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A spectroscopic method to determine the activity of the restriction endonuclease EcoRV that involves a single reaction

Authors
  • Huang, Qing
  • Quiñones, Edwin
Type
Published Article
Journal
Analytical Biochemistry
Publication Date
Jan 01, 2008
Identifiers
DOI: 10.1016/j.ab.2008.04.038
Source
Elsevier
License
Unknown

Abstract

A one-step protocol is presented to determine the activity of EcoRV as a model of restriction enzymes. The protocol involved a molecular beacon as DNA substrate, with the target sequence recognized by EcoRV in the stem. EcoRV cleaved the stem forming two fragments, one of which contained the fluorophore and quencher, initially bound by 3 bp. This shorter fragment rapidly dissociated at 37 °C, causing an increase of fluorescence intensity that was used to gauge the reaction kinetics. The reaction can be described using the Michaelis–Menten mechanism, and the kinetic parameters obtained were compared with literature values involving other protocols.

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