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MBE Advance Access published online on June 15, 2005
Molecular Biology and Evolution, doi:10.1093/molbev/msi195
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© The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org
Accepted June 10, 2005

Research Article

Extensive Human DNA Contamination in Extracts from Ancient Dog Bones and Teeth

Helena Malmström 1*, Jan Storå 2, Love Dalén 3, Gunilla Holmlund 4, and Anders Götherström 1

1 Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Sweden
2 Osteoarchaeological Research Laboratory, Stockholm University, Sweden
3 Department of Zoology, Stockholm University, Sweden
4 The National Board of Forensic Medicine, Department of Forensic Genetics, Faculty of Health Science, Linköping University, Sweden

* To whom correspondence should be addressed.
Helena Malmström, E-mail: helena.malmstrom{at}ebc.uu.se


  Abstract

Ancient DNA (aDNA) sequences, especially those of human origin, are notoriously difficult to analyze due to molecular damage and exogenous DNA contamination. Relatively few systematic studies have focused on this problem. Here we investigate the extent and origin of human DNA contamination in the most frequently used sources for aDNA studies, that is, bones and teeth from museum collections. To distinguish contaminant DNA from authentic DNA we extracted DNA from dog (Canis familiaris) specimens. We monitored the presence of a 148 bp human specific and a 152 bp dog specific mitochondrial DNA (mtDNA) fragment in DNA extracts as well as in negative controls. The total number of human and dog template molecules were quantified using real-time PCR and the sequences were characterized by amplicon cloning and sequencing. Although standard precautions to avoid contamination were taken, we found that all samples from the 29 dog specimens contained human DNA, often at levels exceeding the amount of authentic ancient dog DNA. The level of contaminating human DNA was also significantly higher in the dog extracts than in the negative controls, and an experimental setup indicated that this was not caused by the carrier effect. This suggests that the contaminating human DNA mainly originated from the dog bones rather than from laboratory procedures. When cloned, fragments within a contaminated PCR product generally displayed several different sequences, although one haplotype was often found in majority. This leads us to believe that recognized criteria for authenticating ancient DNA cannot separate contamination from ancient human DNA the way they are presently used.

Keywords: ancient DNA; authentication; mitochondrial DNA; quantitative PCR.
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