Drevna DNK u arheologiji - od arheoloških iskopavanja do laboratorijskih analiza

Abstract

Sa razvojem tehnologije, analize drevne DNK postale su sastavni deo istraživanja u arheologiji, doprinoseći saznanjima vezanim za istoriju populacija. Međutim, analize drevne DNK predstavljaju izazov iz nekoliko razloga. Destruktivne su i skupe, a osim toga, u arheološkom uzorku je prisutna veoma mala količina degradovane i hemijski izmenjene endogene DNK. Nepovoljni sredinski uslovi u kojima je materijal pronađen i skladišten (poput visokih temperatura i vlage) podstiču dalju degradaciju organskog materijala. Dodatni problem je i moguća kontaminacija savremenom DNK, do koje može doći u bilo kom koraku analiza. Optimalno i pažljivije rukovanje uzorcima prilikom iskopavanja i skladištenja istog, dovodi do povećanja količine endogene DNK u uzorku i smanjivanja cene sekvenciranja. Pokazano je da se najveći prinos drevne DNK postiže izdvajanjem iz kalcifikovanog materijala, a posebno unutrašnjeg dela petrozne kosti. Pretvaranje kostiju u prah obavlja se u laboratorijama specijalno posvećenim ovim analizama, u kojima su preduzete određene mere i protokoli kako bi se kontaminacija sprečila. U zavisnosti od istraživačkog pitanja i nakon provere očuvanosti DNK, donosi se odluka da li će se sekvencirati ceo genom i na kojoj dubini ili samo delovi genoma od interesa. U slučaju nedostupnosti jedarne DNK, mitohondrijalna DNK kao i polimorfni genetički markeri na Y hromozomu mogu biti informativni pri određivanju materalnih, tj. pateralnih linija, ali za razliku od jedarne DNK ne mogu pružiti potpunu informaciju o demografskim promenama i populacionoj dinamici. Međutim, kako bismo stvarno razumeli prošlost, neophodna je dobra komunikacija i razmena informacija između arheologa, antropologa, genetičara i lingvista.

With the development of molecular methods, especially Next Generation Sequencing platforms, ancient DNA analyses have been integrated into archaeological analyses so as to help answer the questions about past populations. However, the extraction of ancient DNA (aDNA) from archaeological samples is a very challenging process as the amount of endogenous DNA is often very low due to several reasons. Firstly, aDNA is very badly preserved in samples, especially in warm and humid climates. Even in optimal conditions, such as caves and cold and dry environments, DNA is degraded to shorter fragments and chemically modified. Secondly, besides the DNA of interest, there is a huge amount of exogenous and microbial DNA in samples. And finally, contamination with modern DNA represents a constant threat to the authenticity of the analysis and could be introduced at any step of the analyses, either during excavation, museum storage or laboratory work. Since these methods are destructive and expensive, it is a duty of archaeologists, museum curators as well as biologists, to take certain steps to prevent further degradation of the material and possible contamination. One of the most essential steps is the use of gloves while handling samples. Moreover, samples should not be washed or chemically treated and they should be properly stored in a dry place, without fluctuations in temperature and moisture. The optimal storage and handling could greatly affect the amount of endogenous DNA in samples, which dictates further possibilities of analyses as well as the cost. Researches so far have showed that the inner part of petrous bones yields the highest amount of endogenous DNA, as it is the densest bone in mammals and therefore more resistant to contamination. In the absence of petrous bones, other material such as teeth or hard, but not porous bones could be used as well. Conversion of bones into powder for DNA extraction has to be carried out in special facilities dedicated only to aDNA analysis to prevent contamination. With the development of NGS, it became possible to successfully determine very short and degraded sequences (characteristic for aDNA) even from very small amounts of old and badly preserved samples and to significantly reduce the time of sequencing, as well as its cost. Whether the research includes sequencing of the whole genome at different sequencing depth or capturing specific sequences of the genome depends greatly on DNA preservation, research question and the cost. In addition, mitochondrial DNA and genetic markers on Y chromosome could be very informative of maternal and paternal genetic ancestry, respectively. However, regardless of the research question, it is of the outermost importance that genetic data is observed in context, together with anthropological and archaeological data. It is only in this way that we can obtain reliable information about the past.