The analysis of ancient DNA from MH sites has two goals :
- The first goal is to establish the sex of the
deceased. This method is particularly useful in cases where the traditional
morphological analysis of bones cannot provide sufficient information,
either because the skeletons are poorly preserved or because they are
immature.
This is certainly the case in intramural cemeteries where graves are often disturbed and contain a high proportion of infants and children. Extramural cemeteries are also often poorly preserved because of the later habitation of the sites. - The second
goal is to detect family relationships.
This can only be achieved with DNA analysis.
In the case of the MH period, reconstructing family relationships is particularly interesting for two reasons: Both intramural and extramural MH cemeteries are divided in small groupings - and intramural burials are often associated with specific houses (the graves are dug under or between houses). DNA analysis allows us to establish whether these burial groupings and households were related with kinship ties.
Second, reconstructing the network of kinship relations in MH communities which do not display overt signs of social differentiation, allows us to establish the significance of kinship in social life.
 Grave J2, double burial, Lerna (Courtesy C. Zerner |
The analysis is undertaken by Sofia
Kouidou-Andreou, Leda
Kovatsi and Dimitra Nikou at the Laboratory of Biological Chemistry, Medical School,
Aristotle University of Thessaloniki.
First, a pilot analysis of
12 samples from Lerna has been carried out. The results are extremely encouraging. Samples from Asine have recently been taken, and will be analyzed in the next few months. Depending on the outcome of these analyses, we will apply for permission
to undertake a more extensive sampling programme. We would
like to sample a large number of skeletons which will be selected
on the basis of primarily archaeological criteria. The intention is
to include men, women and children, as well as different wealth categories
and modes of disposal from each burial group. The preservation of the
skeletons is, of course, also an important consideration. This careful approach is necessary because of the significance of skeletal assemblages and the need to preserve them for future generations of researchers. We are planning
to sample teeth as they are a more appropriate source of genetic material,
but we may consider using long bones if teeth are not available.
The following method is used: In order to extract DNA
(mitochondrial and genomic), teeth will be pulverized after their
surface has been extensively cleaned and contaminants have been removed.
DNA
will be isolated from the pulverized sample by means of a technique developed
in our laboratory. This protocol involves extensive digestion of
the sample with proteinase K and extraction of DNA from the digest followed
by purification of the extract.
Genomic DNA is used for the identification
of sex. The amelogenin locus is amplified with the help of specific
commercial kits (Sullivan et al 1993).
In order to establish family relationships,
mitochondrial DNA (mtDNA) is studied. Hypervariable control
regions I and II are amplified, each in two overlapping fragments,
and the amplicons are being sequenced.
In order to overcome
the problem of contamination with contemporary DNA, the samples in
our laboratory are handled and processed under very stringent conditions.
Tight segregation is employed between pre-PCR and post-PCR areas.
DNA extractions and PCR setups are carried out in a dedicated biosafety
hood that is located separately from that used for post-PCR analysis.
The use of positive controls is avoided. The extractions are always
carried out for each sample separately, while one negative extraction
control (extraction blank) is also being included. The sample extract
and the extraction blank, plus one negative reagent control (PCR
blank) are subjected to PCR amplification. The batches (extraction
blank, PCR blank, sample) in which the extraction blank or PCR blank
give a positive result, are rejected.
aDNA analysis is prone to PCR-introduced errors due to both
DNA damage and inappropriate enzyme activity.
In order to ensure that our results are genuine, the target sequences
are amplified and sequenced twice and in some cases the whole procedure
is applied to two different DNA extracts of the same specimen.
 Group of graves (17, 20, 21, 24) Lerna (Courtesy C. Zerner) |
The aDNA analysis from MH Lerna serves a further goal: to develop and promote ancient DNA analysis in Greece, a country which is very rich in archaeological remains and where the analysis of human skeletal remains is gradually becoming an important growth area.
