The aim of this initial ancient DNA analysis was two-fold. Firstly, to assess the biomolecular preservation and the presence of endogenous ancient DNA in a representative number of samples and tissues from the remains found at Palace Green. Secondly, to try to match skeletal elements found in the same trench that were suspected to belong to the same individual during excavation but that came back with very different age assessments during osteological analysis.

The stage of development of the bones of Skeleton 28 suggested the individual was an older adolescent or young adult. The development of the cranium, upper limbs and torso were consistent with an age of 16-20 years. The upper and lower jaws were thought to belong to this skeleton during excavation, but because of the damage to the skull the fragments could not be pieced back together to either prove or disprove this.  The upper molars (wisdom teeth) had erupted, suggesting the individual was over 18 years old.  All the dentition showed a high degree of wear, consistent with an age of between 36 – 45 years of age. The wear on the teeth of this individual was particularly striking considering that all other adolescent and young adult skeletons had a minimal amount of wear.   If these teeth do belong to the rest of Skeleton 28 then this is interesting because of the amount of wear observed on the teeth of such a young person.  However, it is important to verify whether the dentition really belongs to Skeleton 28, or whether it belongs to a different individual. This is what we are hoping to discover by analysing the mitochondrial DNA of the bones.

All the analyses were conducted in the new ancient DNA labs of the Department of Archaeology following strict regulations to avoid contamination. Three master students from our MSc in Archaeological Science, Monica Neff, Frank DiRenno and Ashleigh Simpson, and also our archaeological technicians Dr. Beth Upex and Dr. Steve Robertson helped in the sample preparation, extraction and amplification.

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Our new ancient DNA facility (the extraction room)
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Working in the lab requires strict protocols to prevent our DNA from contaminating the archaeological samples

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Sampling

Three samples were taken from the internal part of the petrous portion (pars petrosa) of the temporal bone. This is the densest bone in the body and previous experimental work has demonstrated that it can provide much higher DNA amounts than other skeletal parts (Gamba et al. 2014). The sampling was conducted following the direct advice of the first author of the paper, Cristina Gamba. The internal part of the petrous bone -the densest- was first isolated using a drill, the surface exposed to UV light for 15 minutes on each side to remove contaminant DNA, and then drilled to obtain three 50mg sets of bone powder. DNA was extracted out of these three sets independently using a modification of the protocol of Dabney et al. 2013.

Two teeth from the mandible were also tested. In this case, 4 x 50mg dentin powder samples were obtained by drilling the tooth at the crown (sample 1) and at the root (sample 2). The DNA was extracted from these samples using the same protocol.

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Sampling the petrous portion

DNA analysis

In this initial analysis, we used a PCR approach to target a fragment of the Hypervariable Region of the mitochondrial DNA (mtDNA HVRI). This is a type of DNA found within some small organelles in the cytoplasm of the cell called mitochondria, which are responsible of producing energy for the cell. The number of mitochondria per cell varies depending on the tissue. For example, cells from metabollically active tissues like the liver can contain thousands of these organelles. Each mitochondrion contains on average between 1000 and 10000 copies of mitochondrial DNA. Because the number of copies of mitochondrial DNA per cell is so high, the analysis of mitochondrial DNA in degraded samples is more feasible.

Another particularity of the mtDNA is that it is transmitted through the maternal line. During fertilisation, the mtDNA is exclusively provided by the egg cell, so the offspring get their mitochondrial DNA from their mother. This allows researchers to trace back in time particular mitochondrial DNA lineages and make inferences about bio-geographical origins of human groups. Moreover, mtDNA can be also used in forensic cases where samples across generations need to be compared, like in the identification of the skeletal remains of the Romanov family (Gill et al. 1994).

The drawback of the use of mitochondrial DNA for forensic identification is that its variability is quite low compared to other genetic markers, plus different unrelated individuals can share the same mitochondrial type due to a shared ancestry in a remote past. The power of the identification will depend, ultimately, on how rare a mitochondrial variant is. However, mtDNA is enough to prove exclusion: if two samples display different mtDNA profiles we can confidently state that they belong to two different people.

In parallel to the ancient DNA analysis, buccal swabs were collected from people involved in the analysis of the human remains and the DNA was also extracted and amplified by PCR. The obtained genetic profiles will be compared against the ones collected from the skeletal samples to discard contamination of the remains. This can occur during their excavation and anthropological analysis as a result of sample handling.

Preliminary results

Results obtained so far look very promising. We managed to get a positive amplification out of all 7 of the tested samples!. This means that, in principle, there is mitochondrial DNA in the samples, but we need to obtain the mitochondrial genetic profile in order to establish the origins of this DNA (from the sample or contaminant). Amplified DNA has been already purified and sent to the Durham Genomic Services (DBS Genomics) for Sanger sequencing, and we are currently awaiting for the results. Depending on the outcome of the sequencing, we may apply more sophisticated methods of DNA fingerprinting.

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Seven positive amplifications indicated by the seven orange bands.

Bibliography cited

Dabney, J., Knapp, M., Glocke, I., Gansauge, M.-T., Weihmann, A., Nickel, B., Valdiosera, C., García, N., Pääbo, S., Arsuaga, J.-L., Meyer, M., 2013. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. PNAS 201314445. doi:10.1073/pnas.1314445110

Gamba, C., Jones, E.R., Teasdale, M.D., McLaughlin, R.L., Gonzalez-Fortes, G., Mattiangeli, V., Domboróczki, L., K?vári, I., Pap, I., Anders, A., Whittle, A., Dani, J., Raczky, P., Higham, T.F.G., Hofreiter, M., Bradley, D.G., Pinhasi, R., 2014. Genome flux and stasis in a five millennium transect of European prehistory. Nat Commun 5, 5257. doi:10.1038/ncomms6257

Gill, P., Ivanov, P.L., Kimpton, C., Piercy, R., Benson, N., Tully, G., Evett, I., Hagelberg, E., Sullivan, K., 1994. Identification of the remains of the Romanov family by DNA analysis. Nat. Genet. 6, 130–135. doi:10.1038/ng0294-130

Eva Fernandez-Dominguez

Author: Eva Fernandez-Dominguez

Eva was born and raised in Barcelona, Spain, where she studied Biological Sciences and did a PhD in Palaeogenetics. For her PhD she investigated the genetic impact of the spread of the Neolithic in Europe, looking at the genetic make-up of the first farming communities of the Levant (Syria). Eva spent 7 years working with the Forensic and Population Genetics research group at Complutense University. She then worked on Portugal a post -doc in the the Institute of Archaeology and Palaeosciences. Eva finally arrived in UK in 2012 when she became a Lecturer in Forensic Anthropology at Liverpool John Moores University. In 2015 Eva joined the Archaeology Department at Durham as a Senior Lecturer in ancient DNA where she has been overseeing the setting up of the new ancient DNA laboratories. Eva is leading several projects on ancient human DNA, focusing on the genetic background of the first Near Easter farmers and the transition from the Middle to the Late Neolithic in Iberia . She is also participating in a pilot project from the University of Edinburg on fossil insect DNA.

Mitochondrial DNA analysis