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Mutation detection by real-time PCR: a simple, robust and highly selective method.

Morlan J, Baker J, Sinicropi D - PLoS ONE (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Genomic Health, Inc., Redwood City, California, United States of America.

ABSTRACT

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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Comparison of mutation detection by ASB-PCR and nucleic acid sequencing.ASB-PCR results are shown for the 44 randomly-selected FFPE colorectal tumor specimens for which sequencing data were available. Genomic DNA extracted from 44 randomly-selected FFPE colorectal tumor specimens was submitted to ASB-PCR assays A) Kras G216T, assay Mut1.1, B) Kras G216A, assay Mut2.1, and C) Kras G219A, assay Mut3.1. In each graph the CT values measured in the wild type Kras assay (x-axis) is plotted vs. the CT values measured in the specified Kras variant allele assay (y-axis). Samples were assayed at 0.4 ng of DNA per well. The solid line represents the classification boundary, which was derived as the lower 95% prediction limit of a linear regression of variant-specific assay CT response on a titration of wild type samples submitted to the variant-specific assay. Error bars represent 95% confidence limits based on a pooled estimate of standard error for all samples with a mean CT less than 35. Note that samples for which the 95% confidence intervals overlapped were designated as wild type. Circles: (Ο) Samples called wild type by both PCR and sequencing; Squares (□): samples called mutant by both PCR and sequencing; Triangles (▵): samples called mutant by PCR but wild type by sequencing.
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pone-0004584-g004: Comparison of mutation detection by ASB-PCR and nucleic acid sequencing.ASB-PCR results are shown for the 44 randomly-selected FFPE colorectal tumor specimens for which sequencing data were available. Genomic DNA extracted from 44 randomly-selected FFPE colorectal tumor specimens was submitted to ASB-PCR assays A) Kras G216T, assay Mut1.1, B) Kras G216A, assay Mut2.1, and C) Kras G219A, assay Mut3.1. In each graph the CT values measured in the wild type Kras assay (x-axis) is plotted vs. the CT values measured in the specified Kras variant allele assay (y-axis). Samples were assayed at 0.4 ng of DNA per well. The solid line represents the classification boundary, which was derived as the lower 95% prediction limit of a linear regression of variant-specific assay CT response on a titration of wild type samples submitted to the variant-specific assay. Error bars represent 95% confidence limits based on a pooled estimate of standard error for all samples with a mean CT less than 35. Note that samples for which the 95% confidence intervals overlapped were designated as wild type. Circles: (Ο) Samples called wild type by both PCR and sequencing; Squares (□): samples called mutant by both PCR and sequencing; Triangles (▵): samples called mutant by PCR but wild type by sequencing.

Mentions: Two samples that typed positive by ASB-PCR, for substitutions G216T and G219A, were negative by sequence analysis (Table S2). Several lines of evidence suggest that these discordant samples did, in fact, harbor Kras mutations. First, the CT values obtained for these samples in the Mut1 and Mut3 assays clearly distinguished them from wild type specimens. The CT values from ASB-PCR analysis of all 44 samples are depicted graphically in Figures 4 A–C. The CT values from assays with the mutant alleles are on the Y-axes and those from assays with the wild type allele are on the X-axes. Both discordant samples (triangles in Figures 4A and 4C) cluster with other mutation positive samples and are well-separated from the classification line for the wild type population. Second, the mutation calls for these samples made from the sequencing electropherograms were complicated by experimental noise that may have resulted from poor sample quality (data not shown). In addition, for one of the discordant samples sequencing data was only obtained from one strand of the sequencing template. Inspection of the electropherograms for these samples in fact revealed minor peaks that are consistent with the presence of the mutations called by ASB-PCR. Third, as described below, ASB-PCR analysis of RNA extracted from the same samples confirmed the mutation calls made by ASB-PCR analysis of DNA. Taken together, these results support the view that the two discordant samples were true positives for Kras mutations that were undetected by sequence analysis.


Mutation detection by real-time PCR: a simple, robust and highly selective method.

Morlan J, Baker J, Sinicropi D - PLoS ONE (2009)

Comparison of mutation detection by ASB-PCR and nucleic acid sequencing.ASB-PCR results are shown for the 44 randomly-selected FFPE colorectal tumor specimens for which sequencing data were available. Genomic DNA extracted from 44 randomly-selected FFPE colorectal tumor specimens was submitted to ASB-PCR assays A) Kras G216T, assay Mut1.1, B) Kras G216A, assay Mut2.1, and C) Kras G219A, assay Mut3.1. In each graph the CT values measured in the wild type Kras assay (x-axis) is plotted vs. the CT values measured in the specified Kras variant allele assay (y-axis). Samples were assayed at 0.4 ng of DNA per well. The solid line represents the classification boundary, which was derived as the lower 95% prediction limit of a linear regression of variant-specific assay CT response on a titration of wild type samples submitted to the variant-specific assay. Error bars represent 95% confidence limits based on a pooled estimate of standard error for all samples with a mean CT less than 35. Note that samples for which the 95% confidence intervals overlapped were designated as wild type. Circles: (Ο) Samples called wild type by both PCR and sequencing; Squares (□): samples called mutant by both PCR and sequencing; Triangles (▵): samples called mutant by PCR but wild type by sequencing.
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Related In: Results  -  Collection

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pone-0004584-g004: Comparison of mutation detection by ASB-PCR and nucleic acid sequencing.ASB-PCR results are shown for the 44 randomly-selected FFPE colorectal tumor specimens for which sequencing data were available. Genomic DNA extracted from 44 randomly-selected FFPE colorectal tumor specimens was submitted to ASB-PCR assays A) Kras G216T, assay Mut1.1, B) Kras G216A, assay Mut2.1, and C) Kras G219A, assay Mut3.1. In each graph the CT values measured in the wild type Kras assay (x-axis) is plotted vs. the CT values measured in the specified Kras variant allele assay (y-axis). Samples were assayed at 0.4 ng of DNA per well. The solid line represents the classification boundary, which was derived as the lower 95% prediction limit of a linear regression of variant-specific assay CT response on a titration of wild type samples submitted to the variant-specific assay. Error bars represent 95% confidence limits based on a pooled estimate of standard error for all samples with a mean CT less than 35. Note that samples for which the 95% confidence intervals overlapped were designated as wild type. Circles: (Ο) Samples called wild type by both PCR and sequencing; Squares (□): samples called mutant by both PCR and sequencing; Triangles (▵): samples called mutant by PCR but wild type by sequencing.
Mentions: Two samples that typed positive by ASB-PCR, for substitutions G216T and G219A, were negative by sequence analysis (Table S2). Several lines of evidence suggest that these discordant samples did, in fact, harbor Kras mutations. First, the CT values obtained for these samples in the Mut1 and Mut3 assays clearly distinguished them from wild type specimens. The CT values from ASB-PCR analysis of all 44 samples are depicted graphically in Figures 4 A–C. The CT values from assays with the mutant alleles are on the Y-axes and those from assays with the wild type allele are on the X-axes. Both discordant samples (triangles in Figures 4A and 4C) cluster with other mutation positive samples and are well-separated from the classification line for the wild type population. Second, the mutation calls for these samples made from the sequencing electropherograms were complicated by experimental noise that may have resulted from poor sample quality (data not shown). In addition, for one of the discordant samples sequencing data was only obtained from one strand of the sequencing template. Inspection of the electropherograms for these samples in fact revealed minor peaks that are consistent with the presence of the mutations called by ASB-PCR. Third, as described below, ASB-PCR analysis of RNA extracted from the same samples confirmed the mutation calls made by ASB-PCR analysis of DNA. Taken together, these results support the view that the two discordant samples were true positives for Kras mutations that were undetected by sequence analysis.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Genomic Health, Inc., Redwood City, California, United States of America.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus