Asymmetric mutant-enriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in...

Identification of mutant genes in tumor tissues and blood plasma (solid and liquid biopsy samples, respectively) is a necessity for individualized treatment of cancer patients. Here we report the use of a novel mutant-enriched PCR - quantitative DNA melting curve analysis (mePCR-qDMA) with TaqMan probes. The TaqMan probes served as blocking agents during PCR and as hybridization probes during DNA melting curve analyses. The end-point measurement of melt peaks areas by PeakFit software, a nonlinear iterative curve-fitting program, permitted quantification of the mutant/wild-type allele ratios. Approximately 6% and 0.1% of mutant KRAS allele in an excess of wild-type allele is detected with the standard and mePCR-qDMA processes, respectively. The application of the approach was tested for detecting the KRAS codon 12/13 mutation in paired tumor and blood plasma samples from 20 colorectal cancer patients. KRAS mutants were detected in 7 and 18 FFPE tumor samples, and in 3 and 7 plasma samples by the standard and mePCR-qDMA process, respectively. The results were confirmed by Sanger sequencing. This simple, rapid, cost-effective, and quantitative method carried out in a closed-tube format could be applied for the clinical analyses of other cancer genes.