Ancient DNA

Abstract

In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets.

Figure 1

Post-mortem DNA modification in fossil remains, with the structures altered by damage shown in red. (a) Formation of strand breaks (single-stranded nicks) by hydrolytic damage. (i) Direct cleavage of the phosphodiester backbone (A). (ii) Depurination resulting in a baseless site (AP-site) (B) followed by breakage of the sugar backbone through β-elimination (C). Strand breaks are believed to be largely responsible for the short amplification length and the high rate of DNA loss in fossil remains. (b) Different types of crosslink formation. (i) Inter-strand crosslinks by alkylation. (ii) Intermolecular crosslinks by Maillard reaction. Crosslinks may prevent the amplification of endogenous template molecules, increasing the risk of contamination. (c) Oxidative and hydrolytic modification of bases resulting in (i) blocking lesions or (ii) miscoding lesions. Some oxidative damage results in lesions blocking the polymerase enzyme, and promoting chimeric sequences through ‘jumping PCR’. Hydrolytic damage of bases may result in miscoding lesions, for example, deamination of cytosine and adenine to uracil and hypoxathine, respectively. These lesions will result in the incorporation of erroneous bases during amplification.

Figure 2

Key aDNA studies through time. Reports are shown with claimed age, date of publication, type of sequence and whether the results were authenticated by independent replication.

Figure 3

The sequential series of null hypotheses necessary for authenticating aDNA sequences, starting from the PCR reaction. Green boxes show the procedures, blue boxes represent an evaluation of results. Yellow boxes are considerations that need to be taken into account, and grey boxes are suggestions for additional procedures. Green and red arrows indicate a positive and negative result respectively. Blue arrows are suggestions for further action.