PCR-induced sequence alterations hamper the typing of prehistoric bone samples for diagnostic achondroplasia mutations

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, approximately 4,890-5,050 BP [before present]), and from an individual from Kirchheim, Germany (Merovingian period, approximately 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 (G-->A/C-->T) 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 G-->A transition in the FGFR3 gene were observed, and it is likely that the false-positive G-->A transitions result from errors introduced during the PCR reaction. Amplifications in the presence of MnCl(2) 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. -->

ISSN:	0737-4038
Přístupová URL adresa:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9e0553fd0a6ad5318ec868b5ae9e1f4a https://pubmed.ncbi.nlm.nih.gov/15254256
Rights:	OPEN
Přírůstkové číslo:	edsair.doi.dedup.....9e0553fd0a6ad5318ec868b5ae9e1f4a
Autor:	Martina Broghammer, N. Blin, Albert Zink, Carsten M. Pusch, I. Kennerknecht, Graeme J. Nicholson, Andreas G. Nerlich, Lutz Bachmann
Rok vydání:	2004
Předmět:	DNA Complementary Sequence analysis Paleopathology Biology medicine.disease_cause DNA Mitochondrial Polymerase Chain Reaction law.invention Achondroplasia DNA Glycosylases Specimen Handling Evolution Molecular chemistry.chemical_compound Skeletal disorder law Complementary DNA Germany Genetics medicine Humans Point Mutation Receptor Fibroblast Growth Factor Type 3 Cloning Molecular Uracil-DNA Glycosidase Molecular Biology Ecology Evolution Behavior and Systematics Polymerase chain reaction Mutation Point mutation Reproducibility of Results DNA Mummies Sequence Analysis DNA Protein-Tyrosine Kinases Biological Evolution Receptors Fibroblast Growth Factor Ancient DNA Phenotype chemistry Egypt
Zdroj:	Molecular biology and evolution. 21(11)
ISSN:	0737-4038
Popis:	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, approximately 4,890-5,050 BP [before present]), and from an individual from Kirchheim, Germany (Merovingian period, approximately 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 (G-->A/C-->T) 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 G-->A transition in the FGFR3 gene were observed, and it is likely that the false-positive G-->A transitions result from errors introduced during the PCR reaction. Amplifications in the presence of MnCl(2) 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.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9e0553fd0a6ad5318ec868b5ae9e1f4a https://pubmed.ncbi.nlm.nih.gov/15254256 Zobrazit plný text záznamu