Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads |
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| Authors: | Laura Zuccaro Cinzia Tesauro Barbara Cerroni Alessio Ottaviani Birgitta Ruth Knudsen Kannan Balasubramanian Alessandro Desideri |
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| Affiliation: | 1. Department of Biology and Interuniversity Consortium, National Institute of Biostructure and Biosystems (INBB), University of Rome Tor Vergata, Rome 00133, Italy;2. Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Rome 00133, Italy;3. Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus 8000, Denmark;4. Max Planck Institute for Solid State Research, D-70569 Stuttgart, Germany |
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| Abstract: | 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 0.3 pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples. |
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| Keywords: | Topoisomerase Activity assay Rolling circle amplification Magnetic beads Confocal microscope |
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