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Restriction Enzymes for Epigenetic Analysis (EpiMark® Validated)

Many restriction enzymes are sensitive to the DNA methylation states. Cleavage can be blocked or impaired when a particular base in the recognition site is modified. Scientists at NEB recently identified the MspJI (NEB #R0661) family of restriction enzymes, which are dependent on methylation and hydroxymethylation for cleavage to occur. 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.

Several of our existing restriction enzymes can also be used to study epigenetic modifications of DNA, such as DpnI (NEB #R0176) and DpnII (NEB #R0543) that recognize the same sequence, but different methylation patterns. McrBC also only cleaves DNA that is methylated cytosine on one or both strands. MspI (NEB #R0106) and HpaII (NEB #R0171) recognize the same sequence (CCGG) but are sensitive to different methylation status. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmCo or 5-ghmC) at either cytosine blocks cleavage. MspI will recognize and cleave 5-hmC and 5-hmC, but not 5-ghmc. These enzymes are included in the EpiMark® 5-hmC and 5-mC Analysis Kit (NEB #E3317).

Simplify DNA Methylation Analysis with MspJI

MspJI (NEB #R0661) 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.


EpiMark® is a registered trademark of New England Biolabs, Inc.


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Protocols for Restriction Enzymes for Epigenetic Analysis (EpiMark® Validated)

    Publications related to Restriction Enzymes for Epigenetic Analysis (EpiMark® Validated)

  1. Mooijman D, Dey S S, Boisset JC, Crosetto N, van Oudenaarden A 2016. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction Nature Biotechnology. 34, PubMedID: 27347753, DOI: 10.1038/nbt.3598