Molecular breeding of polymerases for amplification of ancient DNA (original) (raw)

Nature Biotechnology volume 25, pages 939–943 (2007)Cite this article

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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Ancient DNA analysis

Article 11 February 2021

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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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  1. ph1@mrc-lmb.cam.ac.uk

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK
    Marc d'Abbadie, David Loakes & Philipp Holliger
  2. Department of Evolutionary Genetics, Max Planck Institute for Molecular Anthropology, Deutscher Platz 6, Leipzig, D-04103, Germany
    Michael Hofreiter & Svante Pääbo
  3. Section on DNA Replication, Repair and Mutagenesis, Building 6, Room 1A13, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, 20892-2725, Maryland, USA
    Alexandra Vaisman & Roger Woodgate
  4. Département de Recherche Fondamentale sur la Matière Condensée, Laboratoire Lésions des Acides Nucléiques, SCIB/UMR-E 3 (CEA/UJF), Commissariat à l'Énergie Atomique (CEA)/Grenoble, Grenoble, F-38054, Cedex 9, France
    Didier Gasparutto & Jean Cadet

Authors

  1. Marc d'Abbadie
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  2. Michael Hofreiter
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  3. Alexandra Vaisman
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  4. David Loakes
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  5. Didier Gasparutto
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  6. Jean Cadet
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  7. Roger Woodgate
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  8. Svante Pääbo
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  9. Philipp Holliger
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Contributions

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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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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