Methylation Dependent Restriction Enzymes for Epigenetics

Scientists at NEB recently identified the MspJI family of restriction enzymes (MspJI (NEB #R0661), LpnPI (NEB #R0663), FspEI (NEB #R0662)), which are dependent on methylation and hydroxymethylation for cleavage to occur (1). These enzymes excise ~ 32 base pair fragments containing a centrally located 5-hmC or 5-mC modified residue that can be extracted and sequenced. Due to the known position of this epigenetic modification, bisulfite conversion is not required prior to downstream analysis. These EpiMark® validated, methylation-dependent restriction enzymes expand the potential for mapping epigenetic modifications and simplify the study of DNA methylation. Additionally, they provide an opportunity to better understand the role of 5-hydroxymethylcytosine in the genome.

A variety of our existing restriction enzymes can also be used to study epigenetic modifications of DNA.

  • MspI (NEB #R0106) and HpaII (NEB #R0171) differentially cleave their recognition site CCGG based on methylation of the internal cytosine.
  • DpnI (NEB #R0176) and DpnII (NEB #R0543) differentially cleave their recognition site GATC based on methylation of the adenine.
  • McrBC (NEB #M0272) only cleaves DNA that contains 5-methylcytosine or 5-hydroxymethylcytosine or N4-methylcytosine on one or both strands.
EpiMark® is a registered trademark of New England Biolabs, Inc.


  1. Cohen-Karni D et al. (2011) Proc. Natl. Acad. Sci. USA 108 (27): 11040-5. PMID: 21690366
  1. 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.

Simplify DNA Methylation Analysis with MspJI

MspJI recognizes methylated and hydroxymethylated DNA and cleaves out ~ 32 bp fragments for downstream sequencing analysis. Overnight digestion of 1 µg of genomic DNA from various sources with or without MspJI is shown.
Note: Yeast DNA does not contain methylated DNA and thus, no 32-mer is detected.

Restriction Enzymes for Epigenetics Selection Chart

Several of NEB's restriction enzymes can be used to study epigenetic modifications of DNA.