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Ancient DNA extraction from bones and teeth

Nature Protocols volume 2, pages 1756–1762 (2007) Cite this article

Abstract

This method is designed to maximize recovery of PCR-amplifiable DNA from ancient bone and teeth specimens and at the same time to minimize co-extraction of substances that inhibit PCR. This is achieved by a combination of DNA extraction from bone powder using a buffer consisting solely of EDTA and proteinase K, and purification of the DNA by binding to silica in the presence of high concentrations of guanidinium thiocyanate. All steps are performed at room temperature (20–23 °C), thereby reducing further degradation of the already damaged and fragile ancient DNA and providing an optimal trade-off between DNA release and degradation. Furthermore, the purification step removes most of the various types of PCR inhibitors present in ancient bone samples, thereby optimizing the amount of ancient DNA available for subsequent enzymatic manipulation, such as PCR amplification. The protocol presented here allows DNA extraction from ancient bone and teeth with a minimum of working steps and equipment and yields DNA extracts within 2 working days.

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References

  1. Noonan, J.P. et al. Genomic sequencing of Pleistocene cave bears. Science 309, 597–599 (2005).

    Article  CAS  Google Scholar 

  2. Noonan, J.P. et al. Sequencing and analysis of Neanderthal genomic DNA. Science 314, 1113–1118 (2006).

    Article  CAS  Google Scholar 

  3. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).

    Article  CAS  Google Scholar 

  4. Poinar, H.N. et al. Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA. Science 311, 392–394 (2006).

    Article  CAS  Google Scholar 

  5. Green, R.E. et al. Analysis of one million base pairs of Neanderthal DNA. Nature 444, 330–336 (2006).

    Article  CAS  Google Scholar 

  6. Rogaev, E.I. et al. Complete mitochondrial genome and phylogeny of Pleistocene mammoth Mammuthus primigenius. PLoS Biol. 4, e73 (2006).

    Article  Google Scholar 

  7. Geigl, E.-M. On the circumstances surrounding the preservation and analysis of very old DNA. Archaeometry 44, 337–342 (2002).

    Article  CAS  Google Scholar 

  8. Pääbo, S. Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proc. Natl. Acad. Sci. USA 86, 1939–1943 (1989).

    Article  Google Scholar 

  9. Höss, M., Dilling, A., Currant, A. & Pääblo, S. Molecular phylogeny of the extinct groundsloth Mylodon darwinii. Proc. Natl. Acad. Sci. USA 93, 181–185 (1996).

    Article  Google Scholar 

  10. Hofreiter, M., Jaenicke, V., Serre, D., Haeseler Av, A. & Pääbo, S. DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Res. 29, 4793–4799 (2001).

    Article  CAS  Google Scholar 

  11. Hansen, A.J. et al. Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments. Genetics 173, 1175–1179 (2006).

    Article  CAS  Google Scholar 

  12. Rohland, N. & Hofreiter, M. Comparison and optimization of ancient DNA extraction. Biotechniques 42, 343–352 (2007).

    Article  CAS  Google Scholar 

  13. Höss, M. & Pääbo, S. DNA extraction from Pleistocene bones by a silica-based purification method. Nucleic Acids Res. 21, 3913–3914 (1993).

    Article  Google Scholar 

  14. Hänni, C., Brousseau, T., Laudet, V. & Stehelin, D. Isopropanol precipitation removes PCR inhibitors from ancient bone extracts. Nucleic Acids Res. 23, 881–882 (1995).

    Article  Google Scholar 

  15. Kalmar, T., Bachrati, C.Z., Marcsik, A. & Rasko, I. A simple and efficient method for PCR amplifiable DNA extraction from ancient bones. Nucleic Acids Res. 28, E67 (2000).

    Article  CAS  Google Scholar 

  16. Hofreiter, M. et al. Evidence for reproductive isolation between cave bear populations. Curr. Biol. 14, 40–43 (2004).

    Article  CAS  Google Scholar 

  17. Rompler, H. et al. Nuclear gene indicates coat-color polymorphism in mammoths. Science 313, 62 (2006).

    Article  CAS  Google Scholar 

  18. Hofreiter, M., Serre, D., Poinar, H.N., Kuch, M. & Pääbo, S. Ancient DNA. Nat. Rev. Genet. 2, 353–359 (2001).

    Article  CAS  Google Scholar 

  19. Pääbo, S. et al. Genetic analyses from ancient DNA. Annu. Rev. Genet. 38, 645–679 (2004).

    Article  Google Scholar 

  20. Willerslev, E. & Cooper, A. Ancient DNA. Proc. Biol. Sci. 272, 3–16 (2005).

    Article  CAS  Google Scholar 

  21. Roempler, H. et al. Multiplex amplification of ancient DNA. Nat. Protoc. 1, 720–728 (2006).

    Article  CAS  Google Scholar 

  22. Leonard, J.A., Wayne, R.K. & Cooper, A. Population genetics of ice age brown bears. Proc. Natl. Acad. Sci. USA 97, 1651–1654 (2000).

    Article  CAS  Google Scholar 

  23. Haak, W. et al. Ancient DNA from the first European farmers in 7500-year-old Neolithic sites. Science 310, 1016–1018 (2005).

    CAS  PubMed  Google Scholar 

  24. Shapiro, B. et al. Rise and fall of the Beringian steppe bison. Science 306, 1561–1565 (2004).

    Article  CAS  Google Scholar 

  25. Serre, D. et al. No evidence of neandertal mtDNA contribution to early modern humans. Plos Biol. 2, 313–317 (2004).

    Article  CAS  Google Scholar 

  26. Orlando, L. et al. Revisiting Neanderthal diversity with a 100,000 year old mtDNA sequence. Curr. Biol. 16, R400–R402 (2006).

    Article  CAS  Google Scholar 

  27. Caramelli, D. et al. A highly divergent mtDNA sequence in a Neanderthal individual from Italy. Curr. Biol. 16, R630–R632 (2006).

    Article  CAS  Google Scholar 

  28. Iudica, C.A., Whitten, W.M. & Williams, N.H. Small bones from dried mammal museum specimens as a reliable source of DNA. Biotechniques 30, 732–736 (2001).

    Article  CAS  Google Scholar 

  29. Vigilant, L., Hofreiter, M., Siedel, H. & Boesch, C. Paternity and relatedness in wild chimpanzee communities. Proc. Natl. Acad. Sci. USA 98, 12890–12895 (2001).

    Article  CAS  Google Scholar 

  30. Schander, C. & Halanych, K.M. DNA, PCR and formalinized animal tissue—a short review and protocols. Org. Divers. Evol. 3, 195–205 (2003).

    Article  Google Scholar 

  31. Poinar, H.N. et al. Molecular coproscopy: dung and diet of the extinct ground sloth Nothrotheriops shastensis. Science 281, 402–406 (1998).

    Article  CAS  Google Scholar 

  32. Reynolds, M.M. & Williams, C.G. Extracting DNA from submerged pine wood. Genome 47, 994–997 (2004).

    Article  CAS  Google Scholar 

  33. Ou, C.Y., Moore, J.L. & Schochetman, G. Use of UV irradiation to reduce false positivity in polymerase chain reaction. Biotechniques 10, 442, 444, 446 (1991).

    CAS  PubMed  Google Scholar 

  34. Gilbert, M.T.P., Hansen, A.J., Willerslev, E., Turner-Walker, G. & Collins, M. Insights into the processes behind the contamination of degraded human teeth and bone samples with exogenous sources of DNA. Int. J. Osteoarchaeol. 16, 156–164 (2006).

    Article  Google Scholar 

  35. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

    Google Scholar 

  36. Tuma, R.S. et al. Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators. Anal. Biochem. 268, 278–288 (1999).

    Article  CAS  Google Scholar 

  37. Singer, V.L., Jones, L.J., Yue, S.T. & Haugland, R.P. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal. Biochem. 249, 228–238 (1997).

    Article  CAS  Google Scholar 

  38. Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M. Real time quantitative PCR. Genome Res. 6, 986–994 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the members of the MPI EVA ancient DNA groups and Holger Römpler for discussion. This work was funded by the Max Planck Society.

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  1. rohland@eva.mpg.de

Authors and Affiliations

  1. Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, Leipzig, D-04103, Germany

    Nadin Rohland & Michael Hofreiter

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  1. Nadin Rohland
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  2. Michael Hofreiter
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The authors declare no competing financial interests.

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Anticipated results. (PDF 42 kb)

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Rohland, N., Hofreiter, M. Ancient DNA extraction from bones and teeth. Nat Protoc 2, 1756–1762 (2007). https://doi.org/10.1038/nprot.2007.247

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