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.
(1) Cohen-Karni D et al. (2011) Proc. Natl. Acad. Sci. USA 108 (27): 11040-5. PMID: 21690366
EpiMark® is a registered trademark of New England Biolabs, Inc.
FAQs for Methylation Dependent Restriction Enzymes for Epigenetics
- Are there any published papers in which McrBC has been used?
- Does McrBC cut hemi-methylated DNA?
- How much enzyme should be used for cleaving genomic DNA?
- Does McrBC produce blunt or sticky ends?
- Why does my McrBC cleaved DNA smear when run on an agarose gel?
- Is extended digestion of McrBC recommended?
- Will DpnI cleave hemimethylated DNA?
- What's the difference between DpnI, DpnII, MboI, and Sau3AI?
Protocols for Methylation Dependent Restriction Enzymes for Epigenetics
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using LpnPI (R0663)
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using FspEI
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using MspJI
- Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
- Protocol for Digestion Prior to droplet digital PCR (ddPCR)
- Optimizing Restriction Endonuclease Reactions
- Double Digest Protocol with Standard Restriction Enzymes
Epigenetics - Expanding on Genomic Foundations
The Epigenetics brochure provides information on these unique solutions to study DNA and histone modifications. NEB offers a suite of validated products for epigenetics research.
- Dam-Dcm and CpG Methylation
Other Tools & Resources
Restriction Enzymes for Epigenetics Selection Chart
Simplify DNA Methylation Analysis with MspJI
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.