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

Figure 4

Detection of the KRAS codons 12 and 13 mutations in CRC tissue DNA using snapback primer HRMA. (a) The KRAS mutant allele frequency distribution in TCGA and MSKCC-IMPACT. A number of samples harboring KRAS mutation was dichotomized by mutant allele frequency. The distribution of KRAS mutant samples was skewed in samples with <20% mutant allele. Compared with the primary tumor samples, tissues from metastatic tumors have lower mutant allele burden. (b) Analytical sensitivity of snapback primer HRMA. The system was able to discriminate 1% dilution of KRAS mutant allele from the wild-type sequence. (c) Analytical sensitivity of Sanger sequencing. The direct sequencing can detect KRAS mutation in the plasmid pool with 20% KRAS mutant allele. (d) Patient (P92) with low-abundance G12A mutation was detected using the snapback primer system. (e) Sanger sequencing was determined P92 as KRAS mutation free, while further subclone sequencing of P92 was detected the mutation in the sample.

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