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

摘要

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 three base-pairs. This shorter fragment rapidly dissociated at 37 °C causing an increase of fluorescence intensity, which was used to gauge the reaction kinetics. The reaction can be described using the Michaelis-Menten mecahnism and the kinetic parameters obtained were compared to literature values involving other protocols.