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Anal Biochem. 2010 Jun 1;401(1):168-72. doi: 10.1016/j.ab.2010.02.032. Epub 2010 Mar 1.

Fluorescence-based high-throughput assay for human DNA (cytosine-5)-methyltransferase 1.

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1
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Abstract

We have developed the first economical and rapid nonradioactive assay method that is suitable for high-throughput screening of the important pharmacological target human DNA (cytosine-5)-methyltransferase 1 (DNMT1). The method combines three key innovations: the use of a truncated form of the enzyme that is highly active on a 26-bp hemimethylated DNA duplex substrate, the introduction of the methylation site into the recognition sequence of a restriction endonuclease, and the use of a fluorogenic read-out method. The extent of DNMT1 methylation is reflected in the protection of the DNA substrate from endonuclease cleavage that would otherwise result in a large fluorescence increase. The assay has been validated in a high-throughput format, and trivial changes in the substrate sequence and endonuclease allow adaptation of the method to any bacterial or human DNA methyltransferase.

PMID:
20197058
PMCID:
PMC2854261
DOI:
10.1016/j.ab.2010.02.032
[Indexed for MEDLINE]
Free PMC Article
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