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MBE Advance Access published online on March 10, 2004
Molecular Biology and Evolution, doi:10.1093/molbev/msh107
Molecular Biology and Evolution © Society for Molecular Biology and Evolution 2004; all rights reserved
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Accepted February 7, 2004
© 2004 Molecular Biology and Evolution © Society for Molecular Biology and Evolution 2004; all rights reserved.

Original Articles

Spiking of Contemporary Human Template DNA with Ancient DNA Extracts Induces Mutations Under PCR and Generates Non-Authentic Mitochondrial Sequences

Carsten M. Pusch 1 and Lutz Bachmann 2*

1 Institute of Anthropology and Human Genetics, Division of Molecular Genetics, University of Tübingen, Wilhelmstr. 27, D-72074 Tübingen, Germany
2 Department of Zoology, Natural History Museums and Botanical Garden, University of Oslo, P.O. Box 1172 Blindern, N-0318 Oslo, Norway

* To whom correspondence should be addressed. E-mail: bachmann{at}nhm.uio.no.


  Abstract

The proof of authenticity is the greatest challenge in palaeogenetic research and many safeguards have become standard routine in laboratories specialized on ancient DNA research. We describe here an as yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and non-human sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, namely the absence of long amplifiable fragments, is not sufficient. But even more importantly, we find that the extracts induce mutations in a non-random fashion. We have amplified a 148 bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. The subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total 34 positions of a 103 bp alignment are affected and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution at least as long as the molecular basis for the extract-induced mutations is not understood.

Key Words: ancient DNA extracts, mitochondrial DNA, mutation, PCR errors, PCR inhibition


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