Principle of HeavyMethyl. (A) When the DNA is methylated, the blocker oligonucleotides (solid black) do not bind, leaving the primer- binding site accessible for the primers (gray arrows) to bind and amplify the target. The amplification is detected with a methylation specific oligonucleotide probe [solid black, labeled with fluorescent dye (F) and quencher (Q) in a 5′-exonuclease assay, used here as an example for a real-time detection method]. (B) When the DNA is unmethylated, the blocker oligonucleotides bind, blocking the access of the primers to their binding sites. No PCR product is generated.

Relative and absolute sensitivity of GSTP1 HeavyMethyl assay. GSTP1Exon amplification was performed with and without blockers. (A) 1.5% agarose gel analysis of GSTP1Exon PCRs performed without (–B; lanes 1–6, 13) and with blocker (+B; lanes 7–12) on 50 ng bisulfite-treated unmethylated DNA (–CH₃; lanes 1, 7), different amounts of bisulfite-treated SssI-methylated DNA (CH₃; 1.2 ng, lanes 2 and 8; 0.6 ng, lanes 3 and 9; 0.3 ng, lanes 4 and 10; 0.15 ng, lanes 5 and 11; 75 pg, lanes 6 and 12) and no DNA ( lane 13). (B) Sequence traces of GSTP1Exon products generated on a mixture of 100 pg methylated in a background of 100 ng unmethylated template DNA (relative sensitivity 1:1000) in the presence and absence of blockers. (C) The discrimination of GSTP1Exon products generated by HeavyMethyl assay with (+B) and without (–B) blocker on bisulfite-treated SssI-methylated (+CH₃), unmethylated (–CH₃) and a mixture of 100 pg methylated in 100 ng unmethylated (1:1000) template DNA by melting peak analysis.

HeavyMethyl real-time assays for GSTP1 exon and promoter. (A–C) HeavyMethyl GSTP1 exon assay. Amplification plots of 15 ng SssI-methylated DNA indicated by solid or broken lines and 50 ng of unmethylated DNA indicated by lines with circles (A). (B) 50 ng of unmethylated DNA and 30 pg methylated DNA spiked into 50 ng unmethylated DNA representing a relative sensitivity of 1:1600 (C), which were bisulfite treated and subsequently used as template for the HeavyMethyl assay. The LightCycler detection probes were specific for bisulfite-treated unmethylated (B) or methylated DNA (A, C). HeavyMethyl assays performed with and without blocker are indicated by solid and broken lines, respectively. In (A), the HeavyMethyl assay with and without blocker is also indicated by filled and open circles, respectively. (D) HeavyMethyl GSTP1 promoter assay. The GSTP1 promoter fragment was amplified on 400 ng unmethylated (five replicates indicated by broken lines), 100 pg SssI-methylated DNA (black diamonds) and mixtures containing 100 pg (six replicates indicated by solid lines) or 50 pg (six replicates indicated by lines labeled with open diamonds) methylated DNA in 400 ng unmethylated DNA. All template DNA samples were bisulfite treated prior to real-time PCR experiments. The products were specifically detected via the FRET signal generated by the Fluorescein- and LCRed640-labeled detection probes specific for methylated bisulfite-treated DNA.

Methylation of Calcitonin in colon cancer tissue and serum samples. (A) The PMR is plotted for colon cancer tumor and normal colon tissue samples. The PMR is also plotted for five serum samples from individuals with methylated tumors and 11 serum samples from healthy control donors. (B) Methylation of Calcitonin in normal (n = 43) and adenocarcinoma (n = 33) colon samples was measured by both HeavyMethyl and MSP. ROC curves were generated by plotting the sensitivity against the false-negative rate (1 – specificity). The area under the curve (0.84 for HeavyMethyl and 0.81 for MSP) is used to evaluate the performance of the assay.