Since the seminal paper on bisulfite conversions is celebrating its 20th anniversary this year, I thought it would be nice to feature, over the next few weeks, how the bisulfite conversion technique came to be and the assays that have been developed around this seminal technique.
How It All Began…..
In 1970, Hayatsu et al. discovered a chemical interaction between sodium bisulfite and pyrimidines that would have a tremendous impact on how DNA methylation patterns and changes are studied. This group found that uracil, thymididine, and deoxycydidine were subjected to sulfonation at position six of their pyrimidine rings when treated with sodium bisulfite.1 This model was later extended to 5-methylcytosine (5mC) residue.2
In 1992, Frommer et al. described that the differing rates of converting 5mC to cytosine could be used to analyze DNA methylation patterns in genomic DNA.3 Briefly, DNA was purified from HeLa cells, placenta, liver, and sperm. A plasmid was constructed that contained the promoter and first exon of the human kininogen gene. This plasmid contained known methylation patterns and was used as a standard. The DNAs were linearized with either EcoRI or via fine needle shearing and then treated with sodium bisulfite. The samples were then dialyzed to remove any unreacted bisulfite. Dialyzed samples were dried down, the resuspended in buffer, treated with sodium hydroxide then ammonium acetate. The bisulfite reacted DNA was precipitated and resuspended in buffer. The purified, converted samples were then amplified with strand-specific primers for the human kininogen gene. The group found that it was feasible to identify specific patterns of cytosine methylation by using the discrimination in deamination reactivity of cytosine and 5-methylcytosine by bisulfite conversion.3
Now that you know how the bisulfite movement was born, we will dive into techniques to exploit this reaction. Stay tuned!
- Hayatsu, H. et al. 1970. Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry. 9, 2858–65.
- Wang, Y. et al. 1980. Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res. 8, 4777–90.
- Frommer, M. et al. 1992. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strand. Proc. Natl. Acad. Sci. U.S.A. 89, 1827–31.