New England Biolabs offers a suite of validated products for epigenetics research. These solutions to study DNA and histone modifications are designed to address some of the challenges of current methods.
EpiMark 5-hmC and 5-mC Analysis Kit: The EpiMark® 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-hmC and 5-mC within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. The EpiMark Kit is the first commercially available PCR-based assay to reproducibly identify and quantitate the presence of 5-hmC at a specific locus. Click here to learn more and find ordering information
Restriction Enzymes:
Many restriction enzymes are sensitive to 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 family of restriction enzymes, which are sensitive to methylation and hydroxymethylation. These enzymes will cleave out 32 base pair fragments containing a centrally located 5-hmC or 5-mC modified residue that can be extracted and sequenced providing 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 such as, DpnI and DpnII, that recognize the same sequence, but different methylation patterns. McrBC also only cleaves DNA that is methylated on one or both strands. Click here to learn more and find ordering information
Histones: In eukaryotes, chromatin is organized into nucleosome core particles (NCPs)
that consist of approximately 147 bp of DNA and an octamer complex made up
of two molecules of each histone (H2A, H2B, H3 and H4) (1). The linker histone, H1, can further condense chromatin by binding to linker DNA between the
nucleosome core particles. For histone modification studies, New England Biolabs
offers a selection of unmodified, recombinant human histones that function as
substrates and can assemble into octamers (2,3). Octamer formation can be further
simplified using the EpiMark® Nucleosome Assembly Kit which contains preformed Histone H2A/H2B Dimer and Histone H3.1/H4 Tetramer.
Applications:
Purification & characterization of histone modification enzymes
NEB offers histone, protein and DNA Methyltransferases.
Methyltransferases:
NEB offers a selection of DNA methyltransferases that can be used to generate methylated DNA at specific sites for gene expression studies. Several protein methyltransferases are also available for the specific methylation of histones.
Applications:
Positive controls for methylation specific PCR or bisulfite sequences
Bisulfite treatment and PCR Single/Stranded Conformation Polymorphism Analysis (Bisulfite-PCR-SSCP/BiPS)
Control DNA:
Altered epigenetic patterns are a hallmark of cancer. Many genes are silenced in cancerous cells due to newly acquired de novo methylation of CpG islands (3). Currently, there are several methods that detect methylated CpGs. Methylation-specific PCR (MSP) is a technology used for the sensitive detection of gene methylation in the genome (4). MSP uses an initial bisulfite reaction to modify the DNA by converting unmethylated cytosines to uracils while 5-methylcytosines remain unaltered. This reaction is followed by PCR amplification with specific primers designed to distinguish methylated from unmethylated DNA. Since this is an extremely sensitive PCR-based assay, the use of control DNA is necessary to determine the quality of the bisulfite conversion and to identify artifacts such as primer-dimer pairing and mispriming that can cloud the interpretation of results.
To create a methylation-positive DNA control, all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5´…CG…3´ in HeLa, NIH-3T3 and Jurkat genomic DNA have been enzymatically methylated with CpG Methylase (M.SssI). These modified DNAs are extensively tested for complete methylation by an additional methyl group transfer assay and MSP. A partially demethylated DNA control has also been created by incubating Jurkat cells with a potent methyltransferase inhibitor, 5-aza-2-deoxycytidine (5-Azadc). Bisulfite conversion (5) and sequencing of a section of intergenic (IGS) repetitive DNA (rDNA) that is normally methylated revealed significant CpG demethylation. These DNA controls are powerful tools in the investigation of genomic DNA methylation and epigenomic research.
