Sequencing of Sodium Bisulfite-treated DNA: DNA Methylation Analysis
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).
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.
PCR products are then separated on a 1 % agarose gel, stained with ethidium bromide, and visualized under ultraviolet (UV) light. The PCR bands are subsequently cut from the gel with a sharp razor, pooled together and purified using the QIAGEN gel extraction kit (QIAGEN Inc., Valencia, CA) according to the vendor’s instructions. The purified DNA product is subsequently ligated to a TA cloning vector, TOPO pCR2.1 Sequencing Vector (Invitrogen, Grand Island , NY ). 3 ml of the dilution is then added to DH5 a -T1 competent cells and incubated in ice for 30 minutes. Cells are then heat-shocked for 45 seconds in a 42 o C heat block, and then placed on ice for a further 2 minutes. 250 m l SOC medium is added to each vial of cells. The vials are subsequently shaken at 225 RPM, at 37 o C for one hour, after which the content of each vial is spread on agar plates containing 100 m g/ml of ampicillin. Plates are then incubated overnight at 37 o C for the production of positive colonies.
Five-ten ampicillin resistant colonies grown on agar plates are selected and cultured overnight in Luria-Bertani (LB) medium composed of 1.0% NaCl, 1.0% tryptone and 0.5% yeast extract (DIFCO, MD, USA), pH 7.0 supplemented with 100 m g/ml of ampicillin (Sigma-Aldrich, St. Louis, MO). The ampicillin provides a selective pressure for the exclusive growth of positive colonies. Plasmid DNA is isolated using the QIAGEN Miniprep Kit (QIAGEN Inc., Valencia , CA ), following the manufacturer’s instructions. Screening for positive plasmids is done by a restriction digest. 1 m l of purified plasmid, with 1 m l of EcoRI (Fermentas Inc., MD, USA ), 1 m l of Buffer EcoRI and 17 m l of dH20, are incubated for 2 hours at 37 o C. The reaction mixture is subsequently seperated on a 2.5% agarose gel, stained with ethidium bromide and visualized under UV light. Positive clones are then sequenced using M13 primers (Invitrogen), and analyzed for percentage methylation of specific CpG dinucleotides. Data analsis consists of plotting CpG island dinucleotide positions and percentage of cytosines methylated.