Fig 1.
Schematic representation of the CastPCRTM assay.
(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 AKT1 E17K mutation in this sample.
Table 1.
Individual and mean ΔCt results of two AKT1 E17K DNA reference standards analyzed by castPCR™ to determine analysis parameters.
Table 2.
Interpretation of castPCR™ AKT1 E17K assay results.
Table 3.
Comparison of AKT1 mutation detection by castPCR™, MALDI-TOF MS and BEAMing in 6 AKT1 E17K mutation positive breast cancer samples.
Table 4.
Comparison of AKT1 mutation detection data generated by castPCR™, MALDI-TOF MS and BEAMing in 4 AKT1 E17K gynecological cancer samples.
Fig 2.
Ct values of duplicate assays showing intra-assay agreement.
All samples were performed in duplicate, resulting in two Ct values for the Gene Reference Assay (open circle) and two Ct values for the Mutant Allele Assay (closed circle). The blue circles show the Ct values obtained in the first experiment, and the red circles show the Ct values obtained in the second experiment. Absence of a closed circle for a particular sample indicates that no Ct value was detected. The two samples in which the AKT1 E17K mutation was detected (samples 7 and 41 with a ΔCt <8.1) are indicated with an arrow.
Fig 3.
AKT1 E17K ctDNA reference standard for OncoBEAM™ evaluation.
(A) Representative TapeStation (D1000 screentape) graphs showing the DNA sizes from a wild-type AKT1 (left) and an AKT1 E17K harboring (right) cell line sample upon fragmentation by Covaris prior to spiking into plasma. The arrows demonstrate the average peak size. (B) Graph presenting the AKT1 E17K mutant allele frequencies detected in the ctDNA reference standard samples analyzed in triplicate by the OncoBEAM™ assay (presented on the y-axis) and ddPCR assay (presented on the x-axis).
Table 5.
AKT1 E17K mutation detection by OncoBeam™ digital PCR in ctDNA reference standard.