A real-time DNase assay (ReDA) based on PicoGreen fluorescence.
DNA nucleases (DNases) perform a wide variety of important cellular functions and are also very useful for research and in biotechnological applications. Due to the biological and technological importance of DNases and their use in a wide range of applications, DNase activity assays are essential. Traditional DNase assays employ radiolabeled DNA substrates and require separation of the products of the reaction from the unreacted substrate before quantification of enzyme activity. As a consequence, these methods are discontinuous. In this report, we describe a continuous DNase assay based on the differential fluorescence output of a DNA dye ligand called PicoGreen. The assay was developed to characterize a processive dsDNA exonuclease, lambda exonuclease. The assay appears to have general utility as it is also suitable for measuring the DNA digestion activities of a processive helicase/nuclease, RecBCD, a distributive exonuclease, T7 gene 6 exonuclease, and an endonuclease, DNaseI. The benefits of, and limitations to, the method are discussed.
Tolun
Gökhan
G
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, PO Box 016129, Miami, FL 33101-6129, USA.
Myers
Richard S
RS
D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D005470:Fluorometry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D009930:Organic Chemicals; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013997:Time Factors; D014764:Viral Proteins
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D014764:Viral Proteins; D004247:DNA; D003851:Deoxyribonucleases; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda