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, 3 (5), e2214

Evidence of Authentic DNA From Danish Viking Age Skeletons Untouched by Humans for 1,000 Years


Evidence of Authentic DNA From Danish Viking Age Skeletons Untouched by Humans for 1,000 Years

Linea Melchior et al. PLoS One.


Background: Given the relative abundance of modern human DNA and the inherent impossibility for incontestable proof of authenticity, results obtained on ancient human DNA have often been questioned. The widely accepted rules regarding ancient DNA work mainly affect laboratory procedures, however, pre-laboratory contamination occurring during excavation and archaeological-/anthropological handling of human remains as well as rapid degradation of authentic DNA after excavation are major obstacles.

Methodology/principal findings: We avoided some of these obstacles by analyzing DNA from ten Viking Age subjects that at the time of sampling were untouched by humans for 1,000 years. We removed teeth from the subjects prior to handling by archaeologists and anthropologists using protective equipment. An additional tooth was removed after standard archaeological and anthropological handling. All pre-PCR work was carried out in a "clean- laboratory" dedicated solely to ancient DNA work. Mitochondrial DNA was extracted and overlapping fragments spanning the HVR-1 region as well as diagnostic sites in the coding region were PCR amplified, cloned and sequenced. Consistent results were obtained with the "unhandled" teeth and there was no indication of contamination, while the latter was the case with half of the "handled" teeth. The results allowed the unequivocal assignment of a specific haplotype to each of the subjects, all haplotypes being compatible in their character states with a phylogenetic tree drawn from present day European populations. Several of the haplotypes are either infrequent or have not been observed in modern Scandinavians. The observation of haplogroup I in the present study (<2% in modern Scandinavians) supports our previous findings of a pronounced frequency of this haplogroup in Viking and Iron Age Danes.

Conclusion: The present work provides further evidence that retrieval of ancient human DNA is a possible task provided adequate precautions are taken and well-considered sampling is applied.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Map of Denmark.
The arrow indicates the location of Galgedil in the island of Funen.
Figure 2
Figure 2. Map of the burials at Galgedil.
Galgedil is a large Danish burial ground from the early Viking period. The area in the inner box was excavated from 1999–2002, whereas the outer area was excavated in 2005. The red squares show the graves. The ten subjects analyzed in this work are from the 2005 excavation; subject ID (blue) and mtDNA haplotypes (black) are indicated with arrows. The two areas surrounded by a dotted line have been used as sand pits, which unintentionally may have lead to destruction or removal of graves.
Figure 3
Figure 3. Sampling of teeth for aDNA analysis.
The last layer of soil was removed and two teeth extracted while wearing full body suit, hairnet, gloves, shoe covers, and face masks. The teeth were placed in sealed sterile tubes and transported to the aDNA-lab.
Figure 4
Figure 4. Mini gel patterns of PCR amplified mtDNA.
PCR amplification of a fragment spanning the mtDNA HVR-1 region from nt 16307–16405 (D-fragment) and a fragment (H-fragment) harbouring nt 7028 (nt 6987–7047) using extract from subject G2. Upper row, lanes 1–5, D-fragment: 1, size standard (ΦX174 RF DNA HeaIII-digested); 2, PCR blank; 3, mock extraction; 4, extract; 5, positive DNA control. Lower row, lanes 1–5, H-fragment, same order as for D-fragment. Arrows indicate mtDNA bands.
Figure 5
Figure 5. Alignment of cloned mtDNA sequences (B-segment: nt 16132–16228) from two teeth from subject G10 from Galgedil.
rCRS, revised Cambridge reference sequence ; T1 and T3, tooth #1 and #3 from individual G10. Tooth #1 was untouched by humans prior to sampling while tooth #3 was sampled after archeological and anthropological manipulation. b1-10, clones 1-10 of the B-segment. The transition at nt 16172 was observed with DNA extracts from all three teeth from subject G10 (tooth #2 not shown) and is presumed to be a true substitution in the authentic DNA. Five of the clones from tooth #3 show diverging sequences (b2, b4, b6, b7 and b8) which lack the transition at nt 16172. Four of the diverging sequences harbor a transition at nt 16223 and two of these (b2 and b7) show additional transitions at nt's16187 and 16189. The fifth diverging sequence, b4, is rCRS and lacks any substitutions. The diverging sequences are considered to represent exogenous DNA, but they do not match any of the staffs (Table 2). The substitutions at nt's 16188, 16191 and 16213 are presumed to be the result of scattered post-mortem miscoding lesions, which is a characteristic finding for ancient DNA .
Figure 6
Figure 6. Median-joining network of ancient Danes.
Median-joining network relating 36 HVR-1 sequences (nt 16064–16405) of Danish Viking (Galgedil (10 subjects) and Kongemarken (8 subjects)) and Roman Iron Age (Bøgebjerggård (7 subjects) and Skovgaarde (11 subjects)) samples genotyped for mtDNA haplogroup defining coding region substitutions. Variable sites are shown along the branches of the network. Substitutions at nucleotide positions 11719 and 14766 (shown in parentheses) were inferred from the haplogroup trees drawn using completely sequenced mtDNA genomes , , . Reticulation between haplogroups, e.g. R0a vs JT (16126 parallelism) and U1 vs U7 (16189 parallelism) were solved manually considering phylogenetic analyses based on complete sequence data. L3 is used as the root. Sample codes correspond to Tables 1 and S3; haplogroup labels are shown in grey font.

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