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

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.