Fig. 4
Allele-specific PCR with Ex19_std and wildtype DNA. Real time PCR with allele-specific amplification of Ex19_std and wildtype, in varying MAFs, in a total DNA concentration assay of 0.25 ng/μl. a: Ex19_std alone (50% MAF). b: 10% MAF of mutDNA in mixed DNA assay. Similarly, c: 5% MAF of mutDNA, d: 1% MAF, and e: 0.1% MAF in mixed DNA assays. f: wildtype DNA alone. g: non-template control. a and b are real-time PCR raw amplification profile of the mutant allele in the individual MAF assays depicted as fluorescent intensity (Y-axis) against cycle threshold (Ct) values (X- axis). a is the profile of mutant DNA acquired in the FAM channel. b is the profile of wildtype DNA acquired in the VIC channel. c: Comparison of Ct values of wildtype DNA (left y-axis) in different MAF assays and the relationship between the Ct of wildtype and mutant alleles (right y-axis) obtained from the ΔCt values for individual MAF assay (x- axis). Note that the ΔCt values of upto 1% MAF were less than 12 and detected as mutant positive