Sodium bisulfite is the undisputed superstar of 5mC analysis, and it is just learning to share the spotlight with other techniques. Sodium bisulfite is a chemical that alters the sequence of DNA. Bisulfite causes the deamination of unmethylated cytosine nucleotides to uracil, while leaving methylated cytosine intact. This property allows the discrimination of 5-methyl-C for C (Frommer et al., 1992). The single-nucleotide resolution of this approach has secured bisulfite analyses as the gold-standard for 5mC studies.
Bisulfite is applicable to gene-specific and genome-wide studies. The most common gene-specific approach is bisulfite-sequencing (BS-seq). In BS-seq, PCR is performed on the bisulfite-converted DNA amplifying a small (<500bp) region of interest. The newly produced uracils pair with adenosines, which then pair with thymidines as PCR progresses, effectively changing unmethylated C to T. When the final PCR product is sequenced and compared to the original, C’s that remain C’s were methylated, while C’s converted to T’s were unmethylated (Clark et al., 1994). Pyrosequencing is the best option for this since it provides a percentage methylation at each cytosine (Colella et al., 2003). If sequencing is not needed, several bisulfite PCR approaches are possible. These rely on PCR primers that are designed detect only specific possible outcomes of bisulfite conversion. These techniques allow high-throughput analysis of many methylation sites, but at reduced sensitivity compared to sequencing.
Bisulfite analysis can also interrogate methylation at the genome-wide level (Li et al., 2010). By coupling bisulfite conversion to next-gen DNA sequencing platforms, the percent methylation of any given C in the genome can be detected. Approaches such as whole genome shotgun bisulfite sequencing (WGSBS) and reduced representation bisulfite sequencing (RRBS) utilize next-gen platforms in this way.
Bisulfite Conversion Methods
WGSBS (Whole genome shotgun bisulfite sequencing): Shotgun sequencing is a processes for sequencing a lot DNA, such as a whole genome, and in WGSBS, the genomic DNA is treated with sodium bisulfite before sequencing.
RRBS (Reduced representation bisulfite sequencing):Provides the sensitivity of WGSBS without the cost by only sequencing a reduced, representative sample of the whole genome.
Bislufite Conversion Additional Reading
This review covers many epigenetic techniques including bisulfite and compares the strengths, weaknesses, and applications of each. It also goes into some of the basics of DNA methylation biology and challenges in the study of epigenetic disorders.
This paper gives a good introduction to sodium bisulfite function and use. They authors also give a protocol for bisulfite analysis and discuss some of the challenges that may be faced.
- Clark, S., Harrison, J., Paul, C., and Frommer, M. (1994). High-Sensitivity Mapping of Methylated Cytosines. Nucleic Acids Res. 22, 2990-2997.
- Colella, S., Shen, L., Baggerly, K., Issa, J., and Krahe, R. (2003). Sensitive and quantitative universal Pyrosequencing (TM) methylation analysis of CpG sites. BioTechniques 35, 146-+.
- Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L., and Paul, C.L. (1992). A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. U. S. A. 89, 1827-1831.
- Li, N., Ye, M., Li, Y., Yan, Z., Butcher, L.M., Sun, J., Han, X., Chen, Q., Zhang, X., and Wang, J. (2010). Whole genome DNA methylation analysis based on high throughput sequencing technology. Methods 52, 203-212.