Re-classified specimen. Shown are results from one of the 4 specimens that was re-classified from KRAS-WT to KRAS-mutant by COLD-PCR (sample #3 in Additional file 3). With regular PCR no definite mutant allele is detected by the melting curve assay, or by direct sequencing (left panel). Using COLD-PCR a clear mutant peak is visible in the melting curve assay and confirmed as a G12C mutation by sequencing (c.34 G > C). Arrows point to the position of the mutant melting peak and mutant base pair position. Note that a T
of 79.4°C was used for COLD-PCR sequencing compared to a T
of 81°C for the COLD-PCR melting curve assay (see Methods), which is the likely explanation for the higher proportion of mutant allele observed by COLD-PCR sequencing (bottom right).