Methods
enzymatic digestion, methylation-specific PCR (often combined with TaqMan like oligonucleotide probes (MethyLight)) and co-amplification at lower denaturation temperature PCR (COLD-PCR). E-ice-COLD-PCR (Enhanced-improved and complete enrichment-COLD-PCR) is a sensitive method that takes advantage of a Locked Nucleic Acid (LNA)-containing oligonucleotide probe to block specifically unmethylated CpG sites allowing the strong enrichment of low-abundant methylated CpG sites from a limited quantity of input. E-ice-COLD-PCRs are performed on bisulfite-converted DNA followed by Pyrosequencing analysis. The quantification of the initially present DNA methylation level is obtained using calibration curves of methylated and unmethylated DNA. The E-ice-COLD-PCR reactions can be multiplexed, allowing the analysis and quantification of the DNA methylation level of several target genes. In contrast to the above-mentioned assays, E-ice-COLD-PCR will also perform in the presence of frequently occurring heterogeneous DNA methylation patterns at the target sites. The presented protocol describes the development of an E-ice-COLD-PCR assay including assay design, optimization of E-ice-COLD-PCR conditions including annealing temperature, critical temperature and concentration of LNA blocker probe followed by Pyrosequencing analysis.