Abstract: A rapid, simple and high-throughput activity assay method of non-specific nuclease has been established, according to the fact that under the irradiation of ultraviolet light and through the combination of nucleic acids with DNA stain Gelred.The fluorescent complex was generated and then emited fluorescence. In the study, the change of optical density caused by the decrease of substrate DNA measured by a gel imager, therefore the enzyme activity was obtained. Key factors involved in the activity assay experiment were studied, as well as the linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ) of the method were all verified. The results suggested that the optimal reaction time was 3 min, the linear range of enzyme activity was 4.17-9.01 U/μL, and the linear relationship, precision, accuracy and sensitivity met the evaluation requirements of methodology development. In conclusion, the method exhibited lower LOD and shorter reaction time compared with the UV absorption assay method, and it could be applied for multiple samples analysis.

Key words: non-specific nuclease, enzyme activity, high throughput, optical density, gel imaging