Engineering of polymerases for the amplification of damaged DNA: applications in paleobiology, archaeology and forensic medicine

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In the absence of repair, lesions accumulate in DNA, eventually corrupting the encoded genetic information. Thus, although DNA may persist in specimens of paleontological, archaeological, or forensic interest for considerable periods of time, it is inevitably damaged. However, not all information is irretrievably lost. Much of it is simply inaccessible because the DNA polymerases commonly used for PCR amplification stall at the sites of damage. Here we propose to use protein engineering and directed evolution technologies to modify polymerases in such a way that they are able to bypass DNA lesions and amplify damaged DNA in PCR. The host laboratory has already shown that in principle the polymerases commonly used for PCR and sequencing (such as Taq DNA polymerase) can be endowed with an ability (although currently limited) to bypass template lesions without compromising processivity. The aim of the project is to further engineer and evolve this ability and explore the application of such novel polymerases for the retrieval of previously inaccessible DNA sequences from samples of prehistoric flora and fauna and from specimens of archaeological interest.