HRM analysis is based on the dissociation behavior of dsDNA due to increasing temperature. Melting of dsDNA depends on GC-content. AT-rich regions melt faster.
[A] Genomic DNA — all cytosines that are not part of CpG sites are symbolized by X. CpG sites containing potentially methylated cytosines are indicated as CG. [B] Bisulfite conversion of genomic DNA — all unmethylated cytosine residues are converted to uracil. Cytosines within CpG sites are indicated by Y and are either converted to U (if unmethylated) or remain unmodified (if methylated).
Mixtures of methylated (M) and unmethylated (U) DNA in various ratios were used as template. A CpG island from the promoter region of the PTPN6 gene (protein tyrosine phosphatase, nonreceptor type 6) was amplified, and the degree of methylation was determined by HRM methylation analysis. [A] Difference plot when using the EpiTect HRM PCR Kit and the Rotor-Gene Q. [B] Difference plot and melting peaks of 100% unmethylated and methylated DNA, respectively (inset) when using the EpiTect HRM PCR Kit and the LightCycler 480. [C] Difference plot and melting peaks of 100% unmethylated and methylated DNA, respectively (inset) when using Supplier R's HRM kit (with 3.5 mM MgCl2) on the LightCycler 480. The kit was tested with 1.5 mM and 3.5 mM MgCl2 (within the lower and upper limits of the recommended Mg2+ concentration). Amplification with 1.5 mM MgCl2 failed (data not shown); amplification with 3.5 mM MgCl2 resulted in the generation of nonspecific products when using methylated DNA as template (red double peaks), limiting the HRM analysis.
Mixtures of methylated and unmethylated DNA (EpiTect Control DNA) of varying ratios were used as template. A CpG island from the promoter region of the APC gene (adenomatosis polyposis coli) was amplified and the degree of methylation was determined by HRM methylation analysis on the Rotor-Gene Q using the EpiTect HRM PCR Kit. [A] A standard normalized melt curve and a [B] difference plot normalized to the 50% methylated sample are shown.