Tolun Gökhan, Myers Richard S
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, PO Box 016129, Miami, FL 33101-6129, USA.
Nucleic Acids Res. 2003 Sep 15;31(18):e111. doi: 10.1093/nar/gng111.
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.
DNA核酸酶（DNases）执行多种重要的细胞功能，并且在研究和生物技术应用中也非常有用。由于DNases的生物学和技术重要性及其在广泛应用中的使用，DNase活性测定至关重要。传统的DNase测定使用放射性标记的DNA底物，并且在定量酶活性之前需要将反应产物与未反应的底物分离。因此，这些方法是不连续的。在本报告中，我们描述了一种基于称为PicoGreen的DNA染料配体的差异荧光输出的连续DNase测定方法。该测定方法是为了表征一种连续性双链DNA外切核酸酶λ外切核酸酶而开发的。该测定方法似乎具有普遍适用性，因为它也适用于测量连续性解旋酶/核酸酶RecBCD、分布性外切核酸酶T7基因6外切核酸酶和核酸内切酶DNaseI的DNA消化活性。本文讨论了该方法的优点和局限性。
