Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing™

• DOI: 10.2144/03351md02
• Publication Date: 2018-09-24T15:24:34Z
• Authors: Jörg Tost; Jenny Dunker; Ivo Glynne Gut
• Keywords: Pyrosequencing; CpG site

of the technology is the close correlation between the peak heights in a Pyrogram (Pyrosequencing AB), representing the sequence raw data, and the amount of nucleotides incorporated by the DNA polymerase during the Pyrosequencing reaction that allows for precise allele frequency determination for a SNP in pooled DNA samples (10–12). Very recently, an approach to the analysis of the degree of methylation on a single MVP after bisulfite treatment was shown using mixtures of PCR products for calibration (13). Bisulfite treatment converts unmethylated cytosines to uracils, while methylated cytosines remain unchanged under the appropriate reaction conditions. Therefore, after PCR, methylation sites can be treated as C/T SNPs with an allele frequency spectrum spanning the entire range (0%–100%). Here we demonstrate the Pyrosequencing technology to analyze and precisely quantify the degree of DNA methylation. Up to six MVPs were examined simultaneously in a single Pyrosequencing reaction. The method is amenable to the analysis of bisulfite-treated DNA derived from paraffin-embedded tissue samples, highly reproducible, and accurate if calibration is carried out properly (to detect any biased PCR amplification).