MBE Advance Access originally published online on July 14, 2004
Molecular Biology and Evolution 2004 21(11):2005-2011; doi:10.1093/molbev/msh208
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Research Article |
PCR-Induced Sequence Alterations Hamper the Typing of Prehistoric Bone Samples for Diagnostic Achondroplasia Mutations
* Institute of Anthropology and Human Genetics, Division of Molecular Genetics, University of Tübingen, Tübingen, Germany; Institut für Ur- und Frühgeschichte, Abteilung Ältere Urgeschichte und Quartärökologie, University of Tübingen, Tübingen, Germany; Institute of Organic Chemistry, University of Tübingen, Tübingen, Germany; Institute of Pathology, Division of Palaeopathology, Academic Teaching Hospital München-Bogenhausen, München, Germany; || Institute of Human Genetics, University of Münster, Münster, Germany; and ¶ Department of Zoology, Natural History Museums and Botanical Garden, University of Oslo, Oslo, Norway
E-mail: bachmann{at}nhm.uio.no.
Achondroplasia (ACH) is a skeletal disorder (MIM100800) with an autosomal dominant Mendelian inheritance and complete penetrance. Here we report the screening of ancient bone samples for diagnostic ACH mutations. The diagnostic G
A transition in the FGFR3 gene at cDNA position 1138 was detected in cloned polymerase chain reaction (PCR) products obtained from the dry mummy of the Semerchet tomb, Egypt (first dynasty, 
4,890–5,050 BP [before present]), and from an individual from Kirchheim, Germany (Merovingian period, 1,300–1,500 BP), both of which had short stature. However, these mutations were also reproducibly observed in four ancient control samples from phenotypically healthy individuals (false-positives), rendering the reliable molecular typing of ancient bones for ACH impossible. The treatment of a false-positive DNA extract with uracil N-glycosylase (UNG) to minimize type 2 transitions (GA/CT) did not reduce the frequency of the false-positive diagnostic ACH mutations. Recently, it was suggested that ancient DNA extracts may induce mutations under PCR. Contemporary human template DNA from a phenotypically healthy individual was therefore spiked with an ancient DNA extract from a cave bear. Again, sequences with the diagnostic GA transition in the FGFR3 gene were observed, and it is likely that the false-positive GA transitions result from errors introduced during the PCR reaction. Amplifications in the presence of MnCl2 indicate that position 1138 of the FGFR3 gene is particularly sensitive for mutations. Our data are in line with previously published results on the occurrence of nonrandom mutations in PCR products of contemporary human mitochondrial HVRI template DNA spiked with ancient DNA extracts.
Key Words: ancient DNA extracts • ancient nuclear DNA • diagenesis • extract-induced mutations • manganese • PCR errors
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