title: Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner.
abstract: Paraffin-embedded tissue is an important source of material for molecular pathology and genetic investigations. We used DNA isolated from microdissected formalin-fixed, paraffin-embedded gastric tumors for mutation analysis of a region of the human gene for uracil-DNA glycosylase (UNG), encoding the UNG catalytic domain, and detected apparent base substitutions which, after further investigation, proved to be polymerase chain reaction (PCR) artifacts. We demonstrate that low DNA template input in PCR can generate false mutations, mainly guanine to adenine transitions, in a sequence-dependent manner. One such mutation is identical to a mutation previously reported in the UNG gene in human glioma. This phenomenon was not caused by microheterogeneity in the sample material because the same artifact was seen after amplification of a homogenous, diluted plasmid. We did not observe genuine mutations in the UNG gene in 16 samples. Our results demonstrate that caution should be taken when interpreting data from PCR-based analysis of somatic mutations using low amounts of template DNA, and that methods used to enrich putative subpopulations of mutant molecules in a sample material could, in essence, be a further amplification of sequence-dependent PCR-generated artifacts.
authors.affiliation: Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway.
authors.forename: Mansour
authors.initials: M
authors.lastname: Akbari
authors.forename: Marianne Doré
authors.initials: MD
authors.lastname: Hansen
authors.forename: Jostein
authors.initials: J
authors.lastname: Halgunset
authors.forename: Frank
authors.initials: F
authors.lastname: Skorpen
authors.forename: Hans E
authors.initials: HE
authors.lastname: Krokan
mesh_terms: D016477:Artifacts; D020134:Catalytic Domain / Q000235:genetics; D045647:DNA Glycosylases / Q000235:genetics*; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis*; D006801:Humans; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic; D051981:Uracil-DNA Glycosidase
chemical_list: D004273:DNA, Neoplasm; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase