Abstract
In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged1. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding2 of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication3,4 selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000–60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.
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Acknowledgements
M.d'A. was supported by a Medical Research Council studentship and a Junior Research Fellowship from Trinity College, Cambridge, UK. A.V. and R.W. were supported by funds from the NICHD/NIH Intramural Research Program.
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M.d'A. contributed to library construction, CSR selection and, together with M.H. and S.P., to ancient DNA amplification. M.d'A., P.H., A.V. and R.W. contributed to polymerase characterization using template lesions synthesized by D.L., D.G. and J.C. P.H. contributed to the planning and design of the project and manuscript writing.
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Supplementary Figs. 1–5, Supplementary Table 1, Supplementary Methods (PDF 2554 kb)
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d'Abbadie, M., Hofreiter, M., Vaisman, A. et al. Molecular breeding of polymerases for amplification of ancient DNA. Nat Biotechnol 25, 939–943 (2007). https://doi.org/10.1038/nbt1321
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DOI: https://doi.org/10.1038/nbt1321
