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Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method

BACKGROUND: Molecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characteriza...

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Autores principales: Morlan, John, Baker, Joffre, Sinicropi, Dominick
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2642996/
https://www.ncbi.nlm.nih.gov/pubmed/19240792
http://dx.doi.org/10.1371/journal.pone.0004584
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author Morlan, John
Baker, Joffre
Sinicropi, Dominick
author_facet Morlan, John
Baker, Joffre
Sinicropi, Dominick
author_sort Morlan, John
collection PubMed
description BACKGROUND: Molecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characterization of tumors. A variety of methods exist for mutation analysis but the development of assays with high selectivity tends to require a process of trial and error, and few are compatible with real-time PCR. We sought to develop a real-time PCR-based mutation assay methodology that successfully addresses these issues. METHODOLOGY/PRINCIPAL FINDINGS: The method we describe is based on the widely used TaqMan® real-time PCR technology, and combines Allele-Specific PCR with a Blocking reagent (ASB-PCR) to suppress amplification of the wildype allele. ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules was developed enabling sensitive and selective detection of single point substitutions, insertions, or deletions against a background of wild-type allele in thousand-fold or greater excess. CONCLUSIONS/SIGNIFICANCE: ASB-PCR is a simple and robust method for assaying single nucleotide mutations and polymorphisms within the widely used TaqMan® protocol for real time RT-PCR. The ASB-PCR design rules consistently produce highly selective mutation assays while obviating the need for redesign and optimization of the assay reagents. The method is compatible with formalin-fixed tissue and simultaneous analysis of gene expression by RT-PCR on the same plate. No proprietary reagents other than those for TaqMan chemistry are required, so the method can be performed in any research laboratory with real-time PCR capability.
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spelling pubmed-26429962009-02-25 Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method Morlan, John Baker, Joffre Sinicropi, Dominick PLoS One Research Article BACKGROUND: Molecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characterization of tumors. A variety of methods exist for mutation analysis but the development of assays with high selectivity tends to require a process of trial and error, and few are compatible with real-time PCR. We sought to develop a real-time PCR-based mutation assay methodology that successfully addresses these issues. METHODOLOGY/PRINCIPAL FINDINGS: The method we describe is based on the widely used TaqMan® real-time PCR technology, and combines Allele-Specific PCR with a Blocking reagent (ASB-PCR) to suppress amplification of the wildype allele. ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules was developed enabling sensitive and selective detection of single point substitutions, insertions, or deletions against a background of wild-type allele in thousand-fold or greater excess. CONCLUSIONS/SIGNIFICANCE: ASB-PCR is a simple and robust method for assaying single nucleotide mutations and polymorphisms within the widely used TaqMan® protocol for real time RT-PCR. The ASB-PCR design rules consistently produce highly selective mutation assays while obviating the need for redesign and optimization of the assay reagents. The method is compatible with formalin-fixed tissue and simultaneous analysis of gene expression by RT-PCR on the same plate. No proprietary reagents other than those for TaqMan chemistry are required, so the method can be performed in any research laboratory with real-time PCR capability. Public Library of Science 2009-02-25 /pmc/articles/PMC2642996/ /pubmed/19240792 http://dx.doi.org/10.1371/journal.pone.0004584 Text en Morlan et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Morlan, John
Baker, Joffre
Sinicropi, Dominick
Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method
title Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method
title_full Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method
title_fullStr Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method
title_full_unstemmed Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method
title_short Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method
title_sort mutation detection by real-time pcr: a simple, robust and highly selective method
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2642996/
https://www.ncbi.nlm.nih.gov/pubmed/19240792
http://dx.doi.org/10.1371/journal.pone.0004584
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