Detection and quantitation of the activity of DNA methyltransferases using a biotin/avidin microplate assay.
The biotin-avidin microplate assay is a sensitive method to measure methylation of biotinylated oligonucleotide substrates by DNA methyltransferases (MTases). The methylation reaction is carried out in solution using [methyl-3H]-AdoMet. Afterwards, the oligonucleotides are immobilized on an avidin-coated microplate, where the incorporation of [3H]-labeled methyl groups into the DNA is stopped by addition of unlabeled AdoMet to the binding buffer. Separation of radioactively labeled DNA from unreacted AdoMet and enzyme is performed by washing steps. Subsequently, the radioactivity incorporated into the DNA is released by a nucleolytic digestion of the DNA. By liquid scintillation counting, the amount of DNA methylation can be determined. Advantages of the microplate assay are its high sensitivity which allows the detection of low amounts of DNA methylation, the efficient separation of reaction components resulting in a low background of radioactivity and a high accuracy (+/-10%) and reliability. Furthermore, the assay is very convenient, fast and well suited for automation.
Liebert
Kirsten
K
School of Engineering and Science, International University Bremen, Bremen, Germany.
Jeltsch
Albert
A
D001360:Avidin; D001710:Biotin; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000032:analysis*; D012436:S-Adenosylmethionine / Q000378:metabolism
D001360:Avidin; D001710:Biotin; D012436:S-Adenosylmethionine; D015254:DNA Modification Methylases