DMB (DNMT-magnetic beads) assay: measuring DNA methyltransferase activity in vitro.
DNA methylation is an epigenetic modification of DNA that leads to heritable alterations in transcriptional regulation and conformational changes in chromatin structure of higher eukaryotes. Mammalian DNA methyltransferases, which are the enzymes responsible for DNA methylation, have attracted the attention of both basic and clinical researchers because they appear to participate in embryogenesis and carcinogenesis via chromatin modification. DNA methyltransferase catalyzes the transfer of a methyl group into DNA strands. Since traditional assays for DNA methyltransferase activity in vitro have insufficient reproducibility, there is a need in the art for more sensitive and quantitative methods for measuring enzymatic activity. We report a novel assay system, in which the activity of a DNA methyltransferase is measured as the incorporation of tritium into biotinylated DNA oligonucleotides. The DNA is immobilized onto magnetic beads with streptavidin covalently attached to the bead surface. The radioactive DNA can easily be separated from the unreacted radioactive substrate using a magnet. The radioactivity is counted by the liquid scintillation system. This DMB assay is simple and easy, has very low background, and, most importantly, is highly reproducible for the precise enzymatic analysis of any DNA methyltransferase in vitro.
Yokochi
Tomoki
T
Epigenetic Gene Regulation and Cancer Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Robertson
Keith D
KD
D000818:Animals; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis* / Q000235:genetics; D008280:Magnetics; D008322:Mammals; D008967:Molecular Biology / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics; D012436:S-Adenosylmethionine / Q000737:chemistry; D013379:Substrate Specificity; D014316:Tritium / Q000737:chemistry
D011994:Recombinant Proteins; D014316:Tritium; D012436:S-Adenosylmethionine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases