COBRA: a sensitive and quantitative DNA methylation assay

Nucleic Acids Res. 1997 Jun 15;25(12):2532-4. doi: 10.1093/nar/25.12.2532.


We report here on a quantitative technique called COBRA to determine DNA methylation levels at specific gene loci in small amounts of genomic DNA. Restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA as described previously. We show that methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. In addition, we show that this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples. COBRA thus combines the powerful features of ease of use, quantitative accuracy, and compatibility with paraffin sections.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Colorectal Neoplasms / chemistry
  • Colorectal Neoplasms / pathology
  • DNA / chemistry*
  • DNA Methylation*
  • DNA, Neoplasm / chemistry*
  • Dinucleoside Phosphates / analysis
  • Dinucleoside Phosphates / chemistry
  • Genome, Human
  • Humans
  • Indicators and Reagents
  • Polymerase Chain Reaction / methods
  • Receptors, Estrogen / genetics*
  • Restriction Mapping / methods*
  • Sensitivity and Specificity
  • Sulfites


  • DNA, Neoplasm
  • Dinucleoside Phosphates
  • Indicators and Reagents
  • Receptors, Estrogen
  • Sulfites
  • cytidylyl-3'-5'-guanosine
  • DNA
  • sodium bisulfite