Two-color fluorescent cytosine extension assay for the determination of global DNA methylation

Biotechniques. 2017 Apr 1;62(4):157-164. doi: 10.2144/000114533.


Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA. In addition, TCF-CEA provided an index of global DNA methylation that was sensitive to differences >5%. TCF-CEA results were highly correlated with LUminometric Methylation Assay (LUMA) results using human liver cell lines (HepG2, HepaRG, HC-04) as well as a human liver primary cell culture. Hypomethylation was observed in cells treated with the de-methylating agent 5-aza-2'-deoxycytidine. These results demonstrate that TCF-CEA provides a simple method for measuring relative degrees of global DNA methylation that could potentially be scaled up to higher-throughput formats.

Keywords: accuracy; dual label; methylation sensitive restriction enzyme; precision.

MeSH terms

  • Azacitidine / analogs & derivatives
  • Azacitidine / chemistry
  • Biological Assay / methods*
  • Cytosine*
  • DNA Methylation / genetics*
  • DNA Restriction Enzymes / genetics
  • Decitabine
  • Fluorescence
  • Hep G2 Cells
  • Humans
  • Liver / cytology
  • Liver / metabolism
  • Primary Cell Culture


  • Decitabine
  • Cytosine
  • DNA Restriction Enzymes
  • Azacitidine