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Magnetic bead-based electrochemical assay for determination of DNA methyltransferase activity

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DUDOVÁ Zdenka BARTOŠÍK Martin FOJTA Miroslav

Rok publikování 2017
Druh Článek v odborném periodiku
Časopis / Zdroj Electrochimica Acta
Citace
www http://dx.doi.org/10.1016/j.electacta.2017.02.104
Doi http://dx.doi.org/10.1016/j.electacta.2017.02.104
Klíčová slova DNA methyltransferase; DNA methylation; magnetic beads; restriction endonuclease; carbon electrode
Popis DNA methylation, an epigenetic mechanism playing important role in many cellular processes, is carried out via action of DNA methyltransferase (MTase) enzymes. Increasing evidence shows that altered activity of MTases may cause aberrant DNA methylation, which in turn may lead to malignant transformation, and ultimately to cancer. Assessing MTase activity is thus considered of high importance in early cancer diagnostics. Electrochemistry represents an interesting alternative to current methods of MTase analysis, as it is relatively inexpensive, fast and simple. We describe here a development of electrochemical assay in which DNA probe, containing restriction sites CCGG for HapII endonuclease and bearing biotin label at its distal end, is immobilized at magnetic beads and incubated in MTase solution. Methylation of DNA then blocks restriction by HapII and the terminal biotin moiety is preserved to be available for attachment of alkaline phosphatase producing an electroactive indicator, yielding electrochemical signal from the methylated DNA. On the other hand, without MTase the DNA is not methylated and thus the biotinylated DNA end is cleaved-off, leading to the signal diminution. The assay is simple and quick, requiring less than 3 h to completely assess MTase activity. (C) 2017 Elsevier Ltd. All rights reserved.
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