Multiplex amplification of ancient dna

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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OF HIGHLY DEGRADED DNA Article 01 July 2020 REFERENCES * Krause, J. et al. Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae. _Nature_ 439,

724–727 (2006). Article  CAS  Google Scholar  * Gilbert, M.T.P. Bandelt, H.J., Hofreiter, M. & Barnes, I. Assessing ancient DNA studies. _Trends Ecol. Evol._ 20, 541–544 (2005). Article

  Google Scholar  * Cooper, A. & Poinar, H.N. Ancient DNA: do it right or not at all. _Science_ 289, 1139 (2000). Article  CAS  Google Scholar  * Hofreiter, M., Serre, D., Poinar, H.N.,

Kuch, M. & Pääbo, S. Ancient DNA. _Nat. Rev. Genet._ 2, 353–359 (2001). Article  CAS  Google Scholar  * Pääbo, S. et al. Genetic analyses from ancient DNA. _Annu. Rev. Genet._ 38,

645–679 (2004). Article  Google Scholar  * 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). Article  Google Scholar  * Lambert, D.M. et al. Rates of evolution in ancient DNA from Adelie penguins. _Science_ 295, 2270–2273 (2002).

Article  CAS  Google Scholar  * Rogaev, E.I. et al. Complete mitochondrial genome and phylogeny of Pleistocene mammoth _Mammuthus primigenius_. _PLoS Biol._ 4, e73 (2006). Article  Google

Scholar  * 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). Article

  Google Scholar  * Poinar, H.N., Höss, M., Bada, J.L. & Pääbo, S. Amino acid racemization and the preservation of ancient DNA. _Science_ 272, 864–866 (1996). Article  CAS  Google

Scholar  * Smith, C.I. et al. Neanderthal DNA. Not just old but old and cold? _Nature_ 410, 771–772 (2001). Article  CAS  Google Scholar  * 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). Article  Google Scholar  *

Poinar, H.N. & Stankiewicz, B.A. Protein preservation and DNA retrieval from ancient tissues. _Proc. Natl Acad. Sci. USA_ 96, 8426–8431 (1999). Article  CAS  Google Scholar  * Handt, O.,

Krings, M., Ward, R.H. & Pääbo, S. The retrieval of ancient human DNA sequences. _Am. J. Hum. Genet._ 59, 368–376 (1996). CAS  PubMed  PubMed Central  Google Scholar  * 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  * 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  * Eichinger, L. et al. The genome of the social amoeba _Dictyostelium discoideum_. _Nature_ 435, 43–57 (2005). Article  CAS  Google Scholar  * Greenwood, A., Capelli, C.,

Possnert, G. & Pääbo, S. Nuclear DNA sequences from late pleistocene megafauna. _Mol. Biol. Evol._ 16, 1466–1473 (1999). Article  CAS  Google Scholar  * Hummel, S., Schultes, T.,

Bramanti, B. & Herrmann, B. Ancient DNA profiling by megaplex amplications. _Electrophoresis_ 20, 1717–1721 (1999). Article  CAS  Google Scholar  * Schultes, T., Hummel, S. &

Herrmann, B. Amplification of Y-chromosomal STRs from ancient skeletal material. _Hum. Genet._ 104, 164–166 (1999). Article  CAS  Google Scholar  * Margulies, M. et al. Genome sequencing in

microfabricated high-density picolitre reactors. _Nature_ 437, 376–380 (2005). Article  CAS  Google Scholar  * Poinar, H.N. et al. Metagenomics to paleogenomics: Large-scale sequencing of

mammoth DNA. _Science_ 311, 392–394 (2006). Article  CAS  Google Scholar  * 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). Article  CAS  Google Scholar  * Hofreiter, M. et al. Evidence for reproductive isolation between cave bear populations.

_Curr. Biol._ 14, 40–43 (2004). Article  CAS  Google Scholar  * 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  * 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  * 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). Article  CAS  Google Scholar  * 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). Article  CAS  Google Scholar  Download references 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.

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Molecular Biochemistry, Institute of Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, Leipzig, 04103, Germany Holger

Römpler & Torsten Schöneberg * MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK Paul H Dear & Helen Spriggs * Department of Evolutionary Genetics, Max Planck

Institute for Evolutionary Anthropology, Deutscher Platz 6, Leipzig, 04103, Germany Johannes Krause, Matthias Meyer, Nadin Rohland, Mathias Stiller & Michael Hofreiter Authors * Holger

Römpler View author publications You can also search for this author inPubMed Google Scholar * Paul H Dear View author publications You can also search for this author inPubMed Google

Scholar * Johannes Krause View author publications You can also search for this author inPubMed Google Scholar * Matthias Meyer View author publications You can also search for this author

inPubMed Google Scholar * Nadin Rohland View author publications You can also search for this author inPubMed Google Scholar * Torsten Schöneberg View author publications You can also search

for this author inPubMed Google Scholar * Helen Spriggs View author publications You can also search for this author inPubMed Google Scholar * Mathias Stiller View author publications You

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Correspondence to Michael Hofreiter. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS

ARTICLE CITE THIS ARTICLE 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 Download

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