SODIUM BISULFITE INDUCED OXIDATIVE DEAMINATION OF GENOMIC DNA
In single-stranded DNA, sodium bisulfite preferentially deaminates cytosine residues to uracil, compared with a very slow rate of deamination of 5-methylcytosine to thymine (Shapiro et al., 1973). Frommer et al. (1992) utilized this difference in bisulfite reactivity for genomic sequencing of 5-methylcytosine residues, by fully denaturing total genomic DNA and treating total genomic DNA with sodium bisulfite under conditions such that cytosine is converted stoichiometrically to uracil, but 5-methylcytosine remains nonreactive.
The DNA is initially denatured by alkali treatment prior to treatment with bisulfite. The second part of the procedure involves PCR amplification of the region of interest in the bisulfite-reacted DNA to yield a fragment in which all uracil, formerly cytosine, and thymine residues have been amplified as thymine and only 5-methylcytosines have been amplified as cytosine.
The bisulfite reaction yields DNA strand products, which are no longer complementary. PCR primers can therefore be designed, such that a specific pair can only bind to one of the bisulfite-reacted DNA strands. Primers for each strand will differ in every position where there is a C or G in the original sequence (Frommer et al., 1992).
If the PCR products from the bisulfite-treated DNA are cloned and individual clones are sequenced, the sequences will provide methylation maps of single DNA strands from individual DNA molecules in the original genomic DNA sample. The procedure yields a sequence and methylation pattern specific for each strand of the original genomic DNA. The position of each 5-methylcytosine will be given by a positive band on a sequencing gel (Frommer et al., 1992).
METHODS and MATERIALS: A Protocol for the Analysis of DNA Methylation by Sequencing of Sodium Bisulfite-treated DNA
Genomic DNA is isolated using the Qiagen DNA KitTM and subjected to sodium bisulfite treatment to modify unmethylated cytosine to uracil, using the CpGenome TM DNA Modification Kit (Intergen Company, Oxford , UK ) and following the manufacturer’s instructions.
The conditions for PCR were as follows: 1 cycle at 95 0 C for 15 min; 40 cycles of 94 0 C for 1 min, 60 0 C for 1 min and 72 0 C for 1 min; and 1 cycle of 72 0 C for 10 min. QIAGEN has recently developed an excellent kit for methylation analsys that should be sought if you are able to.