Snapback Primer Mediated Clamping PCR for Detection of EGFR and KRAS Mutations in NSCLC Patients by High Resolution Melting Analysis
Figure 2
A schematic of genotyping by snapback primer HRM after normal PCR in detecting EGFR E746-A750 del (Figure 1(a)) and KRAS exon 2, codon 13(1-G>A). In detection of EGFR E746-A750 del (15 bp), probe (22 bp) cannot cover the mutant site, so there is no peak of intramolecular snapback hairpins for mutant-type samples, while the peak value of wild type is 74.5°C. Besides this, difference also exists in peaks of intermolecular duplexes. When there is an obviously small peak 2°C below the main peaks of intermolecular duplexes, it means this sample just consists of wild sequence (peak a); then if 4°C-below, mutant exists (peaks b, c, and d). Mixed samples (peak b, 10% mutation, and peak c, 20% mutation) showed that the more mutant template the lower probe peak. In detection of KRAS, as for point mutation detection, wild-type (peak a) probe peak value is 74°C and about 5°C below is the mutation (peak d). Mixed samples (peak c, 50% mutation, and peak b, 20% mutation) showed that the higher the percentage, the higher the peak standing for.
