Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method (original) (raw)
Figure 2
Optimization of allele-specific primer and blocker Tm.
CT 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 Tm of 60.8°C and a variant-specific primer with a Tm of 50°C. A. The effect of primer Tm on variant-specific assay CT. B. The effect of blocker oligonucleotide Tm on variant-specific assay CT.