Optimization of COLD-PCR Conditions. A G12C (G GT-T GT) KRAS-mutant colorectal cancer sample with approximately 10% mutant allele was tested at progressively lower critical denaturation temperatures (T
). As the T
was progressively lowered, the height of the mutant melting peak at 62.5°C increased relative to the height of the wild-type melting peak at 70°C. DNA sequencing performed on COLD-PCR products corroborated an increase in the peak height of the mutant T base (c.34 G > T) at the first position of codon 12 (compare top and bottom panel insets).