J Forensic Sci, 2014 doi: 10.1111/1556-4029.12568 Available online at: onlinelibrary.wiley.com

TECHNICAL NOTE PHYSICAL ANTHROPOLOGY; ODONTOLOGY

Montserrat Hervella,1,† Ph.D.; Maitane G. I~niguez,2,† M.Sc.; Neskuts Izagirre,1 Ph.D.; on de-la-R ua,1 Ph.D. Alberto Anta,2 Ph.D.; and Concepci

Nondestructive Methods for Recovery of Biological Material from Human Teeth for DNA Extraction*

ABSTRACT: The extraction of DNA from human skeletal remains applied to forensic, and evolutionary studies do not exclude risks, which

are to be evaluated when working with unique specimens that could be damaged or even destroyed. In the present study were evaluated several nondestructive methods for recovering DNA instead of the most currently used pulverization method. Three different procedures to access inside the dental pieces (occlusal perforation, cervical perforation, and cervical cut) have been compared with the aim of recovering as many cell remains as possible to carry out a DNA extraction. Given the DNA quantitation results, a method was proposed that consists of a cervical cut to facilitate the access to the pulp cavity and a subsequent filing of the root canals down to the apex of the dental root. This methodology allows the recovery of both mitochondrial and nuclear DNA, with the minimum deterioration for the dental pieces.

KEYWORDS: forensic science, human teeth, ancient DNA, DNA recovery, mitochondrial DNA, nuclear DNA In the fields of anthropological and forensic research, DNA is potentially obtainable from any tissue. However, the teeth are the skeletal remains of choice due to their high resistance to the action of external physical and chemical agents, reason why they have a preferential preservation in relation to other tissues (1–5). Teeth are therefore an exceptional source of DNA, sometimes the only possible one, such as in certain legal-medical identification processes or evolutionary studies (6–17). In a tooth, DNA is present both in cells composing the dental pulp (fibroblasts, macrophages, and lymphocytes, among others) and in those inside the cementum and the dentin (cementoblasts and odontoblasts). This last array of cells is surrounded by a mineral matrix, which constitutes a protective element against any degrading environment, thus favoring a better preservation of the DNA (18,19). Different techniques have been described to obtain DNA from dental tissue, being the two described here the most frequently used: • Complete crushing or pulverization. This is a destructive technique, as tooth is broken through manual or automated

1

Faculty of Science and Technology, Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Barrio Sarriena s/n, Leioa, Spain. 2 Faculty of Medicine and Odontology, Department of Stomatology I, University of the Basque Country UPV/EHU, Barrio Sarriena s/n, Leioa, Spain. *Supported in part by grant CGL-2007-65515 and CGL-2011-29057 from the Spanish Ministry of Science and Innovation; grant IT542-10 from the Basque Government for Research Groups; and UFI11/09 for Formation and Research Unit of the UPV/EHU. † These authors contributed equally to this work. Received 3 May 2013; and in revised form 22 Oct. 2013; accepted 2 Nov. 2013. © 2014 American Academy of Forensic Sciences



milling (freezer mill), to extract DNA from all cells of dental tissue (20–23). Such technique presents several disadvantages: the big amount of waste produced, which can be a source of contamination on one hand, and on the other hand, of inhibition in the reaction of amplification of DNA and lost of the whole tooth. Longitudinal cut through the teeth, using a very thin disc or saw. The genetic material is extracted after revealing the pulp cavity of the dental piece (24–27). Such technique presents the advantage of being more conservative than the previous one and facilitating the access to the cells of the dental pulp (source of the genetic material) (6,18); but correspondingly, the efficiency in DNA recovery is lower (28).

The knowledge of the anatomy of each tooth is a great help in the selection of the specimens under study and to achieve the maximum yield from the DNA extraction. The pulp cavity of a tooth is generically divided in two parts: the pulp chamber and the root canal. The pulp chamber corresponds to the coronary portion of the pulp cavity; it is single, voluminous, and houses the coronary pulp. The root canal corresponds to the radicular portion of the teeth and has a conical shape, similar to that of the root (29). The widest root canal in the upper molars is the palatine one, and in the lower molars, it is the distal one or the most subsequent. The canine tooth presents only one canal, usually greater, at least in length, than in any other tooth from the same arch, and for this reason, it is an optimum tooth for DNA extraction. The goal of the present study was to develop the conservative techniques for obtaining DNA from dental material, aiming to minimize the damage for specimens with forensic, anthropological, and archaeological value, and to achieve, at the same time, maximum yield in DNA recovery from degraded samples. We 1

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propose the use of techniques based on the specific anatomy of each tooth, accessing to the pulp cavity to recover the existent cells both in the pulp chamber and in the root canals. Besides, the proposed approach makes it easier to recover DNA from subjects with a pulp cavity of a small size due to the dentin deposit in its walls or the building of nodules and calcium needles because of age or other physical, chemical, or bacterial processes. Materials and Methods The study was carried out in a set of 20 teeth from twenty individuals (15 from the present-day and 5 from prehistoric times). The teeth from the present-day, corresponding to two incisors, two canines, and 11 third molars, were recently extracted (one week previous to ADN isolated) in accordance with the corresponding ethic requirements and having the favorable report from the Ethics Board for Researching of the University of the Basque Country (UPV/EHU). Regarding the prehistoric teeth, five-third molars were recovered from the Chalcolithic site of Longar (Navarre) (4456  70 YBP) (30), representing a sample containing a fragmented genetic material. Methodology consisted of the comparison of the three procedures detailed below: (a) Occlusal perforation; a procedure consisting of the perforation of the occlusal surface (upper part of the crown) to gain access to the pulp chamber and the root canals with the endodontic files. This methodology is currently used in odontology, and it is an easy procedure (Fig. 1). (b) Cervical perforation; it is a procedure that also allows to gain access to the pulp cavity, but in this case, it is performed through the tooth neck (the anatomic portion between the crown and the root), thus keeping the dental crown. In this case, it is required to slightly bend the file to gain access to the pulp and root canals (Fig. 1). (c) Cervical cut; it is a less conservative procedure compared to the previous ones, but it allows to gain access to the pulp cavity in a more direct way, as the cut at the neck level divides the tooth in two anatomically well-differentiated parts: crown and root (Fig. 1).

In the first experiment, methods (a) and (b) were compared in a sample set of 10 teeth from present-day, from which five were perforated at coronary level and five were perforated at cervical level. In the second experiment, methods (a) and (c) were compared in a sample set of 10 teeth (five from present-day and five prehistoric times). In each group, two teeth were cut at cervical level (method c) and three were perforated through the crown (method a). Due to the use of prehistoric samples for this study, a minimum number of authentication criteria were observed to ensure the endogenous origin of the recovered DNA (31–34). Besides, a number of precautions were taken in this analysis to avoid the possibility of contamination. In the first place, the DNA extraction and the preparation of the amplification were conducted in a sterile chamber with positive pressure, in which no work with human DNA from present samples had been previously carried out. Likewise, the sterility of the research material and the working areas dedicated to work exclusively with aDNA was maintained through a routine treatment with bleach and UV irradiation. Finally, proper clothes were used at every moment when working with aDNA: cap, gloves, laboratory coat, and face mask for one use only. Aiming to eliminate superficial contamination on dental pieces, they were subjected to a clean process with acids and UV irradiation (35). After such decontamination phase, the pulp chamber was opened using turbine or micro-motor with a tungsten carbide bur, a pyriform bur, or a small circular one (Proclinic, Barcelona, Spain). Once the opening of the pulp chamber was performed, root canals were filed to extract as much cell remains as possible. Ktype endodontic files with different lengths (21, 25, and even 28 mm; Proclinic, Hospitalet de Llobregat, Spain) were used to manipulate curved canals, thus having the option to choose a file according to the length of the tooth being handled. As to the files diameter, they were used gradually from the smallest to the biggest size (with a numbering from 10 to 140, which represents the diameter for the instrument in hundreds of millimeters, in compliance with the ISO encoding) according to the size of the root canals. The canals of the tooth were permeabilized with a lysis buffer (5 mL; 0.5 M EDTA pH 8.0–8.5; 0.5% SDS; 50 mM Tris–HCl pH 8.0; 0.01 mg/mL proteinase K), using an endodontic irrigation syringe used in odontological treatments (Proclinic, Barcelona, Spain). After permeabilization of the tooth, this was incubated in the lysis buffer with agitation for 2 h at 56°C, followed by DNA organic extraction through phenol-chloroform. The obtained DNA extract was concentrated and purified through Centricon-30 (Amicon Bioseparations, Millipore, Bedford, MA).

TABLE 1––mtDNA haplotype (hypervariable segment-I, HVS-I) obtained from five prehistorical samples (Longar site) and from the researchers and archaeologists.

FIG. 1––Scheme of the three DNA recovery procedures tested in the present paper: occlusal perforation, cervical perforation, and cervical cut.

Sample

mtDNA Haplotype (HVS-I)

Researchers and Archaeologists

LO-1 LO-2 LO-3 LO-4 LO-5

16176 16320 16319 16270–16319 16270–16311

#1 #2 #3 #4 #5

mtDNA Haplotype (HVS-I) rCRS 16189 16092–16224–16311 16304 16291

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NONDESTRUCTIVE METHODS FOR DNA RECOVERY FROM TEETH

3

300000 CERVICAL PER. OCCLUSAL PER.

250000

No. molec/μl

200000

150000

100000

50000

0 Incisors (I-1, I-2)

Canines (C-1, C-2)

Third molars ( M-a1, M-a2)

Third molars (M-b1, M-b2)

Third molars (M-c1, M-c2)

FIG. 2––qPCR quantitation: graphical representation of the number of molecules per microliter (No. molec/lL) of a fragment from the HVS-I of the mtDNA, in the DNA extracts recovered through the procedures of occlusal perforation and cervical perforation carried out on teeth from present-day (see i.d. samples in Table 2).

TABLE 2––qPCR quantitation of the number of molecules/lL and ng/lL of a fragment from the HVS-I of the mtDNA, in the DNA extracts recovered through the procedure of occlusal and cervical perforation carried out on teeth from present-day. I.D. Sample I-2 C-1 M-a2 M-b1 M-c2 I-1 C-2 M-a1 M-b2 M-c1

Teeth

Perforation

Incisor (42) Canine (33) 3rd molar 3rd molar 3rd molar Incisor (42) Canine (33) 3rd molar 3rd molar 3rd molar

Cervical Cervical Cervical Cervical Cervical Occlusal Occlusal Occlusal Occlusal Occlusal

HVS-I (No. molec/lL)

HVS-I (ng/lL)

256.879 103.446 143.990 133.990 99.687 165.897 156.485 139.587 156.988 140.090

0.46727 0.188171 0.261922 0.243731 0.181333 0.301771 0.28465 0.253913 0.285565 0.254828

After DNA extraction of dental tissue, teeth were subjected to a very simple odontological restoration (36), only using a selfetching adhesive (AdheSE OneF VivaPen by Ivoclar Vivadent, Ref.73930, Proclinic, Hospitalet de Llobregat, Spain) and a syringe for standard color A3 composite-fluid (Wave MV by SDI, Ref.99496, Proclinic, Barcelona, Spain). The number of amplifiable molecules obtained from the DNA extracts was quantified through quantitative PCR (qPCR), using a TaqMan probe specific for a fragment of 113 bp of length from the hypervariable segment I (HVS-I) of the mitochondrial DNA (mtDNA). The applied methodology is described in detail

in (17). On the other hand, this fragment from the HVS-I of the mtDNA was sequenced (15), and the sex of the individuals was identified through the PCR amplification of the amelogenin gene (AMEL) (16). Finally, the amplified fragments obtained were analyzed on a Bioanalyzer (Agilent Technologies, Santa Clara, CA) to determine the concentration of the final PCR product. To demonstrate the DNA authenticity, the HVS-I haplotypes of the mtDNA obtained in the ancient samples were compared to the haplotypes corresponding to researchers handling them. Results and Discussion In a set of 20 teeth from 20 individuals (15 from the present and five from prehistoric times), a comparison of different procedures to access to the pulp cavity of a tooth was carried out, aiming to extract as much cell remains as possible and to obtain an optimum yield in DNA recovery, preserving the integrity of teeth. Authenticity of the DNA Results in Ancient Samples The DNA authenticity of the prehistoric samples has been demonstrated by the analysis of the mtDNA haplotypes, which were different in all the cases (Table 1). Besides, the mitochondrial haplotypes of the ancient samples are not coincident with the researchers and archaeologists that handled them (Table 1). Finally, some nuclear polymorphisms (Caspase-12 and LCT genes) have been obtained from those ancient samples, whose results were published somewhere else (37,38).

TABLE 3––Bioanalyzer quantitation of the amplified DNA from ten present-day teeth subjected to occlusal and cervical perforation. Amplified DNA from a   SD). fragment of the HVS-I of the mtDNA and the amelogenin (AMEL) gene. Average and standard deviation for both procedures (X I.D. Sample I-2 C-1 M-a2 M-b1 M-c2 I-1 C-2 M-a1 M-b2 M-c1

Teeth Incisor (42) Canine (33) 3rd molar 3rd molar 3rd molar Incisor (42) Canine (33) 3rd molar 3rd molar 3rd molar

Perforation Cervical Cervical Cervical Cervical Cervical Occlusal Occlusal Occlusal Occlusal Occlusal

HVS-I (ng/lL)

  SD HVS-I X

AMEL (ng/lL)

  SD AMEL X

32.56 27.82 25.41 26.26 22.96 44.89 23.56 27.34 20.39 18.41

27  3.5

(XX) 11.36 (XX) 10.36 (XX) 14.46 (XX) 4.44 (XY) 6.35 (XX) 12.36 (XX) 9.44 (XX) 10.20 (XY) 6.47 (XY) 7.95

9.4  4.0

27  10.6

9.3  2.3

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TABLE 4––Bioanalyzer quantitation of the amplified DNA from present-day and prehistoric teeth, subjected to occlusal perforation and cervical cut. Amplified   SD). (OD, DNA from a fragment of the (HVS-I) of the mtDNA and the amelogenin (AMEL) gene. Average and standard deviation of both procedures (X odontology samples; LO, Longar prehistoric site). I.D. Sample

Age

OD-1 OD-2 OD-3 OD-4 OD-5 LO-1 LO-2 LO-3 LO-4 LO-5

Present-day

Prehistoric

Tooth 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd

molar molar molar molar molar molar molar molar molar molar

Procedure

HVS-I (ng/lL)

  SD HVS-I X

AMEL (ng/lL)

  SD AMEL X

Occlusal perforation Occlusal perforation Occlusal perforation Cervical cut Cervical cut Occlusal perforation Occlusal perforation Occlusal perforation Cervical cut Cervical cut

15.90 14.73 20.22 16.19 29.26 3.07 2.48 3.66 4.66 3.65

16.95  9.2

(XX) 9.59 (XY) 10.53 (XX) 9.76 (XX) 8.56 (XY) 12.69 (XY) 0.86 (XY) 0.14 (XY) 0.53 (XX) 1.46 (XX) 2.34

9.96  0.5

22.72  9.2 3.07  0.6 4.15  0.7

a)

TABLE 5––qPCR quantitation of the No. of molecules/lL and ng/lL of a fragment from the (HVS-I) of the mtDNA, from the DNA recover in the extract of present-day and prehistoric teeth, subjected to occlusal perforation and cervical cut.

10.62  2.9 0.51  0.3 1.9  0.6

PRESENT-DAY SAMPLES

250000 OCCLUSAL PER. CERVICAL CUT

OD-1

Age Presentday

Tooth

Procedure

3rd molar

Occlusal perforation Occlusal perforation Occlusal perforation Cervical cut Cervical cut Occlusal perforation Occlusal perforation Occlusal perforation Cervical cut Cervical cut

OD-2

3rd molar

OD-3

3rd molar

OD-4 OD-5 LO-1

3rd molar 3rd molar 3rd molar

Prehistoric

LO-2

3rd molar

LO-3

3rd molar

LO-4 LO-5

3rd molar 3rd molar

HVS-I (No. molec/lL)

AMEL (ng/lL)

105.560

0.19202

134.560

0.24477

145.689

0.26501

143.589 234.566 436.33

0.26119 0.42668 0.00794

343.3

0.00624

367.4

0.00668

150000

100000

50000

0 Third molars (OD-1, OD-4)

Third molars (OD-1, OD-5)

1200

OCCLUSAL PER. CERVICAL CUT

1000

847.3 967.3

0.01541 0.01759

Third molars (OD-3)

PREHISTORIC SAMPLES

b)

800 No. molec/μl

I.D. Sample

No. molec/μl

200000

600 400

Occlusal Perforation Versus Cervical Perforation 200

Occlusal perforation through the crown was carried out in five teeth from present-day individuals, and cervical perforation was performed in another five individuals, aiming to access to the pulp cavity. The number of molecules of the mtDNA from the DNA extracts was quantified through qPCR (Fig. 2 and Table 2). Besides, amplification and subsequent quantitation of amplified DNA were carried out for a fragment from the HVS-l of the mtDNA and for the nuclear marker of the AMEL gene (Table 3). Figure 2 graphically represents the number of molecules of mtDNA recovered with both procedures, having variable results with regards to the yield of DNA. Only in the case of incisors, cervical perforation gives a better recovery of mtDNA molecules, whereas in the rest of the teeth, the yield of the mtDNA molecules is in favor of the occlusal perforation, although the differences were not significant. On the other hand, the amplified PCR products from the DNA extracts were quantified through the Bioanalyzer (Table 3), and the results were very similar for both the 113-pb fragment from the HVS-I of the mtDNA and the AMEL gene; the averages for both procedures did not present significant differences. Having obtained a similar yield in both procedures, occlusal perforation would be the method of choice, as the access to the pulp cavity is easier and implies less tooth handling by the

0 Third molars (LO-1, LO-4)

Third molars (LO-2, LO-5)

Third molars (LO-3)

FIG. 3––qPCR quantitation: graphical representation of the number of molecules per microliter (No. molec/lL) of a fragment from the (HVS-I) of the mtDNA, present in the DNA extracts recovered through the occlusal perforation and cervical cut procedures. (a) Present-day samples and (b) prehistoric samples (see i.d. samples in Table 4).

researcher, both very important issues when considering the need to avoid the contamination of the samples. Occlusal Perforation Versus Cervical Cut The occlusal perforation as a method to access the pulp cavity is compared to the procedure consisting of a cervical cut of the tooth and an access to the pulp cavity through the root canals. In this case, ten teeth were used from individuals with different chronologies (five from present-day and five from prehistoric times) (Tables 4 and 5). Two different procedures were applied for each group of the same chronology: occlusal perforation was

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carried out on six teeth and cervical cut on four teeth. As in the previous methodological comparison, quantitation of the number of molecules of the mtDNA was carried out through qPCR (Fig. 3 and Table 5). In addition, the Bioanalyzer quantitation of the PCR product (HVS-I fragment and nuclear marker of the AMEL gene) was carried out (Table 4). As expected, a greater number of molecules of the mtDNA were detected in the DNA extracts from present-day samples than in those of the prehistoric samples (Fig. 3 and Table 5). Analyzing the results within each of the two groups of samples (present-day and prehistoric times), it can be observed that the number of molecules of the mtDNA yield is greater when accessing to the pulp cavity through cervical cut than when accessing through occlusal perforation (Fig. 3 and Table 5). Regarding the amplified DNA quantitation obtained by the Bioanalyzer, we observe that the average results point to the same direction (Table 4). However, in the case of samples containing degraded DNA (in the present study, the prehistoric teeth), differences in the DNA amplified yield are significantly in favor of cervical cut. Although recovery obtained after DNA extraction is substantially adequate in both procedures as much in ancient as in present-day samples, the cervical cut procedure seems to be more suitable for samples containing degraded DNA, because of the bigger number of DNA molecules recovered and less handling of the dental piece (reducing the possibility of contamination and/or destruction). For dental pieces which are important due to their anthropological and forensic value, the extraction of a sufficient amount of DNA to allow the analysis of nuclear and mitochondrial markers in a reproducible way, minimizing the damage, it is suggested the use of the cervical cut of the tooth and the access to the pulp cavity through endodontic files as the most optimum nondestructive method for DNA recovery. The results obtained for the amount of DNA extracted through different procedures allow us to suggest the use of a cervical cut to facilitate the access to the pulp cavity, and a subsequent filing of the root canals down to the apex of the dental root. The combination of these two methods for the recovery of cells and the extraction of DNA has been applied in several studies by our group with satisfactory results, having obtained not only sequences of the mtDNA (17), but also the genotyping of the SNP for the Caspase-12 (37) and Lactase (38) genes too. This technique to gain access to the inside of the tooth allows to maintain the integrity of the dental pieces and, on the other hand, facilitates the extraction of the cell remains even in dental pieces with a pulp cavity of a small size. Acknowledgment We are grateful to the archaeologists for providing the skeletal remains used in this study. References 1. Kurosaki K, Matsushita T, Ueda S. Individual DNA identification from ancient human remains. Am J Hum Genet 1993;53:638–43. 2. Ricaut FX, Keyser-Tracqui C, Crubezy E, Ludes B. STR-genotyping from human medieval tooth and bone samples. Forensic Sci Int 2005;151:31–5. 3. Woodward S, Weyand NJ, Bunnell M. DNA sequence from Cretaceous period bone fragments. Science 1994;266:1229–32.

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30. Armendariz J, Irigary S. El sepulcro megalıtico de Longar (Viana, Navarra). In: Navarra G, editor. La tierra te sea Leve. Arqueologia de la muerte en Navarra. Navarra, Spain: Gobierno de Navarra Prensa Publicac, 2007;73–7. 31. Cooper A, Poinar HN. Ancient DNA: do it right or not at all. Science 2000;289:1139. 32. Gilbert MT, Willerslev E. Authenticity in ancient DNA studies. Med Secoli 2006;18:701–23. 33. Hofreiter M, Jaenicke V, Serre D, Haeseler AvA, P€a€abo S. DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Res 2001;29:4793–9. 34. P€a€abo S, Poinar H, Serre D, Jaenicke-Despres V, Hebler J, Rohland N, et al. Genetic analyses from ancient DNA. Annu Rev Genet 2004;38:645–79. 35. Ginther C, Issel-Tarver L, King MC. Identifying individuals by sequencing mitochondrial DNA from teeth. Nat Genet 1992;2:135–8. 36. Barbero JG. Conceptos generales sobre obturacion. In: Barbero JG, editor. Patologıa y terapeutica dental. Madrid, Spain: Sintesis Editorial, 2005;323–31.

37. Hervella M, Plantinga TS, Alonso S, Ferwerda B, Izagirre N, Fontecha L, et al. The loss of functional caspase-12 in Europe, is a Pre-Neolithic Event. PLoS ONE 2012;7:e34417. 38. Plantinga TS, Alonso S, Izagirre N, Hervella M, Fregel R, van der Meer JW, et al. Low prevalence of lactase persistence in Neolithic South-West Europe. Eur J Hum Genet 2012;20:778–82. Additional information and reprint requests: Montserrat Hervella, Ph.D. Department of Genetics Physical Anthropology and Animal Physiology University of the Basque Country UPV/EHU Barrio Sarriena s/n 48940 Leioa, Bizkaia Spain E-mail: [email protected]

Nondestructive methods for recovery of biological material from human teeth for DNA extraction.

The extraction of DNA from human skeletal remains applied to forensic, and evolutionary studies do not exclude risks, which are to be evaluated when w...
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