Research Article

Comprehensive Validation of Snapback Primer-Based Melting Curve Analysis to Detect Nucleotide Variation in the Codon 12 and 13 of KRAS Gene

Figure 3

Establishment of a snapback primer PCR based HRMA detecting KRAS codons 12 and 13 mutations. (a) Design of snapback primer HRMA for KRAS genotyping. A probe’s element with 18 nucleotides was designed totally complementary to the target sequence. In addition, a 2-bp mismatch at the 5′end of snapback primer was intentionally used to block further extension of the snapback hairpin. When asymmetric PCR conditions were optimized, the stem-loop hairpins and double-strand DNA amplicons were formed. The melting transitions were processed by plotting the negative derivative of fluorescence versus the melting temperature. At the moment, snapback melting peaks signified targeted genotyping. (b) Snapback probe assay for the detection of KRAS codons 12 and 13 mutations. The negative derivative (dF/dT) plot of melting curve consists of two melting regions. Stem-loop hairpin melting peaks for mutation identification were located in the region between 66°C to 70°C, and the double-strand amplicon as DNA template amplification control was situated at ( 84±0.5°C). (c) Discriminating power of the snapback primer assay. Melting curve profile of three plasmids (G13D, GGA>GCA, and GTG>CAC) revealed the specificity of the snapback primer system for detecting single base variation (66°C-70°C), with the at least 2-degree difference in the melting peaks. (d) Enrichment of snapback primer amplification. 1% dilution of the KRAS G12A or KRAS G13D mutant allele can be detected by Sanger sequencing, while the same sequencing signal of a mutant allele can only be observed in the PCR products with 10% mutant allele load.