Biotin-avidin microplate assay for the quantitative analysis of enzymatic methylation of DNA by DNA methyltransferases

An assay is described to measure methylation of biotinylated oligonucleotid e substrates by DNA methyltransferases using [methyl-H-3]-AdoMet. After the methylation reaction the oligonucleotides are immobilized on an avidin-coa ted microplate, The incorporation of [H-3] into the DNA is quenched by addi tion of unlabeled AdoMet to the binding buffer. Unreacted AdoMet and enzyme are removed by washing. To release the radioactivity incorporated into the DNA, the wells are incubated with a non-specific endonuclease and the radi oactivity determined by liquid scintillation counting. As an example, we ha ve studied methylation of DNA by the EcoRV DNA methyltransferase. The react ion progress curves measured with this assay are linear with respect to tim e, Methylation rates linearly increase with enzyme concentration, The rates are comparable to results obtained with the same enzyme using a different assay. The biotin-avidin assay is inexpensive, convenient, quantitative, fa st and well suited to process many samples in parallel. The accuracy of the assay is high, allowing to reproduce results within +/- 10%. The assay is very sensitive as demonstrated by the detection of incorporation of 0.8 fmo l methyl groups into the DNA. Under the experimental conditions, this corre sponds to methylation of only 0.03% of all target sites of the substrate. U sing this assay, the DNA methylation activity of some M.EcoRV variants coul d be detected that was not visible by other in vitro methylation assays.