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.

Start this procedure by denaturing the isolated DNA in 0.1 molar sodium hydroxide for 10 minutes at 95 degrees Celsius. Neutralize the DNA with 1 molar ammonium acetate on ice, and then dilute two-fold with double-distilled water.

The most critical step in the dot blot is the spotting of the samples onto the membrane, do it slowly, and carefully. Try to minimize each spotted area to 3 to 4 millimeters in diameter.

Using a narrow-mouth pipette tip, carefully spot 2 microliters of the serial diluted genomic DNA onto a positively-charged nylon membrane at the center of the grid. Blot the membrane at 80 degrees Celsius for 30 minutes. Next, block the non-specific antibody binding sites by soaking the membrane in 5% BSA in TBST in a 10-centimeter Petri dish for 1 hour at room temperature with gentle shaking.

After 1 hour, wash the membrane three times in TBST for 5 minutes each time. Incubate the membrane with a mouse anti-5-methylcytosine monoclonal antibody in TBST at 4 degrees Celsius overnight. On the following day, wash the membrane three times in TBST for 5 minutes each time.

Then, incubate with a secondary antibody — horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin G in TBST for one hour at room temperature. After washing the membrane in TBST as before, add the enzyme substrate to the membrane, and incubate for 5 to 10 minutes. Finally, visualize the secondary antibody signal using a chemiluminescence kit according to the manufacturer's instructions.