logo
AAT Bioquest

How is DNA methylation detected?

Posted January 22, 2021


Answer

Three methods are commonly used to identify and quantitate DNA methylation, these include:

  • Bisulfite genomic sequencing – this technique is considered to be the gold standard for detecting DNA methylation from unmethylated DNA using PCR amplification. First, DNA is treated with bisulfite. This converts cytosine residues to uracil while leaving methylated cytosines unaffected. After bisulfite conversion, DNA is amplified using PCR which converts uracil residues to thymine. Finally the degree of DNA methylation can be determined either by direct PCR sequencing or cloning sequencing.
  • Differential enzymatic cleavage of DNA – this technique uses methylation-sensitive restriction enzymes to analyze methylated DNA. This class of enzymes cleaves DNA at specific unmethylated-cytosine residues while leaving methylated DNA intact. Following digestion, DNA is then amplified using PCR, and because unmethylated DNA was digested previously only methylated DNA amplification products are detected.
  • Methylated DNA immunoprecipitation – this technique uses monoclonal antibodies raised against 5-methylcytosine (5-mC) to extract or enrich methylated DNA. In this method, purified genomic DNA extracted from cells is sonicated into random fragments ranging from 300 to 1000 base pairs in length. The DNA fragments are then denatured and incubated first with monoclonal 5mC antibodies and then anti-mouse IgG antibodies conjugated with magnetic beads. Magnets are used to separate methylated DNA from the solution and unbound DNA is removed in the supernatant. After isolation and purification, the methylated DNA is ready for sequencing or microarray studies.
Additional resources

DNA methylation detection: bisulfite genomic sequencing analysis

Portelite™ Fluorimetric High Sensitivity DNA Quantitation Kit *Optimized for CytoCite™ and Qubit™ Fluorometers*