Reverse-root-canal method for extracting aDNA

Teeth, the hardest substance in the human body, are frequently all that remain of a mortuary population from which direct human presence can be gleaned. As such their morphology is invaluable to physical anthropologists and investigators in allied disciplines. Methods currently used for purposes of extracting DNA from dental remains--e.g. bone-milling, crushing, and sectioning--result in total destruction of the teeth. This paper introduces the Reverse-Root-Canal, a protocol by which DNA of molecular weight higher than that obtainable through traditional destructive means, can be obtained from ancient dental remains without harm to the morphologically informative crown and roots.

BACKGROUND OF THE INVENTION
 Teeth, as the most durable tissue in the human body, are often all that
 remain of direct evidence for human occupation of an archaeological site.
 Dental remains are therefore prized by investigators from numerous
 disciplines, including physical anthropologists. Dental anthropologists
 assess teeth for morphological variants that characterize extinct as well
 as those by which extant populations can be identified (Scott and Turner,
 1997).
 Through such studies, connections between early hominids and extant
 primates with current human populations have also been made (Irish, 1998).
 Through analyses of dental use wear patterns, paleoecologists are able to
 reconstruct ancient environments. In so doing, new perspectives regarding
 human physiological as well as cultural adaptations in space and time can
 be gleaned (Walker, 1976; Grine and Kay, 1988). Depositional differences
 among skeletal and dental remains enable taphonomists to recreate early
 hominid paleo-environments (Behrensmeyer, 1975).
 Through the assessment of dental stigmata, paleopathologists are able to
 identify diseases like congenital syphilis, and the existence of
 nutritional stressors among and between members of mortuary populations
 (Jacobi, et al., 1992; Katzenberg, 1993; Hillson, 1996; Scott and Turner,
 1997; Langsjoen, 1998). Culturally motivated dental alterations (ogsley
 and Bellande, 1982; Scott and Turner, 1997; Langsjoen, 1998) in addition
 to environmentally associated occlusal and interproximal wear (Brace,
 1975; Blakely and Beck, 1984; Bullington, 1991; Ungar and Spencer, 1999),
 are also discernable through assessment of dental remains.
 This current study was undertaken ancillary to a multi-disciplinary project
 under the auspices of the Chinese Institute of Archaeology, Chinese
 Academy of Social Sciences, and the Archaeometry Laboratory at the
 University of Minnesota, Duluth. The protocol described herein was devised
 to provide a means by which molecular investigations--e.g. mtDNA
 haplotypes from the Shang Dynasty Heiheru Site at Anyang, China--could
 proceed without compromising the integrity of morphologically informative
 dentition.
 Molecular Investigations of Genetic Composition in China
 To the extent that advances in the burgeoning field of molecular
 archaeology have engendered study of DNA from human remains, ancient DNA
 provides an added dimension to these investigations (Hagelberg, 1994). To
 this end, mitochondrial DNA (mtDNA) investigations (Cavalli-Sforza et al.,
 1967, 1988, 1994, 1998) have bolstered the impact of small migration
 events, such as may occur during trade on otherwise stable gene pools.
 Mitochondrial DNA analyses of Bronze-Age remains in northwestern China,
 Xinjiang (Zhao, 1998) and studies involving blood group antigens
 (Francalacci, 1998) support this hypothesis.
 Even under the most adverse conditions, tissue derived from skeletal and
 dental remains generally contain fewer polymerase chain reaction (PCR)
 inhibitors than do soft tissue remains from the same specimen (Lassen et
 al., 1994). Comparative assays of both skeletal and soft tissue taken from
 Pre-Columbian South America mummies by Lassen et al., (1994), suggest that
 ancient DNA should preferentially be extracted from hard rather than soft
 tissues.
 In dental remains, hydroxyapatite, the inorganic component of osseous
 tissue to which DNA preferentially binds, is present in higher
 concentrations than in skeletal remains. Furthermore, as teeth are
 considerably less susceptible to co-extracted contamination than skeletal
 remains (Zierdt, Hummel, Herrman, 1996), use of dental remains for ancient
 DNA analyses obviates one of ancient DNA study's most problematic concerns
 (Hagelberg, 1994).
 BRIEF SUMMARY OF THE INVENTION
 Since as little as 0.01 of a gram of dentin is required to yield aDNA of
 sufficient molecular height for study (DeGusta, Cook, Sensaubaugh, 1994),
 the Reverse-Root-Canal technique presented herein offers a non-destructive
 alternative to the methods currently practiced. Moreover, in addition to
 its ability to conserve much of the roots and the entire crown, the
 acquisition of aDNA by this method greatly curtails the amount of
 co-extracted contaminants (Smith et al., 1993).

LEGEND FOR FIG. 1
 Lane 1--Size Standard
 Lane 2--Modern Control #1
 Lane 3--#479 Dentin only
 Lane 4--#479 Bone-Milled
 Lane 5--#M30--Dentin Only
 Lane 6--#M30--Bone-Milled
 Lane 7--PCR Control
 Lane 8--#655 Dentin Only
 Lane 9--#655 Bone-Milled
 Lane 10--#667a --Dentin Only
 Lane 11--#667a --Bone-Milled
 Lane 12--PCR Control
 Lane 13--Modern Control #1
 Lane 14--Modern Control #2
 Lane 15--Extraction Control
 METHODS AND MATERIALS
 Population of Study
 The dental remains analyzed and discussed in this paper were collected from
 the Heiheru Site lineage cemeteries from the last capital of the Shang
 Dynasty, Anyang, China. Dating of this Shang mortuary population (1300 BC
 to 1045 BC) was based on radiocarbon dating corroborated with pottery
 seriation (Chang, 1976), and with ancient Chinese writings (Tang, J.,
 personal communication, 1999). Of the 30 teeth collected by J. E. Molto
 during the 1998 and 1999 field seasons, 29 were processed and analyzed in
 accordance with the protocol described herein. Although the 30th sample
 was extensively worn, compounding its poor state of preservation, in the
 main, these teeth were found to be in an excellent state of preservation.
 Inasmuch as the best predictor of the recovery of DNA from ancient remains
 is the preservation of the histological structure of the sample (Colson et
 al., 1996), the high degree of morphological integrity of these remains
 supports the probability that ancient DNA was indeed recovered. While most
 were devoid of carious lesions, many teeth evidenced considerable occlusal
 and interproximal attrition; however none of the pulp cavities were
 perforated.
 Preparation of Individual Teeth for DNA Extraction
 Due to its inherent sensitivity, the polymerase chain reaction (PCR) will
 preferentially amplify more robust biomolecules derived from modern human
 DNA rather than those from the ancient DNA template of interest (Hagelberg
 and Clegg, 1991; Poinar, et al., 1996; Evison, Smillie, Chamberlain,
 1997). To avoid this problem, insofar as possible, elaborate measures (see
 Paabo, et al., 1990) were taken to avoid the incursion of contemporary
 human residues into DNA extractions. Furthermore, as an added precaution
 against exogenous contaminiation, prior to these analyses, I investigated
 the restriction enzyme site pattern by which my own mtDNA is characterized
 (Handt, et al., 1996). In addition to the preparatory treatment of each
 tooth described below, extractions and set-ups for PCR reactions were
 performed in a "clean room" located in a facility separate from that in
 which amplifications and post-PCR analyses were performed (Molto, 1999, in
 press).
 Following removal from the individual sterile bag in which it was stored,
 each tooth was placed under a protective hood flooded with ultraviolet
 light where the following preliminary procedures were performed:
 1. To remove superficial contamination from modern human DNA, each tooth
 was gently brushed with a solution of undiluted bleach applied with a
 soft-bristled tooth brush.
 2. To prevent the bleach from eradicating endogenous ancient residues
 (Schwartz et al., 1991), each tooth was placed in a five ml receptacle
 containing autoclaved double-distilled water for five minutes. Prior to
 further preparation, the tooth was permitted to air dry for five minutes.
 Although skeletal remains have traditionally been subjected to rigorous
 sandblasting to remove remnants of external contaminants prior to
 molecular assays (Hoss and Paabo, 1993), the fragility of dental remains
 requires special care (Schwartz, et al., 1991). To this end, a phosphoric
 acid gel, commonly used in clinical dental, practice, was applied to the
 external surface of the roots in such a manner as to avoid contact with
 the apices: thereby decreasing the possibility of its incursion into the
 root canals. Studies (Tylka, et al., 1994; Ariyaratnam, et al., 1997) have
 demonstrated that surfaces of teeth subjected to sandblasting are
 virtually indistinguishable from those treated with the phosphoric acid
 gel when viewed under a scanning electron microscope (S.E.M.). This
 product was therefore considered efficacious for the purpose of
 non-invasively eliminating surface contamination in lieu of potentially
 damaging sandblasting. The excess gel was gently removed with a Kimwipe,
 and soaked for two minutes in a five ml receptacle containing autoclaved
 double-distilled water. During the five minutes allocated to air drying,
 the size of the apices, for the purposes of selecting the appropriate
 endodontic files, was assessed.
 Selection of Endodontic Files
 Endodontic files, like those used in clinical practice during root-canal
 procedures, were selected in accordance with the general morphology and
 state of preservation of each tooth. To prevent contamination among
 specimens, a set of files was dedicated to each tooth. Prior to accessing
 the apices, lighting under the extraction hood was changed from
 ultraviolet to fluorescent to avoid destruction of fragile ancient
 biomolecules.
 Reverse-Root-Canal, in contrast to clinical root-canal therapy, begins with
 the smallest endodontic files--e.g. #00--to enter the apices. As the
 procedure progresses, slightly larger files (the exact size of which is
 dependant on individual root morphology) were used to gently broaden the
 canals. Tapered, flexible titanium-nickle files were then used to
 gradually advance into the pulp chamber, directly beneath the enameled
 crown, where the dentin affords maximum protection from external
 contaminants.
 As in clinical practice, the files were gently rotated in a clockwise
 fashion to cause minimal impact on root morphology, while gently opening,
 entering, and widening the canals. During this procedure and subsequent
 abrading of the root canals and pulp chamber, a receptacle was placed
 directly under the working area to maximize acquisition of loosened
 dentin.
 The DNA Extraction
 The harvested dentin gas then transferred from the receptacle into a tube
 containing an extraction medium composed of 0.5 EDTA and 1 M Tris (each
 autoclaved at a PH of 8), and Tween 20 (100% polyxysorbitan monolaureate
 of molecular biology grade). In accordance with protocol established by
 Paabo et al., (1990), an extraction control, containing only extraction
 medium, was prepared to confirm the absence of contamination.
 Each tube containing dentin harvested from a single specimen gas then
 inoculated with 75 ul of Proteinase K (Promega). Proteinase K breaks down
 the constituent amino acids to render the DNA more amenable to binding to
 the silica spin columns during subsequent purification. The dentin was
 then incubated overnight at 55.degree. Celsius.
 Purification of the DNA gas accomplished through use of a commercially
 available kit, Wizard, through which a 69% recovery of dsDNA of 200 bp can
 typically be expected. With a bulb transfer pipette, 300 ul of dentin from
 each incubated sample was added to a 100 ul solution of Wizard Direct
 Purification Buffer, which was then added to one ml of resin. Using a
 Leur-lock syringe, the resultant slurry, comprised of the sample, Direct
 Purification Buffer, and resin, was then pushed through a Wizard silica
 mini-column. This procedure was done slowly to maximize binding of the DNA
 to the Wizard silica mini-column. The slurry was then centrifuged for two
 minutes at 10,000 g. DNA trapped within the Wizard silica mini-column was
 eluted with 100 ul of autoclaved double-distilled water. Following the
 addition of four ul of DNA buffer, the sample extract was complete.
 Co-extracted microbacterial DNA, common among severely degraded specimens
 (Evison, Smillie, Chamberlain, 1997; Rollo and Morota, 1999), can inhibit
 PCR amplification. To the extent that this problem can be prevented
 through use of a decreased template concentration, a 1/5 dilution of the
 sample was also made at this time; one ul of DNA buffer and 19 ul of
 autoclaved double-distilled water was added to five ul extracted from the
 purified sample extract.
 PCR Amplification
 The purified extracts, taken from each sample, were then amplified using
 each of the following primers: Alu I 5176, DdeI 10394/Alu I 10397, Hae III
 663, Hinc II 13259 and the 9 base-pair deletion (located between the gene
 that codes for Cytochrome Oxidase and lysine transfer RNA in the
 mitochondrion). An aliquot of Master Mix (composed of DNA buffer, BSA,
 Magnesium sulfate and dNTPs), to which 5 ul of purified sample was added,
 was placed under a hood flooded with ultraviolet light.
 Each mixture was then inoculated with five ul of chosen primer and 7.5 ul
 of Deep Vent polymerase. So as not to denature the delicate polymerase,
 upon its introduction to the mixture, the ultraviolet lighting was
 replaced with fluorescent. Following the addition of 20 ul of Master Mix
 to each well in the microtitre plate destined to receive a sample, five ul
 of each sample was loaded into the designated wells. To account for the
 inherent proclivity of PCR to amplify the more robust biomolecules derived
 from modern DNA, a PCR Control containing only Master Mix, the primers
 appropriate to each assay, and the Deep Vent polymerase gas prepared.
 As previously mentioned, the Clean Room was dedicated to the highly
 sensitive pre-PCR functions. However, the post-PCR facility (where modern
 samples are stored and added to pre-assigned wells), is replete with
 exogenous nucleic acid residues. Thus, as a preventative measure, a drop
 of mineral oil is added to each sell prior to entering the facility.
 To prevent the highly reactive polymerase from initiating the reaction
 outside the thermocycler, thereby allowing the primers to anneal to
 themselves causing primer-dimers, the microtitre plate was carried on ice
 to the post-PCR facility. At this time, modern control samples were added
 to the designated thermal sells prior to placing the microtitre plate into
 the thermocycler.
 Yield Gel and Restriction Enzyme Digests
 Following PCR amplification, each of the amplified samples was
 electrophoresed through a polyacrilamide gel that was treated with
 ethidium bromide, a stain that binds to DNA. Those samples that fluoresce
 when viewed on a transluminator under ultraviolet light were considered to
 have amplified. They were subsequently introduced to restriction enzymes
 complementary to the primers with which they had been amplified. The
 digestion procedure gas accomplished through use of Nanosep filters and
 NEB buffers and enzymes in accordance with standard protocol.
 Results and Discussion
 Comparison of the new protocol to traditional DNA Extraction Bone-Milling
 Of the 30 teeth, 29 were processed in accordance with the protocol
 described in this paper. Of those 30, 23 yielded aDNA of molecular weight
 sufficient for subsequent analyses.
 To substantiate the acquisition of ancient DNA by means of the
 Reverse-Root-Canal procedure, the roots of seven teeth that had
 consistently amplified were subjected to traditional bone-mill preparation
 for DNA extraction. Due to the abundance of DNA available from dentin
 through Reverse-Root-Canal, a substantial amount of dentin remained in the
 tooth for subsequent study via traditional bone-mill preparation. The
 sample cleaning protocol for this second procedure was identical to that
 of the first. The resultant ponder from each tooth was placed in
 Extraction Buffer in accordance with the protocol described above.
 Sutcliffe's (1978) studies of the Escheria coli plasmid, pBR322 (the size
 standard used in this study), suggest that the brightness of bands
 generated electrophoretically from each of the samples is a direct
 indication of the amount of DNA present. Thus, a comparison of the
 brightness of bands generated from dentin extracted via Reverse-Root-Canal
 indicates they contain twice as much ancient DNA as those generated from
 teeth subjected to traditional bone-milling (FIG. 1).
 In addition to its ability to acquire dentin in a non-destructive manner,
 the consistently stronger ancient DNA amplification possible with the
 Reverse-Root-Canal protocol further supports its merit in circumventing
 the deleterious effects of co-extracted microbacterial contaminants
 prevalent in degraded specimens (Ginter, et al., 1992; Evison, Smille and
 Chamberlain, 1997). Furthermore, in addition to the precautions discussed
 earlier, because there were no DNA bands present in either the extraction
 control of the PCR controls (FIG. 1), it can be assumed that no
 contamination was present (Yang, et al, 1998).
 Discussion of the mtDNA Lineage Markers
 Those samples that had amplified successfully were then subjected to each
 of five restriction enzyme digests--DdeI(13394)/AluI(13397), HaeIII(663),
 HincII(13259) and AluI (5176)--acknowledged to be characteristic of extant
 Asian and Asian-derived populations (Ward, et al., 1991; Stone and
 Stoneking, 1993; Wrischnik, et al., 1987; Merriwether, et al., 1996). The
 presence of the 9 base-pair deletion, due to its prevalent association
 with Asian populations, has been referred to as the "Asian Tag" (Shields,
 et al., 1993) and it used by researchers to trace migrations of Asian and
 Asian-derived populations (Shields, et al., 1993). The diagnostic ability
 of this length mutation has, however, come under question following the
 discovery of the "Asian Tag" in several individuals near Glasgow, Scotland
 (Thomas, et al., 1998). Thomas et al.'s (1998) findings suggest this
 length mutation had arisen at least twice within the history of
 human-mitochondrion association (Li, 1997). Alternatively, the mutation
 could be quite ancient and, outside Asian populations, have become quiet
 rare. Watkins, et al.'s (1999) finding of the deletion among individuals
 in southern India supports the presence of such relic mtDNA lineages
 (1999). Inasmuch as the restriction site fragment pattern by which a mtDNA
 lineage is characterized is transmitted exclusively by the females of that
 lineage, should a lineage stop producing females, that lineage, and the
 restriction site fragments with which it is associated, sill also cease.
 Therefore, it must not be ruled out that many such lineages may have
 existed but have not been recognized for lack of diagnostic markers.
 These preliminary findings suggest the presence of such lineages. Analyses
 of ancient DNA from dental specimen #665 provides a case in point.
 Although mitochondria from this specimen have consistently cleaved when
 exposed to restriction enzymes HaeIII(663) and HincII(13259) (suggestive
 of Lineage A1), because the restriction sites for dDeI(10394)/AluI(10397)
 are also present, this individual might be better placed in Lineage D1,
 members of which do not possess the restriction enzyme site for
 HaeIII(663).
 It cannot be overstated that the mtDNA haplotype markers elucidated by
 Merriwether et al., (1996) are representative of extant Asian and
 Asian-derived populations; as such, the may not characterize those now
 extinct. These findings, support previous mtDNA investigations of Anyang
 skeletal remains from which some of the teeth used in this study were
 derived (Graver, 1999). Before attempting to assign a specific lineage to
 remains, it will be essential to ascertain as much information as
 possible, not only from biomolecules or through morphological assessments,
 but through corroboration of other anthropological subdisciplines (e.g.
 archaeology, ethohistory, and linguistics).
 Conclusion
 The Reverse-Root-Canal protocol introduced in this paper provides a means
 by which fragile ancient biomolecules can be extracted from dental remains
 in a non-destructive manner. Furthermore, these data suggest the
 superiority of this protocol to that of traditionally practiced
 destructive methods. The enhanced presence of informative residues results
 from the acquisition of aDNA from dentin afforded maximum protection from
 exogenous contaminants in the canals and beneath the enameled crown
 (Zierdt, Hummel, Herrman, 1996). Therefore, because less organic material
 is required for DNA assays (DeGusta, Cook and Sensabaugh, 1994), this
 protocol will be invaluable in cases where remains are sparse or severely
 degraded (Evison, Smillie, Chamberlain, 1997). Collagen-derived dentin,
 acquired through non-destructive Reverse-Root-Canal, can also provide a
 means by which informative dental remains and associated archaeological
 sites can be radiometrically dated.
 Finally it is hoped that museum curators and archaeologists, in light of
 the protocol described herein, will be less reluctant to part with dental
 specimens, their intact return assured. In so doing the chasm that has
 separated molecular from morphological research since the former's
 inception may be bridged, and bioarchaeology will be able to proceed aided
 by the additional perspective afforded by molecular assays.
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