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Real-time kinetics of restriction-endonuclease cleavage monitored by fluorescence resonance energy-transfer

Academic Article
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Overview

authors

  • Ghosh, S. S.
  • Eis, P. S.
  • Blumeyer, K.
  • Fearon, K.
  • Millar, David

publication date

  • 1994

journal

  • Nucleic Acids Research  Journal

abstract

  • The kinetics of PaeR7 endonuclease-catalysed cleavage reactions of fluorophor-labeled oligonucleotide substrates have been examined using fluorescence resonance energy transfer (FRET). A series of duplex substrates were synthesized with an internal CTCGAG PaeR7 recognition site and donor (fluorescein) and acceptor (rhodamine) dyes conjugated to the opposing 5' termini. The time-dependent increase in donor fluorescence resulting from restriction cleavage of these substrates was continuously monitored and the initial rate data was fitted to the Michaelis-Menten equation. The steady state kinetic parameters for these substrates were in agreement with the rate constants obtained from a gel electrophoresis-based fixed time point assay using radiolabeled substrates. The FRET method provides a rapid continuous assay as well as high sensitivity and reproducibility. These features should make the technique useful for the study of DNA-cleaving enzymes.

subject areas

  • Base Sequence
  • Deoxyribonucleases, Type II Site-Specific
  • Fluorescein
  • Fluoresceins
  • Fluorescence
  • Fluorescent Dyes
  • Kinetics
  • Molecular Sequence Data
  • Oligonucleotides
  • Rhodamines
  • Spectrometry, Fluorescence
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Identity

International Standard Serial Number (ISSN)

  • 0305-1048

Digital Object Identifier (DOI)

  • 10.1093/nar/22.15.3155

PubMed ID

  • 8065930
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Additional Document Info

start page

  • 3155

end page

  • 3159

volume

  • 22

issue

  • 15

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