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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)168-172
Number of pages5
JournalAnalytical biochemistry
Volume401
Issue number1
DOIs
StatePublished - Jun 2010

Keywords

  • Fluorescence detection
  • High-throughput assay
  • Human cytosine DNA methyltransferase 1

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Fluorescence-based high-throughput assay for human DNA (cytosine-5)-methyltransferase 1'. Together they form a unique fingerprint.

Cite this