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Published online on August 25, 2006, 10.1073/pnas.0605327103
PNAS | September 12, 2006 | vol. 103 | no. 37 | 13578-13584


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Inaugural Article
BIOLOGICAL SCIENCES / GENETICS
Patterns of nucleotide misincorporations during enzymatic amplification and direct large-scale sequencing of ancient DNA

M. Stiller*, R. E. Green*, M. Ronan{dagger}, J. F. Simons{dagger}, L. Du{dagger}, W. He{dagger}, M. Egholm{dagger}, J. M. Rothberg{dagger}, S. G. Keates{ddagger}, N. D. Ovodov§, E. E. Antipina, G. F. Baryshnikov||, Y. V. Kuzmin**, A. A. Vasilevski{dagger}{dagger}, G. E. Wuenschell{ddagger}{ddagger}, J. Termini{ddagger}{ddagger}, M. Hofreiter*, V. Jaenicke-Després*, and S. Pääbo*,§§

*Max Planck Institute for Evolutionary Anthropology, D-04103 Leipzig, Germany; {dagger}454 Life Sciences, Branford, CT 06405; {ddagger}P.O. Box 350, London WC1, United Kingdom; §Institute of Archaeology and Ethnography, Siberian Branch of the Russian Academy of Sciences, Krasnoyarsk 660049, Russia; Institute of Archaeology, Russian Academy of Sciences, Moscow 117036, Russia; ||Zoological Institute, Russian Academy of Sciences, St. Petersburg 199034, Russia; **Pacific Institute of Geography, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690041, Russia; {dagger}{dagger}Sakhalin State University, Yuzhno-Sakhalinsk 693008, Russia; and {ddagger}{ddagger}Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010

Contributed by S. Pääbo, June 26, 2006

Whereas evolutionary inferences derived from present-day DNA sequences are by necessity indirect, ancient DNA sequences provide a direct view of past genetic variants. However, base lesions that accumulate in DNA over time may cause nucleotide misincorporations when ancient DNA sequences are replicated. By repeated amplifications of mitochondrial DNA sequences from a large number of ancient wolf remains, we show that C/G-to-T/A transitions are the predominant type of such misincorporations. Using a massively parallel sequencing method that allows large numbers of single DNA strands to be sequenced, we show that modifications of C, as well as to a lesser extent of G, residues cause such misincorporations. Experiments where oligonucleotides containing modified bases are used as templates in amplification reactions suggest that both of these types of misincorporations can be caused by deamination of the template bases. New DNA sequencing methods in conjunction with knowledge of misincorporation processes have now, in principle, opened the way for the determination of complete genomes from organisms that became extinct during and after the last glaciation.

pyrosequencing | deamination | DNA damage | wolves | mammoth

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 20, 2004.

Author contributions: M.H., V.J.-D., and S.P. designed research; M.S., M.R., J.F.S., L.D., W.H., M.E., and J.M.R. performed research; G.E.W. and J.T. contributed new reagents/analytic tools; M.S., R.E.G., and S.P. analyzed data; M.S., R.E.G., and S.P. wrote the paper; and S.G.K., N.D.O., E.E.A., G.F.B., Y.V.K., and A.A.V. contributed paleontological specimens.

Conflict of interest statement: No conflicts declared.

Data deposition: The sequences reported in this paper have been deposited in the GenBank (accession nos. DQ852634–DQ852662) and EMBL (accession nos. CAAM01000001–CAAM01073172) databases.

See accompanying Profile on page 13575.

§§To whom correspondence should be addressed. E-mail: paabo{at}eva.mpg.de

© 2006 by The National Academy of Sciences of the USA


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