Editing Methylation PCR

7. Detection of Hydroxymethyl Cytosine Finally, we would like to briefly describe methods that aim to detect 5-hydroxymethylcytosine (5-hmC) and distinguish it from 5 mC. Essentially, there are two types of approaches for which this can be achieved: digestion- and antibody-based techniques.

It has been determined that the enzyme glucosyltransferase will modify 5-hmC, but not 5 mC bases. Based on this intrinsic difference in their properties as substrates, both New England Biolabs and Zymo Research have developed assay kits for the specific detection of glycosylated 5-hmC.

Zymo Research’s Quest hmC Detection Kit utilizes hydroxymethylcytosine glucosyltransferase and the substrate uridine diphosphoglucose to selectively glucosylate 5-hmC bases. Importantly, modification of the 5-hmC bases renders these sites of the DNA strand resistant to cleavage by the nuclease MspI. Digested DNA is then analysed by either NGS or the qPCR assay. The EpiMark 5-hmC and 5-mC Analysis Kit by New England Biolabs uses the action of T4 beta-glucosyltransferase, followed by MspI digestion to accomplish the same task.

Enrichment for 5-hmC-containing DNA could be done with anti-5-hmC antibodies. They are available from several companies.

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810160/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810160/]

DNA Methylation Enzymes
enzymes for the analysis of DNA methylation variants
Active Motif offers enzymes to study not only the mechanism of the conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC), but also enzymes that enable differentiation between these two forms of DNA methylation.

Recombinant Tet1 protein
Tet1 is a member of the TET family of cytosine oxygenases that convert 5-methylcytosine into 5-hydroxymethylcytosine1. For researchers interested in studying the mechanism of how 5-hydroxymethylcytosine is generated, Active Motif's Recombinant Tet1 protein is a valuable tool. This active enzyme can be used to convert 5-mC containing DNA into 5-hmC containing DNA. To see details, please visit our Recombinant Tet1 protein product page.

β-Glucosyltransferase
β-Glucosyltransferase enzyme is a T4 bacteriophage enzyme that is capable of modifying 5-hydroxymethylcytosine residues with the addition of a glucose moiety. The β-Glucosyltransferase enzyme works on double-stranded DNA and the glucosylation is independent of DNA sequence or structural context2. The glucosylation occurs when DNA containing 5-hmC residues is incubated in the presence of β-Glucosyltransferase and a UDP-Glucose (uridine diphopshoglucose) donor. This highly efficient enzyme will modify the 5-hmC residues, while leaving unmethylated and 5-methylcytosine residues untouched. This high specificity of the enzyme enables clear differentiation of 5-hmC and 5-mC DNA for downstream analysis with glucosyl-sensitive restriction enzymes (e.g. MspI, MspJI or GlaI). Alternatively, the use of a labeled [14C] UDP-Glucose donor allows for direct detection and quantification of 5-hmC residues2. Click to learn more about our β-Glucosyltransferase enzyme.

PvuRts1I enzyme
Unlike glucosyl-sensitive restriction enzymes that require prior addition of a glucose moiety to the 5-hydroxymethylcytosine residue before they can distinguish 5-mC and 5-hmC, Active Motif's unique PvuRts1I restriction enzyme is able to directly discriminate between 5-methylcytosine and 5-hydroxymethylcytosine DNA methylation. PvuRts1I will directly cleave hydroxymethylated DNA in its non-glucosylated form, but will not digest 5-methylctyosine or unmethylated cytosine residues3. The enzyme will also cleave glucosyl-5-hydroxymethylcytosine DNA, but at a lower efficiency. The PvuRts1I enzyme has a cleavage recognition sequence of hmCN11-12/N9-10G. This versatile sequence simply requires the presence of a guanine residue 20, 21 or 22 nucleotides after the presence of a hydroxymethylated cytosine residue. The enzyme will cleave in the middle of the sequence separating the hydroxymethylated cytosine and guanine to generate a two nucleotide 3' overhang. The ends of the overhang are not defined as the sequence of the intervening residues are not specifically defined. The intervening residues also do not affect the enzyme's recognition or cleavage. PvuRts1I digestion is ideal for use in regions of relatively high local concentrations of hydroxymethylcytosine, such as gene bodies. Click to learn more abou


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