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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.

SUBMITTER: Akbari M

PROVIDER: S-EPMC1867510 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner.

Akbari Mansour M Hansen Marianne Doré MD Halgunset Jostein J Skorpen Frank F Krokan Hans E HE

The Journal of molecular diagnostics : JMD 20050201 1

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 i ...[more]

PMID: 15681472

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Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner.

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Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner.

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