Methylation Assays: Measuring DNA Methylation and Demethylation Processes

The functioning of DNA methylation and demethylation processes can be measured using a number of different kits.

In recent decades, DNA’s four bases have been expanded to include newly discovered cytosine variants.

5-methylcytosine (5-mC) is the single variant that has been most extensively studied. Working to silence gene expression, it is an epigenetic mark which performs a vital role in such processes as cell differentiation, neurodegeneration and cancer.

Other forms can be derived from the oxidation of 5-mC. These include 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC), which are being investigated with great interest presently – particularly concerning their influences on human health and disease.

DNA Methylation: DNMTs

Methyltransferases (DNMTs) catalyze the methylation of the C-5 position of cytosine. The family of these DNMT enzymes found in humans includes de novo DNMT3A and DNMT3B, as well as DNMT1 – a maintenance DNA methyltransferase.

DNMT activity can be quantified by certain assays in less than four hours. These assays also allow the screening of potential inhibitors of DNMT activity.

DNA Demethylation: TET Enzymes

The DNA demethylation reaction is catalyzed by the ten-eleven translocation (TET) enzymes. The oxidized derivatives 5hmC, 5-fC, and 5-caC, are converted from 5-mC with TET1, TET2 and TET3.

TET enzyme activity can be quantified by certain assays in less than four hours. These assays also allow the screening of potential inhibitors of TET enzyme activity.

​​​5-mC, 5-hmC and 5-fC

Rather than being just intermediate products of the demethylation pathway as previous thought, it is becoming increasingly apparent that 5-hmC, 5-fC, and 5-caC are epigenetic marks in themselves. Each modification is stable, and has different interacting proteins, as well as varied genomic distributions and differing effects on gene expression.

Assays for the global quantification of these three modifications are available in a range of kits.

Methylated DNA Immunoprecipitation: MeDIP and hMeDIP

Allowing the enrichment of methylated or hydroxymethylated DNA in a sample, MeDIP and hMeDIP are antibody-based techniques. The sample then obtained is usable in downstream qPCR, microarrays or next-generation sequencing.

Experiments can be made more efficient with the use of kits for MeDIP and hMeDIP.

Bisulfite Conversion and Bisulfite Sequencing

The benchmark against which all DNA methylation analyses are measured is bisulfite sequencing, which can provide single-base resolution, quantitative measurement of methylated cytosines.

When DNA is treated with sodium bisulfite, cytosine is converted to uracil, however methylated cytosine remains unconverted. By subsequently sequencing the untreated and bisulfite-treated DNA in parallel, the methylation status of individual cytosine residues in a sample can be provided.

There are kits available for bisulfite conversion, as well as bisulfite conversion coupled with library preparation, and post-bisulfite library preparation.

About Abcam

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Last updated: Apr 1, 2019 at 6:43 AM

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