Bisulfite-mediated Deamination of Cytosine in DNA under Near-neutral Conditions

Abstract

In DNA methylation analysis for epigenetic studies, the most frequently used procedure involves bisulfite-mediated conversion of cytosine to uracil. Resistance of 5-methylcytosine against this bisulfite reaction is the basis for identifying the methylated sites. This chemical modification consists of a treatment of single-stranded nucleic acids with high-concentration bisufite at pH 5-6. We report here that the treatment can be performed at pH > 6 by supplementary presence of tetramethylammonium ion. Quaternary ammonium cation appears to be essential in this unique enhancement of the reaction in the near-neutral pH range, as betaine was also effective but trimethylamine was not. Using a tetramethylammonium-bisulfite reagent at pH 6.4, we achieved a complete deamination of cytosine residues in a DNA molecule, without any significant changes taking place for 5-methylcytosine in it. These findings have opened up the possibility of improving and expanding the DNA methylation analysis technology.