Zuccaro Laura, Tesauro Cinzia, Cerroni Barbara, Ottaviani Alessio, Knudsen Birgitta Ruth, Balasubramanian Kannan, Desideri Alessandro
Department of Biology and Interuniversity Consortium, National Institute of Biostructure and Biosystems (INBB), University of Rome Tor Vergata, Rome 00133, Italy.
Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Rome 00133, Italy.
Anal Biochem. 2014 Apr 15;451:42-4. doi: 10.1016/j.ab.2014.02.003. Epub 2014 Feb 11.
A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage-religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.
已开发出一种高灵敏度检测方法，用于以单分子分辨率检测人类拓扑异构酶I。该方法使用磁珠琼脂糖来浓缩滚环产物，这些产物由拓扑异构酶I环化的DNA分子扩增产生，一旦与荧光探针反应，就可以用共聚焦显微镜检测为单个离散点。每个点对应于由该酶介导的单个切割-连接事件，由于其高信噪比，可以进行计数，从而能够检测到0.3pM的酶，这代表了一种在高度稀释的样品中检测酶活性的有效方法。
