DNA Methylation and HydroxymethylationTools & Technology

The Basics of Bisulfite Conversion for DNA Methylation Analysis

image1Bisulfite conversion is one of the most popular and powerful methods available to study cytosine methylation in DNA (also known as 5-methylcytosine or 5-mC).  The use of sodium bisulfite to convert unmethylated cytosines to uracil was first reported in 1970 by two groups independently, Shapiro et al. at New York University and Hayatsu et al. at the University of Tokyo.  The fundamental principle behind bisulfite treatment is that sulfite can be reversibly added to cytosine to mediate the deamination of cytosine to uracil.  The mechanism by which this process is driven begins with the nucleophilic addition of bisulfite to the C-6 position of cytosine, which allows the rapid deamination of cytosine into 5,6-dihydrouracil-6-sulfonate.  Subsequent treatment with an alkaline solution swiftly eliminates the sulfonate group and regenerates the double bond, yielding uracil (see image for details).  When cytosines are methylated, this reaction is “blocked” due to the extremely low reaction rates for the formation of 5-methyl-6-dihydrocytosine-6-sulfonate, and they are therefore not converted to uracil.

A couple decades after the bisulfite conversion chemistry was developed, it was used by Frommer et al. in an innovative study to detect 5-methylcytosine in DNA.  Following bisulfite conversion, the DNA methylation status can be quickly and easily investigated by routine molecular biology techniques such as direct DNA sequencing, PCR-based methods, or Next-Gen sequencing.  Unmodified cytosines are deaminated to uracil, and read as thymines when sequenced.  Cytosines that are methylated are resistant to the bisulfite conversion, and therefore are still read as cytosines when sequenced.


Methylated cytosine (position 5) is not converted, while unmodified cytosines (positions 7, 9, 11, 14, and 15) are converted to uracil and read as thymine when sequenced.

Methylated cytosine (position 5) is not converted, while unmodified cytosines (positions 7, 9, 11, 14, and 15) are converted to uracil and read as thymine when sequenced.

Bisulfite conversion is a very efficient method to investigate DNA methylation, and is often considered to be the “gold standard” for DNA methylation analysis.  The main benefit of bisulfite treatment for interrogating DNA methylation status is that the location of the 5-mC modification can be determined at single-nucleotide resolution, meaning that the precise cytosine residue that is methylated can be pinpointed.

The bisulfite conversion chemistry and workflow have come a long way since first utilized in 1970.  Early methods to investigate DNA methylation using bisulfite conversion required an overnight bisulfite treatment step, and the DNA was severely damaged during the treatment process.  Today, the entire bisulfite conversion process, including cleanup and purification of bisulfite-converted DNA, can be completed in less than two hours and Next-Gen sequencing-based approaches allow investigation of DNA methylation levels across entire genomes.

While the chemistry might sound complicated, the bisulfite conversion workflow in commercially available kits, such as those from Zymo Research, make the process simple and provide >99.5% conversion efficiency of unmodified cytosines, with virtually no conversion of 5-methylcytosine.  To read more about the industry-leading bisulfite conversion technology offered by Zymo Research, click here.



Shapiro, R., Servis, R.E. and Welcher, M. (1970) Reactions of uracil and cytosine derivatives with sodium bisulfite.  A specific deamination method.  J. Am. Chem. Soc. 92, 422–424.

Hayatsu H, Wataya Y, & Kazushige K (1970). The addition of sodium bisulfite to uracil and to cytosine. Journal of the American Chemical Society, 92 (3), 724-6 PMID: 5411063

Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, & Paul CL (1992). A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proceedings of the National Academy of Sciences of the United States of America, 89 (5), 1827-31 PMID: 1542678

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Kevin B.

Kevin B.

Kevin grew up in Northern California and has also spent several years living on the East Coast. When he is not in the lab, Kevin enjoys snowboarding, watching NFL games (and playing fantasy football!), spending time outdoors, and exploring Southern California.

  • Thank you for your information about bisulfite conversion. I also think that bisulfite sequencing is an imortant method for our DNA research.
    CD Genomics