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.
- Frommer et al. (1992) Proc. Natl. Acad. Sci. 89. 1827-1831. PMID: 1542678
- What types of sample can be processed using the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
- How should EM-seq sequencing data be analyzed?
- No PCR product is detected when I amplify more than 300 bp amplicon.
- Does my sample need to be free of RNA for bisulfite conversion?
- Do I need extra purification of my mammalian DNA prep in order to use it in a bisulfite conversion reaction?
- My thermocycler does not allow to set up the reaction volume to 140 μl, what I should do?
- Which DNA polymerase do you recommend for amplification of converted DNA?
- How should I store my bisulfite converted DNA after it is eluted from the column?
- What is the shelf life of the reagents supplied?
- Capture and Elute Step for Control DNA (E2600)
- Capture Methylated CpG DNA (E2600)
- Cycling Protocol Using EpiMark™ Bisulfite Conversion Kit (E3318)
- DNA Fragmentation (E2600)
- Double Digest Protocol with Standard Restriction Enzymes
- Downstream Analysis (NEB #E2600)
- Elute Captured Methylated CpG DNA (E2600)
- End-point PCR Using EpiMark Bisulfite Conversion Kit (E3318)
- Optimizing Restriction Endonuclease Reactions
- Prebind MBD2a-Fc to Protein A Magnetic Beads (NEB #E2600)
- Protocol for use with NEBNext ChIP-seq Library Prep Reagent Set for Illumina (E6200)
- Reagent Preparation Using EpiMark® Bisulfite Conversion Kit (E3318)
- Wash Off Unbound DNA (E2600)
- 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, PubMedID: 26544880, DOI: 10.1371/journal.pone.0142529
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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.