Identifying individuals by sequencing mitochondrial DNA from teeth. Nat Genet 1992;2:135-8.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

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