Abstract
This method is designed to assemble long, continuous DNA sequences using minimal amounts of fragmented ancient DNA as template. This is achieved by a two-step approach. In the first step, multiple fragments are simultaneously amplified in a single multiplex reaction. Subsequently, each of the generated fragments is amplified individually using a single primer pair, in a standard simplex (monoplex) PCR. The ability to amplify multiple fragments simultaneously in the first step allows the generation of large amounts of sequence from rare template DNA, whereas the second nested step increases specificity and decreases amplification of contaminating DNA. In contrast to current protocols using many template-consuming simplex PCRs, the method described allows amplification of several kilobases of sequence in just one reaction. It thus combines optimal template usage with a high specificity and can be performed within a day.
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References
Krause, J. et al. Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae. Nature 439, 724–727 (2006).
Gilbert, M.T.P. Bandelt, H.J., Hofreiter, M. & Barnes, I. Assessing ancient DNA studies. Trends Ecol. Evol. 20, 541–544 (2005).
Cooper, A. & Poinar, H.N. Ancient DNA: do it right or not at all. Science 289, 1139 (2000).
Hofreiter, M., Serre, D., Poinar, H.N., Kuch, M. & Pääbo, S. Ancient DNA. Nat. Rev. Genet. 2, 353–359 (2001).
Pääbo, S. et al. Genetic analyses from ancient DNA. Annu. Rev. Genet. 38, 645–679 (2004).
Malmstrom, H., Stora, J., Dalen, L., Holmlund, G. & Gotherstrom, A. Extensive human DNA contamination in extracts from ancient dog bones and teeth. Mol. Biol. Evol. 22, 2040–2047 (2005).
Lambert, D.M. et al. Rates of evolution in ancient DNA from Adelie penguins. Science 295, 2270–2273 (2002).
Rogaev, E.I. et al. Complete mitochondrial genome and phylogeny of Pleistocene mammoth Mammuthus primigenius. PLoS Biol. 4, e73 (2006).
Höss, M., Jaruga, P., Zastawny, T.H., Dizdaroglu, M. & Pääbo, S. DNA damage and DNA sequence retrieval from ancient tissues. Nucleic Acids Res. 24, 1304–1307 (1996).
Poinar, H.N., Höss, M., Bada, J.L. & Pääbo, S. Amino acid racemization and the preservation of ancient DNA. Science 272, 864–866 (1996).
Smith, C.I. et al. Neanderthal DNA. Not just old but old and cold? Nature 410, 771–772 (2001).
Smith, C.I., Chamberlain, A.T., Riley, M.S., Stringer, C. & Collins, M.J. The thermal history of human fossils and the likelihood of successful DNA amplification. J. Hum. Evol. 45, 203–217 (2003).
Poinar, H.N. & Stankiewicz, B.A. Protein preservation and DNA retrieval from ancient tissues. Proc. Natl Acad. Sci. USA 96, 8426–8431 (1999).
Handt, O., Krings, M., Ward, R.H. & Pääbo, S. The retrieval of ancient human DNA sequences. Am. J. Hum. Genet. 59, 368–376 (1996).
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).
Haak, W. et al. Ancient DNA from the first European farmers in 7500-year-old Neolithic sites. Science 310, 1016–1018 (2005).
Eichinger, L. et al. The genome of the social amoeba Dictyostelium discoideum. Nature 435, 43–57 (2005).
Greenwood, A., Capelli, C., Possnert, G. & Pääbo, S. Nuclear DNA sequences from late pleistocene megafauna. Mol. Biol. Evol. 16, 1466–1473 (1999).
Hummel, S., Schultes, T., Bramanti, B. & Herrmann, B. Ancient DNA profiling by megaplex amplications. Electrophoresis 20, 1717–1721 (1999).
Schultes, T., Hummel, S. & Herrmann, B. Amplification of Y-chromosomal STRs from ancient skeletal material. Hum. Genet. 104, 164–166 (1999).
Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).
Poinar, H.N. et al. Metagenomics to paleogenomics: Large-scale sequencing of mammoth DNA. Science 311, 392–394 (2006).
Kwok, S. et al. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 18, 999–1005 (1990).
Hofreiter, M. et al. Evidence for reproductive isolation between cave bear populations. Curr. Biol. 14, 40–43 (2004).
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).
Leonard, J.A., Wayne, R.K. & Cooper, A. Population genetics of ice age brown bears. Proc. Natl Acad. Sci. USA 97, 1651–1654 (2000).
Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).
Magnuson, V.L. et al. Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning. Biotechniques 21, 700–709 (1996).
Acknowledgements
We thank our lab members, especially C. Stäubert and I. Böselt, for comments that improved the manuscript. This work was funded by the Max Planck Society, the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung.
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Römpler, H., Dear, P., Krause, J. et al. Multiplex amplification of ancient DNA. Nat Protoc 1, 720–728 (2006). https://doi.org/10.1038/nprot.2006.84
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DOI: https://doi.org/10.1038/nprot.2006.84
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