A spectroscopic method to determine the activity of the restriction endonuclease EcoRV that involves a single reaction.
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.
Huang
Qing
Q
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico 00931-3346.
Quiñones
Edwin
E
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico 00931-3346. Electronic address: equinones@uprrp.edu.
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases