Principles of bi-sulfite conversion of DNA and methylation-specific PCR (MSP) in biological research

Abstract Epigenetics is a connection between genotype and phenotype, where the phenotypic changes are observed without hampering genomic sequences of an organism. Among the various epigenetic alterations, DNA methylation is relatively stable and well-established form of covalent modification, where a methyl group is attached to the 5th carbon of the cytosine ring in a gene, whereby altering the function of that gene. It directly affects gene transcription by regulating the accessibility of the gene promoter to transcription factors (TFs) toward their binding sites (TFBSs) that leads to changes in chromatin structure, thereby restricting access of TFs to the gene promoter. In order to detect the pattern of DNA methylation, bi-sulfite conversion-based method is generally considered as the “gold-standard,” which converts unmethylated cytosine (C) into uracil (U) and leaving methylated cytosine (5mC) unchanged. The methylation-specific PCR (MSP) is the most commonly used technique that relies on this conversion method, where modified DNA samples are subjected to PCR amplification using methylated and unmethylated specific primers, which has been developed as the most commonly used minimally invasive method for early diagnosis or prognosis of different types of cancer. This chapter presents a comprehensive review on the principles of bisulfite conversion of dsDNA along with its certain modifications for improving the conversion rate of C to U with a moderate degree of DNA degradation and various applications of MSP in biological research.