Bisulfite Conversion

Sodium bisulfite conversion of genomic DNA to differentiate and detect unmethylated versus methylated cytosines is the gold standard for DNA methylation analysis. It provides single nucleotide resolution map of 5-mC of the genome (1). Bisulfite conversion involves the deamination of unmodified cytosines to uracil, leaving the modified bases 5-mC and 5-hmC. This procedure can then be followed either by PCR amplification or massively parallel sequencing methods to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.

Treatment of denatured DNA (i.e., single-stranded DNA) with sodium bisulfite leads to deamination of unmethylated cytosine residues to uracil, leaving 5-mC or 5-hmC intact. The uracils are amplified in subsequent PCR reaction as thymines, whereas 5-mC or 5-hmC residues get amplified as cytosines. Comparison of sequence information between the reference genome and bisulfite-treated DNA can provide information about cytosine methylation patterns. Other applications, such as Methylation-Specific PCR (MSP), involve analyzing untreated and bisulfite treated DNA using two different sets of PCR primer pairs, one pair that targets the unaltered, methylated sequence and the other that targets the converted unmethylated sequence.

A new method for methylome analysis at the single base level, NEBNext® Enzymatic Methyl-seq (EM-seq™) (NEB #E7120), is now available. This enzyme-based technology minimizes damage to DNA and produces high-quality libraries that enable superior detection of 5mC and 5hmC from fewer sequencing reads. 

  1. Frommer et al. (1992) Proc. Natl. Acad. Sci. 89. 1827-1831. PMID: 1542678

Choose Type:

FAQs for Bisulfite Conversion
Protocols for Bisulfite Conversion
Application Notes for Bisulfite Conversion
    Publications related to Bisulfite Conversion
    • Chernov AV., Reyes L., Peterson S., Strongin AY. (2015) Depletion of CG-Specific Methylation in Mycoplasma hyorhinis Genomic DNA after Host Cell Invasion PLoS One; 10, e0142529. PubMedID: 26544880, DOI: 10.1371/journal.pone.0142529
Overview of EpiMark® Bisulfite Conversion Kit
Overview of NEBNext® Enzymatic Methyl-seq
Legal Information

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.


  • What is Epigenetics?

    What Is Epigenetics?

    If all cells are created from the same genetic material, why are there so many different cell types? Listen to Sriharsa Pradhan, Senior Scientist, RNA Biology at NEB, as he describes how DNA is methylated and how this affects the path of reading the DNA code the same way an obstruction would derail a train off its tracks.