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

Journal
Analytical Biochemistry
Publisher
Elsevier
Identifiers
DOI: 10.1016/j.ab.2008.04.038
Disciplines
  • Biology
License
Unknown

Abstract

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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