Snapback Primer Mediated Clamping PCR for Detection of EGFR and KRAS Mutations in NSCLC Patients by High Resolution Melting Analysis
Figure 1
Snapback primer mediated clamping PCR and HRM analysis. We designed probe’s sequence that matches wild type to make sure that the melting temperature of wild hairpin stem structure will be higher than mutant type. (a) HRM analysis by snapback primer genotyping after normal PCR. Tsp value is the middle melting temperature of wild and mutant snapback hairpin, which is the temperature chosen for extending in SPACE-PCR cycles. (b) The principle of SPACE-PCR. When extended in Tsp (°C), mutant can be enriched. (c) HRM analysis with snapback primer after SPACE-PCR (SPACE-HRM). After SPACE-PCR, both the melting curves of wild snapback hairpin and amplicon are reduced. Heterozygous samples intend to be pure mutant.
