Comparison of the mutation detection sensitivity of high resolution melting (HRM) and sequencing. The sensitivity of KRAS HRM and dideoxynucleotide sequencing was tested using four HCT116 DNA dilutions. Based on qPCR data, the HCT116 DNA was mixed with wild-type DNA to dilute the mutant allele to 20%, 10%, 5%, and 1% of the total alleles. Sequencing was only sensitive to 10-20% whereas HRM readily detected 5% mutant sequence. Panel A: Sequencing traces of four HCT116 DNA dilutions. The mutant A allele was readily detectable at a 20% mutant frequency. However, when the mutant allele was present at 10%, it was barely distinguishable from the sequencing background. When the frequency of the mutant was below 10%, the mutation was not detectable by dideoxynucleotide sequencing. Panel B: Using HRM, the mutation was readily detectable down at 5% mutation frequency. The 1% dilution was not distinct from the normal DNA. The melting curves of each dilution are shown in orange (20%), brown (10%), green (5%), red (1%) and blue (wild-type control).