Schematic representation of the CastPCR^TM assay.

<p>(A) Two independent amplification reactions are required for mutation detection: the Mutant Allele Assay, in which the mutant allele is amplified via the mutation-specific primer whilst amplification of the wild-type DNA is suppressed by the blocker containing the minor groove binder (MGB) (left), and the Gene Reference Assay, in which a DNA region located within the same gene but outside the mutant region is amplified (right). Both assays include an internal positive control (IPC) to control for PCR failure. (B) Example of Amplification Plots from a breast cancer sample analysis. The first curve to cross the signal threshold line (green line) represents the signal generated by the IPC reaction (blue line). The second curve (red line) represents the signal generated by the Mutant Allele Assay (left) or Gene Reference Assay (right). In this example, the Ct values for the Gene Reference Assay and the Mutant Allele Assay were 23.5 and 27.5 respectively. The resulting ΔCt is 4, indicating that the castPCR™ assay detected the <i>AKT1</i> E17K mutation in this sample.