Ancient DNA can be defined as DNA extracted from dead organisms or their body parts, independently of their age and storage period.

The invention of polymerase chain reaction (PCR) in late eighties revolutionized molecular biology and molecular archaeology. The main concept of this process is the possibility of enzymatic in vitro synthesis of practically unlimited copies number of any DNA fragment, using only a few copies of target DNA as a template.

The high sensitivity of PCR results in the main problem of easy contamination of specimens being studied with modern DNA. All stages of the work with ancient DNA must be supplied with blank reactions. To avoid possible contamination, all procedures were carried out in laminar flow hood, using a separate set of pipettes and plasticware. All reagents were routinely tested for probable contamination. For the exact modeling of ancient DNA extraction, we drilled holes in plastic test tubes in the same manner as we did in bones. The "negative" templates were extracted from this polypropylene powder with the same reagents as the ancient DNA from bones. These "negative" templates were used for negative controls setup in PCR.

For the extraction of ancient DNA, the femoral bones of three individuals with the best preservation level were used - Sunghir 1, Sunghir 2 and Sunghir 3.

We successfully amplified the fragments of mtDNA HVSI of individuals S2 and S3. This indicates the excellent preservation of SUNGHIR bone remains. The results of our preliminary analysis showed the nucleotide sequences of mtDNA HVSI for the two buried SUNGHIR adolescents are identical. Their sequence is a derivative of so-called "Cambridge" mtDNA sequence, differing from it probably only in one position (G-A transversion in position 16129). The identity of individuals' S2 and S3 mtDNA HVSI nucleotide sequences might point to their kinship by maternal lineage. However, this probability cannot be evaluated without the knowledge of this mtDNA pattern type frequency in SUNGHIR paleopopulation.

Gender typing

For the identification of individuals S1, S2 and S3 gender amplification of locus DYZ1, linked with chromosome Y, was performed. This locus is present in the cell in large copies - about 800-1500. The scheme of gender typing with this method is very simple: if, after the PCR reaction, the Y-specific amplification product is present in the sample, then the Y chromosome is present in ancient DNA of the individual and hence, the individual is of male gender. In case of absence of this specific signal the individual is probably a woman.

The unambiguous identification of the studied remains' gender by this method seems to be impossible, because the possibility of the absence of Y chromosome residue due to high degradation of the target sequence is not avoided. Some methods are described, allowing the simultaneous amplification of both X and Y chromosome specific loci. These loci are the parts of the unique X-Y homological nuclear gene amelogenin. Our previous results showed that the system based on amplification of highly repeated locus DYZ1 is much more effective for work with ancient material than the systems based on amplification of gender chromosomes unique sequences (unpublished data).

The results of electrophoretic separation of DYZ1 locus amplification products in ancient DNA specimens S1, S2 and S3 are shown in picture 1.
CDRThe Y chromosome specific fragment is visible only in lanes 3 and 4 (specimens S1 and S2 respectively). In lane 5 (specimen S3) the product of expected length is absent.

The obtained data suggests that adult S1 and adolescent S2, buried on Sunghir station, are males.

The absence of a Y chromosome - specific signal in DNA from the S3 child indicates its XX karyotype. Good levels of DNA conservation in this sample is confirmed by successful amplification of a 453 b.p. - long mtDNA HVSI fragment from the same ancient DNA template. Thus, the absence of Y chromosome specific amplification product is connected with the absence of chromosome Y in used biological material, but not with its complete degradation. These facts allow us to assert that the individual S3 was a young female.