A human A/T SNP in the AHRR7 gene was analyzed using genomic DNA from wild-type (blue), homozygous mutant (green), and heterozygous (red) samples. Experiments were performed using the Type-it HRM PCR Kit and a Rotor-Gene Q cycler with a HRM channel. Data analysis was performed with the unsupervised mode of Rotor-Gene ScreenClust HRM Software. A/T polymorphisms (class IV SNPs) are most difficult to discriminate due to minute differences between homozygote alleles (in this example, less than 0.1°C). [A] HRM raw data, [B] the normalized melting curve, [C] the residual plot, and [D] the cluster plot are shown. All pseudo-unknowns were correctly clustered according to genotype.
Gene mutations (insertions/deletions) in EGFR exon 19 were analyzed using Rotor-Gene ScreenClust HRM Software in supervised mode. Experiments were performed using the Type-it HRM PCR Kit, a Rotor-Gene Q cycler with a HRM channel, and plasmid DNA templates. [A] A normalized melt plot shows very similar curve shape with only minute differences in melting points, making genotyping challenging. [B-D] Cluster plots using 3 principal components show the correct assignment of 6 different mutations and the wild type sample: M1: c.2235_2249del15, M3: c.2237_2252del16insT, c.2237_2238ins18, M4: c.2237_2238ins18, M5: c.2239_2248del10insC, M6: c.2240_2254del15, M7: c.2240_2257del18.