Figure 3

Melting profiles indicating that the DNA is heterogeneously methylated at the RARB promoter. If molecules, in which different CpG positions are methylated and unmethylated are amplified, the observed melting profile will be complex. These different molecules will form heteroduplexes and homoduplexes after the PCR and prior to the HRM step. The very early melting observed is likely to be the melting of various heteroduplexes. The intermediate and later melting are likely to be the melting of various homoduplexes with those having the highest number of CpG positions methylated melting the latest. The 100% methylated standards are indicated in red, the 10% methylated standards in blue, the 1% methylated standards in green, the 0.1% methylated standards in brown, the 0% methylated standard in orange and non template controls are indicated in black. A sample (in triplicate) judged to be heterogeneously methylated is indicated in turquoise.