Analysis of In Vivo Methylation

Abstract

The presence of methylated cytosines (as 5-methylcytosine, 5-MeC) in eukaryotic DNA was established nearly 50 years ago. Nevertheless, the function of methylated nucleotides in DNA has not yet been fully established. They have been proposed to play a role in regulation of gene expression, in genome imprinting, in X chromosome inactivation, in DNA repair mechanisms, tumorigenesis, and aging. A number of methods are available to detect 5-MeC in DNA and most are based on the use of methylation-sensitive restriction enzymes or genomic sequencing protocols. Although technically simple, the use of methylation-sensitive restriction enzymes has the disadvantage that only 5-MeCs that are part of the recognition sequences can be detected and analyzed. In addition, hemimethylated DNA cannot normally be detected. One genomic sequencing protocol (1) is based on the chemical sequencing method developed by Maxam and Gilbert (2), but takes advantage of the different reactivity of hydrazine toward C and 5-MeC. Recently, a simple and efficient genomic sequencing technology has been developed (3). Unlike other methods, this novel approach gives a positive display of 5-MeC residues in the DNA. It is based on the observation that sodium bisulfite, followed by alkaline treatment, converts cytosine residues in single-stranded DNA to uracil under conditions where 5-MeC is unreactive (4). This deamination process is outlined in Fig. 1.