COBRA: a sensitive and quantitative DNA methylation assay

TLDR

COBRA is a quantitative technique for measuring DNA methylation at specific gene loci from small amounts of genomic DNA. It uses restriction enzyme digestion of PCR products from sodium bisulfite‑treated DNA to expose methylation‑dependent sequence differences. The digested/undigested PCR product ratio linearly reflects methylation levels over a wide range, and the method works reliably on DNA from microdissected paraffin‑embedded tissue, offering ease of use, quantitative accuracy, and compatibility with such samples.

Abstract

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 methylationdependent 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.