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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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Standard

Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner. / Akbari, Mansour; Hansen, Marianne Doré; Halgunset, Jostein; Skorpen, Frank; Krokan, Hans E.

I: The Journal of molecular diagnostics : JMD, Bind 7, Nr. 1, 02.2005, s. 36-9.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

Harvard

Akbari, M, Hansen, MD, Halgunset, J, Skorpen, F & Krokan, HE 2005, 'Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner', The Journal of molecular diagnostics : JMD, bind 7, nr. 1, s. 36-9.

APA

Akbari, M., Hansen, M. D., Halgunset, J., Skorpen, F., & Krokan, H. E. (2005). Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner. The Journal of molecular diagnostics : JMD, 7(1), 36-9.

Vancouver

Akbari M, Hansen MD, Halgunset J, Skorpen F, Krokan HE. Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner. The Journal of molecular diagnostics : JMD. 2005 feb;7(1):36-9.

Author

Akbari, Mansour ; Hansen, Marianne Doré ; Halgunset, Jostein ; Skorpen, Frank ; Krokan, Hans E. / Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner. I: The Journal of molecular diagnostics : JMD. 2005 ; Bind 7, Nr. 1. s. 36-9.

Bibtex

@article{b0792386992f46888109e2d0e7865729,
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.",
keywords = "Artifacts, Catalytic Domain/genetics, DNA Glycosylases/genetics, DNA Mutational Analysis, DNA, Neoplasm/analysis, Humans, Mutagenesis, Mutation/genetics, Polymerase Chain Reaction, Templates, Genetic, Uracil-DNA Glycosidase",
author = "Mansour Akbari and Hansen, {Marianne Dor{\'e}} and Jostein Halgunset and Frank Skorpen and Krokan, {Hans E}",
year = "2005",
month = "2",
language = "English",
volume = "7",
pages = "36--9",
journal = "Journal of Molecular Diagnostics",
issn = "1525-1578",
publisher = "Elsevier",
number = "1",

}

RIS

TY - JOUR

T1 - Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner

AU - Akbari, Mansour

AU - Hansen, Marianne Doré

AU - Halgunset, Jostein

AU - Skorpen, Frank

AU - Krokan, Hans E

PY - 2005/2

Y1 - 2005/2

N2 - 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.

AB - 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.

KW - Artifacts

KW - Catalytic Domain/genetics

KW - DNA Glycosylases/genetics

KW - DNA Mutational Analysis

KW - DNA, Neoplasm/analysis

KW - Humans

KW - Mutagenesis

KW - Mutation/genetics

KW - Polymerase Chain Reaction

KW - Templates, Genetic

KW - Uracil-DNA Glycosidase

M3 - Journal article

C2 - 15681472

VL - 7

SP - 36

EP - 39

JO - Journal of Molecular Diagnostics

JF - Journal of Molecular Diagnostics

SN - 1525-1578

IS - 1

ER -

ID: 213362626