A simple method for high-throughput quantification of genome-wide DNA methylation by fluorescence polarization

Abstract

To rapidly determine DNA methylation levels from a large number of biological or clinical samples, we have developed an accurate and sensitive method for high-throughput quantification of global methylation of 5′-Cm⁵CGG-3′ sites in the genome, visualized by fluorescence polarization (FP) based measurement of DNA methylation (FPDM). In FPDM, the methyl-sensitive HpaII and methyl-insensitive MspI restriction enzymes were employed to achieve DNA cleavage, followed by incorporation of fluorescent dCMP into the enzyme-cleavage products through polymerase chain extension, yielding an FP-ratio between the HpaII- and MspI-restricted preparations as a measure of methylation. FPDM provided stable estimates of methylation level of submicrograms of lambda or human DNA, and of a 255-bp DNA segment containing a single HpaII/MspI restriction site in accord with, and more accurate than, determination by gel electrophoresis. FPDM was also applied to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA-methyltransferase inhibitor 5-aza-dC.

Keywords::

• epigenetics
• HpaII
• fluorescent dCTP
• global CpG methylation
• methyl-sensitive restriction enzyme