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Biotin-Avidin Microplate Assay for the Quantitative Analysis of Enzymatic Methylation of DNA by DNA Methyltransferases by Markus Roth; Albert Jeltsch is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

What is Biotin-Avidin Microplate Assay for the Quantitative Analysis of Enzymatic Methylation of DNA by DNA Methyltransferases about?

An assay is described to measure methylation of biotinylated oligonucleotide substrates by DNA methyltransferases using [methyl-3 H]-AdoMet. After the methylation reaction the oligonucleotides are immobilized on an avidin-coated microplate. The incorporation of [ 3 H] into the DNA is quenched by addition 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 radioactivity determined by liquid scintillation counting. As an example, we have studied methylation of DNA by the EcoRV DNA methyltransferase. The reaction progress curves measured with this assay are linear with respect to time. 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, fast 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 fmol

Who reads Biotin-Avidin Microplate Assay for the Quantitative Analysis of Enzymatic Methylation of DNA by DNA Methyltransferases?

Author

Markus Roth; Albert Jeltsch

Publisher

Walter de Gruyter GmbH

Published

2000

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)