Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method
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{"title"=>"Mutation detection by real-time PCR: A simple, robust and highly selective method", "type"=>"journal", "authors"=>[{"first_name"=>"John", "last_name"=>"Morlan", "scopus_author_id"=>"6507162801"}, {"first_name"=>"Joffre", "last_name"=>"Baker", "scopus_author_id"=>"56327790700"}, {"first_name"=>"Dominick", "last_name"=>"Sinicropi", "scopus_author_id"=>"6603927609"}], "year"=>2009, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84887212479", "scopus"=>"2-s2.0-84887212479", "doi"=>"10.1371/journal.pone.0004584", "isbn"=>"1932-6203 (Electronic)", "pui"=>"354309080", "issn"=>"19326203", "pmid"=>"19240792"}, "id"=>"71b1c200-a5c2-33e3-9824-4367b1dded56", "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.\\n\\nMETHODOLOGY/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.\\n\\nCONCLUSIONS/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.", "link"=>"http://www.mendeley.com/research/mutation-detection-realtime-pcr-simple-robust-highly-selective-method-4", "reader_count"=>125, "reader_count_by_academic_status"=>{"Unspecified"=>3, "Professor > Associate Professor"=>6, "Librarian"=>1, "Researcher"=>43, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>24, "Student > Postgraduate"=>4, "Other"=>8, "Student > Master"=>16, "Student > Bachelor"=>7, "Lecturer"=>3, "Professor"=>9}, "reader_count_by_user_role"=>{"Unspecified"=>3, "Professor > Associate Professor"=>6, "Librarian"=>1, "Researcher"=>43, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>24, "Student > Postgraduate"=>4, "Other"=>8, "Student > Master"=>16, "Student > Bachelor"=>7, "Lecturer"=>3, "Professor"=>9}, "reader_count_by_subject_area"=>{"Unspecified"=>5, "Engineering"=>3, "Environmental Science"=>2, "Biochemistry, Genetics and Molecular Biology"=>16, "Mathematics"=>1, "Medicine and Dentistry"=>17, "Agricultural and Biological Sciences"=>70, "Neuroscience"=>2, "Veterinary Science and Veterinary Medicine"=>2, "Chemistry"=>4, "Computer Science"=>1, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>3}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>17}, "Neuroscience"=>{"Neuroscience"=>2}, "Chemistry"=>{"Chemistry"=>4}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>70}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>16}, "Mathematics"=>{"Mathematics"=>1}, "Unspecified"=>{"Unspecified"=>5}, "Environmental Science"=>{"Environmental Science"=>2}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>2}}, "reader_count_by_country"=>{"United States"=>4, "China"=>1, "United Kingdom"=>3, "South Africa"=>2, "France"=>1, "Australia"=>1, "Switzerland"=>1, "Germany"=>1, "Indonesia"=>1, "India"=>1}, "group_count"=>4}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/907840"], "description"=>"a<p>ΔC<sub>T</sub> is the difference in C<sub>T</sub> obtained from wild type and mutant templates in the allele-specific mutation assay.</p>b<p>Assay names ending in “.1” were designed with the forward primer to be specific for the mutant sequence and assay names ending in “.2” were designed with the reverse primer specific for the mutant sequence.</p>c<p>ΔC<sub>T</sub> obtained when an allele-specific primer with T<sub>m</sub> about 60° was used without a blocker.</p>d<p>ΔC<sub>T</sub> obtained when a blocker was used in combination with an allele-specific primer.</p>e<p>ΔC<sub>T</sub> obtained when a low T<sub>m</sub> allele-specific primer was used.</p>f<p>ΔC<sub>T</sub> obtained when both a blocker and low T<sub>m</sub> allele-specific primer were used in combination.</p>g<p>Synthetic mutant DNA was used for Assay Mut6.1 due to the unavailability of cell lines carrying this mutation. Wild type DNA was obtained from HeLa cells.</p>", "links"=>[], "tags"=>["reagent", "mutation", "assay", "rna"], "article_id"=>578298, "categories"=>["Molecular Biology", "Biochemistry", "Infectious Diseases", "Pharmacology", "Cancer", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004584.t002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_Reagent_Design_on_Mutation_Assay_Performance_in_Cell_Line_RNA_or_DNA_/578298", "title"=>"Effect of Reagent Design on Mutation Assay Performance in Cell Line RNA or DNA.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2009-02-25 02:18:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/907632"], "description"=>"<p>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 C<sub>T</sub> values measured in the wild type Kras assay (x-axis) is plotted vs. the C<sub>T</sub> 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 C<sub>T</sub> 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 C<sub>T</sub> 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.</p>", "links"=>[], "tags"=>["mutation", "detection", "asb-pcr", "nucleic"], "article_id"=>578088, "categories"=>["Molecular Biology", "Biochemistry", "Infectious Diseases", "Pharmacology", "Cancer", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004584.g004", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_mutation_detection_by_ASB_PCR_and_nucleic_acid_sequencing_/578088", "title"=>"Comparison of mutation detection by ASB-PCR and nucleic acid sequencing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-02-25 02:14:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/907806"], "description"=>"<p>ASB-PCR Design Rules.</p>", "links"=>[], "tags"=>["Biochemistry", "biotechnology", "evidence-based healthcare", "genetics and genomics", "molecular biology", "oncology", "pharmacology"], "article_id"=>578261, "categories"=>["Molecular Biology", "Biochemistry", "Infectious Diseases", "Pharmacology", "Cancer", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004584.t003", "stats"=>{"downloads"=>3, "page_views"=>67, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ASB_PCR_Design_Rules_/578261", "title"=>"ASB-PCR Design Rules.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2009-02-25 02:17:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/448576", "https://ndownloader.figshare.com/files/448584", "https://ndownloader.figshare.com/files/448595"], "description"=>"<div><h3>Background</h3><p>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.</p><h3>Methodology/Principal Findings</h3><p>The method we describe is based on the widely used TaqMan® real-time PCR technology, and combines <u>A</u>llele-<u>S</u>pecific PCR with a <u>B</u>locking 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.</p><h3>Conclusions/Significance</h3><p>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.</p></div>", "links"=>[], "tags"=>["mutation", "detection", "Real-time", "robust", "selective"], "article_id"=>148404, "categories"=>["Molecular Biology", "Biochemistry", "Cancer", "Pharmacology", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0004584.s001", "https://dx.doi.org/10.1371/journal.pone.0004584.s002", "https://dx.doi.org/10.1371/journal.pone.0004584.s003"], "stats"=>{"downloads"=>10, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Mutation_Detection_by_Real_Time_PCR_A_Simple_Robust_and_Highly_Selective_Method/148404", "title"=>"Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2009-02-25 02:20:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/907552"], "description"=>"<p>A. Detection of cell line RNA containing Kras G216T mutant diluted into wild type cell line RNA using the Kras Mut1.2 assay. ΔR<sub>n</sub> is the difference between the normalized fluorescence of the TaqMan reporter probe at each PCR cycle and the background fluorescence measured during the first 15 PCR cycles. Each curve represents the time course of PCR assays (average of triplicate measurements) at each dilution. The horizontal line at ΔR<sub>n</sub> = 0.2 represents the threshold for determination of C<sub>T</sub> for the individual amplification curves. B. Serial-dilutions of RNA extracted from wild type COLO320 (filled squares) and mutant SW480 (filled circles) cell lines submitted to the Kras Mut1.2 assay. Error bars represent 2 times the standard deviation of triplicate determinations.</p>", "links"=>[], "tags"=>["selectivity"], "article_id"=>578018, "categories"=>["Molecular Biology", "Biochemistry", "Infectious Diseases", "Pharmacology", "Cancer", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004584.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sensitivity_and_selectivity_of_the_Mut1_2_assay_/578018", "title"=>"Sensitivity and selectivity of the Mut1.2 assay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-02-25 02:13:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/907772"], "description"=>"a<p>The C<sub>T</sub> and standard deviation (SD) comparing a 50/50 wild type/mutant cell line mixture (32 ng each) with wild type alone (32 ng) using the indicated assay. Wild type cell lines used were either COLO 320 or HeLa, depending on availability at the time. Cell line SW480 is homozygous for Mut1 (G216T); all other cell lines are heterozygous for the indicated mutations. Linearity of all assays ranged from 0.992–0.999. Efficiency for all assays ranged from 92%–116%. ΔC<sub>T</sub> is the difference between the C<sub>T</sub> obtained from Wild Type Cell Line C<sub>T</sub> – Mixed Cell Line C<sub>T</sub>. Selectivity was measured as described in Results.</p>b<p>The C<sub>T</sub> and standard deviation (SD) comparing a 50/50 wild type cell line DNA/mutant synthetic DNA (15,625 copies each) mixture with wild type DNA alone (15,625 copies) using the Mut6.1 assay. Synthetic mutant DNA was used for Assay Mut6.1 as no cell line carrying this mutation was available. Limiting Dilution Assay analysis was used to determine the number of copies of synthetic template as well as the number of wild type Kras alleles in HeLa DNA.</p>", "links"=>[], "tags"=>["mutation", "assays", "rna", "dna", "extracted"], "article_id"=>578223, "categories"=>["Molecular Biology", "Biochemistry", "Infectious Diseases", "Pharmacology", "Cancer", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004584.t004", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Performance_of_Mutation_Assays_using_RNA_or_DNA_Extracted_from_Cell_Lines_/578223", "title"=>"Performance of Mutation Assays using RNA or DNA Extracted from Cell Lines.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2009-02-25 02:17:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/907382"], "description"=>"<p>Diagram to illustrate the assay method.</p>", "links"=>[], "tags"=>["assay"], "article_id"=>577840, "categories"=>["Molecular Biology", "Biochemistry", "Infectious Diseases", "Pharmacology", "Cancer", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004584.g001", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Diagram_to_illustrate_the_assay_method_/577840", "title"=>"Diagram to illustrate the assay method.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-02-25 02:10:40"}
  • {"files"=>["https://ndownloader.figshare.com/files/907481"], "description"=>"<p>C<sub>T</sub> values are for the Kras Mut1.1 assay (G216T). A mutant-specific primer was used to assay either a wild type or mutant RNA template. Circles represent response to wild type synthetic RNA. Squares represent response to mutant synthetic RNA. Error bars represent 95% confidence intervals. The final version of this assay included a blocker oligonucleotide with a T<sub>m</sub> of 60.8°C and a variant-specific primer with a T<sub>m</sub> of 50°C. A. The effect of primer T<sub>m</sub> on variant-specific assay C<sub>T</sub>. B. The effect of blocker oligonucleotide T<sub>m</sub> on variant-specific assay C<sub>T</sub>.</p>", "links"=>[], "tags"=>["allele-specific", "primer", "blocker"], "article_id"=>577938, "categories"=>["Molecular Biology", "Biochemistry", "Infectious Diseases", "Pharmacology", "Cancer", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004584.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Optimization_of_allele_specific_primer_and_blocker_Tm_/577938", "title"=>"Optimization of allele-specific primer and blocker Tm.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-02-25 02:12:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/907862"], "description"=>"a<p>Data derived from Samowitz et al. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004584#pone.0004584-Samowitz1\" target=\"_blank\">[28]</a>. Table values represent the frequency of the specified mutation as a percentage of total observed Kras mutations. The total is not 100% because Samowitz et al. reported an additional mutation at a frequency of 0.4%.</p>b<p>The frequency of the specified mutation calculated as a percentage of total tumor specimens tested by Samowitz et al. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004584#pone.0004584-Samowitz1\" target=\"_blank\">[28]</a>. Four of 449 tumors had two mutations.</p>c<p>Determined in DNA extracted from 82 formalin-fixed paraffin-embedded colorectal cancer specimens.</p>", "links"=>[], "tags"=>["mutations", "colon"], "article_id"=>578319, "categories"=>["Molecular Biology", "Biochemistry", "Infectious Diseases", "Pharmacology", "Cancer", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004584.t001", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Kras_mutations_and_their_frequency_in_Colon_Cancer_/578319", "title"=>"Kras mutations and their frequency in Colon Cancer.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2009-02-25 02:18:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/907715"], "description"=>"<p>Circles: (Ο) Samples called wild type by both PCR and DNA sequencing; Squares (□): samples called mutant by both PCR and DNA sequencing; Triangles (▵): samples called mutant by ASB-PCR but wild type by sequencing. A: Kras G216T, assay Mut1.1. B: Kras G216A, assay Mut2.1. C: Kras G219A, assay Mut3.1.</p>", "links"=>[], "tags"=>["mutation", "assay", "dna", "rna", "72", "fpe", "specimens", "was"], "article_id"=>578162, "categories"=>["Molecular Biology", "Biochemistry", "Infectious Diseases", "Pharmacology", "Cancer", "Biotechnology", "Genetics"], "users"=>["John Morlan", "Joffre Baker", "Dominick Sinicropi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0004584.g005", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Concordance_of_mutation_assay_results_in_DNA_and_RNA_from_72_FPE_tissue_specimens_for_which_both_RNA_and_DNA_data_was_available_/578162", "title"=>"Concordance of mutation assay results in DNA and RNA from 72 FPE tissue specimens for which both RNA and DNA data was available.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-02-25 02:16:02"}

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