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Restriction Enzymes for Epigenetic Analysis

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).

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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Publications related to Restriction Enzymes for Epigenetic Analysis

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 Nat Biotechnol. 34, PubMedID: 27347753, DOI: 10.1038/nbt.3598

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Videos

Interactive Tutorial Explaining the Phenomenon of Epigenetics at a Molecular Level

Watch an interactive tutorial explaining the different molecular mechanisms by which epigenetic change influences gene expression. Learn about how NEB’s reagents are targeted to the various enzymes and DNA elements that are altered by epigenetic change.
What Does Epigenetics Have to Do with Honeybees?

Rick Feehery, Research Associate at New England Biolabs, Inc, explains the mechanisms of epigenetic DNA modification. To drive home the power of such tiny changes, he uses the example of honeybee colonies, wherein DNA methylation is the only factor that determines whether a bee is born a worker, drone or queen.
What are Restriction Enzymes?

Watch as Geoff Wilson, Emeritus Scientist, describes what restriction enzymes are and how they revolutionized molecular biology.
How Complex is Epigenetics?

Epigenetic changes to DNA impact a wide range of biological processes such as organism development and disease progression. Watch as Sriharsa enthusiastically describes this exciting and complex field that has the power to "re-write" the course of DNA transcription.
How Can Epigenetics Help Us Treat Diseases?

Cancer cells have epigenetic changes to their DNA. One of these causes the turning off (silencing) of tumor suppressor genes, a small set of genes involved in cell cycle control. Sriharsa discusses drugs currently in the clinic that are able to demethylate DNA and improve the lives of cancer patients.
EpiMark 5-hmC and 5-mC Analysis Kit Protocol Animation

New England Biolabs is pleased to introduce the EpiMark® 5-hmC and 5-mC Analysis Kit, a simple and robust method for the identification and quantitation of 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) within a specific DNA locus.

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