Fluorometric determination of deoxyribonuclease I activity with PicoGreen.
A rapid and sensitive assay for the detection of deoxyribonuclease I (DNase I) activity is described. This method is based on the ability of PicoGreen dye to enhance its fluorescence when bound to double-stranded DNA. In the standard assay, reaction mixtures containing the DNase I sample and 0.2 microg of the substrate DNA were prepared in a fluorescence microtiter plate and incubated at 37 degrees C. At the end of the reaction, the diluted PicoGreen reagent was added to each well and fluorescence intensity was measured with a fluorescence plate reader. By this assay, it was possible to determine precisely as little as 5 pg of DNase I within an hour. Moreover, using a small amount of the substrate DNA, the method was shown to be suitable for the sensitive detection of DNase I inhibitor activity.
Choi
S J
SJ
Department of Chemistry, Kangnung National University, Korea. sjchoi@knusun.kangnung.ac.kr
Szoka
F C
FC
D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D009930:Organic Chemicals; D011817:Rabbits; D012015:Reference Standards
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA; D003850:Deoxyribonuclease I