5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

DNA methylation is an epigenetic modification that involves the addition of a methyl group to the cytosine base of the DNA to form 5-methylcytosine. This modification alters the gene expression.

To quantify DNA methylation via dot-blot, take genomic DNA samples derived from human chondrocytes at various stages of de-differentiation that exhibit different levels of methylated cytosine.

Treat the DNA samples with sodium hydroxide — a strong alkali — and heat them. This treatment breaks the hydrogen bonds between the two DNA strands, resulting in DNA denaturation. Add ammonium acetate to neutralize the alkali, preventing excessive DNA degradation.

Take a nylon membrane and spot denatured DNA samples as dots. The negatively-charged DNA binds to the positively-charged nylon membrane via electrostatic interactions, resulting in its blotting on the solid support.

Treat the blotted membrane with a blocking buffer to prevent non-specific binding. Next, add anti-5-methylcytosine antibodies to the membrane, and incubate. These antibodies exclusively bind to the methylated cytosine on the DNA.

Wash to remove the unbound antibodies, and add chemiluminescent enzyme-conjugated secondary antibodies that specifically bind to the primary antibodies.

Add a chemiluminescent substrate onto the membrane. The enzyme on the antibody reacts with the substrate to produce chemiluminescence — yielding dots of various intensities.

Image the membrane and measure the dots' intensities which corresponds to extent of DNA methylation in each sample.