Open Access Highly Accessed Research article

Genotyping human ancient mtDNA control and coding region polymorphisms with a multiplexed Single-Base-Extension assay: the singular maternal history of the Tyrolean Iceman

Phillip Endicott1*, Juan J Sanchez2, Irene Pichler3, Paul Brotherton4, Jerome Brooks5, Eduard Egarter-Vigl6, Alan Cooper4 and Peter Pramstaller37

Author Affiliations

1 Museèe de l'Homme, 17 place du Trocadero, 75116 Paris, France

2 Instituto Nacional de Toxicología y Ciencias Forenses, Campus de Ciencas de la Salud,38320 La Laguna, Tenerife, Spain

3 Institute of Genetic Medicine, European Academy of Genetics, 39100 Bolzano-Bozen, Italy

4 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, UK

5 Australian Centre for Ancient DNA, University of Adelaide, Adelaide SA 5005, Australia

6 Department of Pathology, General Regional Hospital, Bolzano-Bozen, Italy

7 Department of Neurology, Lübeck, Germany

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BMC Genetics 2009, 10:29  doi:10.1186/1471-2156-10-29

Published: 19 June 2009



Progress in the field of human ancient DNA studies has been severely restricted due to the myriad sources of potential contamination, and because of the pronounced difficulty in identifying authentic results. Improving the robustness of human aDNA results is a necessary pre-requisite to vigorously testing hypotheses about human evolution in Europe, including possible admixture with Neanderthals. This study approaches the problem of distinguishing between authentic and contaminating sequences from common European mtDNA haplogroups by applying a multiplexed Single-Base-Extension assay, containing both control and coding region sites, to DNA extracted from the Tyrolean Iceman.


The multiplex assay developed for this study was able to confirm that the Iceman's mtDNA belongs to a new European mtDNA clade with a very limited distribution amongst modern data sets. Controlled contamination experiments show that the correct results are returned by the multiplex assay even in the presence of substantial amounts of exogenous DNA. The overall level of discrimination achieved by targeting both control and coding region polymorphisms in a single reaction provides a methodology capable of dealing with most cases of homoplasy prevalent in European haplogroups.


The new genotyping results for the Iceman confirm the extreme fallibility of human aDNA studies in general, even when authenticated by independent replication. The sensitivity and accuracy of the multiplex Single-Base-Extension methodology forms part of an emerging suite of alternative techniques for the accurate retrieval of ancient DNA sequences from both anatomically modern humans and Neanderthals. The contamination of laboratories remains a pressing concern in aDNA studies, both in the pre and post-PCR environments, and the adoption of a forensic style assessment of a priori risks would significantly improve the credibility of results.