Sensitivity threshold of PCR/sequencing, TheraScreen and LNA-PCR assays. DNA from HCT 116 cells carrying the gac codon 13 KRAS mutation (G13D) was diluted with the KRAS wild-type DNA from Colo 320 cells in proportions ranging from 50% to 0.125% mutant DNA. (A) Sequencing electropherograms of a 212-bp amplicon are reported for the different DNA mixtures indicated. (B) Correlation between ACt values and dilutions of mutant DNA in a TheraScreen kit, ACt: difference between Ct of mutation assay and Ct of control assay. The blue diamond indicates the 1% cutoff ACt value for the G13D probe (corresponding to the 9 value). (C) The melting profiles corresponding to the gac codon 13 KRAS change (Tm = 57.6°C) are generated with the mutated DNA/wt DNA dilutions (range from 4% to 0.125%) by the LightCycler software.