Enhanced--COLD-PCR for the Sensitive Detection of Rare DNA Methylation Patterns in Liquid Biopsies.

In the context of precision medicine, the identification of novel biomarkers for the diagnosis of disease, prognosis, predicting treatment outcome and monitoring of treatment success is of great importance. The analysis of methylated circulating-cell free DNA provides great promise to complement or replace genetic markers for these applications, but is associated with substantial challenges. This is particularly true for the detection of rare methylated DNA molecules in a limited amount of sample such as tumor released hypermethylated molecules in the background of DNA fragments from normal cells, especially lymphocytes. Technologies for the sensitive detection of DNA methylation have been developed to enrich specifically methylated DNA or unmethylated DNA using among other 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--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--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--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--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--COLD-PCR assay including assay design, optimization of E--COLD-PCR conditions including annealing temperature, critical temperature and concentration of LNA blocker probe followed by Pyrosequencing analysis.