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Optimized Allele-Specific Real-Time PCR Assays for the Detection of Common Mutations in KRAS and BRAF  Alois H. Lang, Heinz Drexel, Simone Geller-Rhomberg,

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Presentation on theme: "Optimized Allele-Specific Real-Time PCR Assays for the Detection of Common Mutations in KRAS and BRAF  Alois H. Lang, Heinz Drexel, Simone Geller-Rhomberg,"— Presentation transcript:

1 Optimized Allele-Specific Real-Time PCR Assays for the Detection of Common Mutations in KRAS and BRAF  Alois H. Lang, Heinz Drexel, Simone Geller-Rhomberg, Nicole Stark, Thomas Winder, Kathrin Geiger, Axel Muendlein  The Journal of Molecular Diagnostics  Volume 13, Issue 1, Pages (January 2011) DOI: /j.jmoldx Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 Primer and probes used for KRAS and BRAF real-time PCR. The figure illustrates positions of primer and probes used for KRAS and BRAF real-time PCR. Reference and mutation-specific PCRs share the same probe and the opposite PCR primer. Solid arrows display mutation-unspecific primers; and dotted arrows, mutation-specific primers. Codons affected by the mutation are underlined. The Journal of Molecular Diagnostics  , 23-28DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 Amplification curves obtained from allele-specific real-time PCR assays. DNA from mutant plasmids was diluted into wild-type plasmids. Based on a total DNA amount of 1000 copies per reaction, the proportion of mutant plasmid DNA was gradually reduced to obtain decreasing ratios of mutant to wild-type DNA. R, reference PCR, representative for all dilutions; a, b, c, and d, 10%, 2.5%, 1%, and 0%, respectively, proportion of mutant DNA; 0, nontemplate control. The Journal of Molecular Diagnostics  , 23-28DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 Supplemental Figure S1 Amplification curves from KRAS and BRAF PCRs and from respective internal control PCRs. First row: amplification curves of KRAS 12Ala, 12Val, 12Cys, and BRAF 600Glu target-specific real-time PCRs (ie, the fluorescein channel). Second row: amplification curves of respective internal control PCRs (ie, the VIC channel). Ten copies of mutant plasmid DNA were diluted into 990 copies of genomic wild-type DNA. R, reference PCR reaction mix; a, allele-specific PCR reaction mix. The Journal of Molecular Diagnostics  , 23-28DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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