PCR techniques in characterizing DNA methylation

Abstract

DNA methylation was the fi rst epigenetic mark to be discovered, involving the addition of a methyl group to the 5′ position of cytosine by DNA methyltransferases, and can be inherited through cell division. DNA methylation plays an important role in normal human development and is associated with the regulation of gene expression, tumorigenesis, and other genetic and epigenetic diseases. Differential methylation is now known to play a central role in the development and outcome of most if not all human malignancies. Bisulfi te conversion is a commonly used approach for gene-specifi c DNA methylation analysis. Treatment of DNA with bisulfi te converts cytosine to uracil while leaving 5-methylcytosine intact, allowing for single-nucleotide resolution information about the methylated areas of DNA. PCR-based methods are routinely used to study DNA methylation on a gene-specifi c basis, after bisulfi te treatment. Variations of this method include bisulfi te sequencing, methylation-specifi c PCR, real-time PCR-based MethyLight, and methylation-sensitive high-resolution melting PCR. Several whole-epigenome profi ling technologies such as MethylC-seq reduced representation bisulfi te sequencing (RRBS) and the Infi nium Human methylation 450 K bead chip are now available allowing for the identifi cation of epigenetic drivers of disease processes as well as biomarkers that could potentially be integrated into clinical practice.