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Rolling circle amplification based detection of human topoisomerase I activity on magnetic beads

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  • Laura Zuccaro, Department of Biology and Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy. Electronic address: laura.zuccaro@gmail.com.
  • ,
  • Cinzia Tesauro
  • Barbara Cerroni, Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy. Electronic address: b.cerroni@gmail.com.
  • ,
  • Alessio Ottaviani, Denmark
  • Birgitta Ruth Knudsen
  • Kannan Balasubramanian, Max Planck Institute for Solid State Research, Heisenbergstrasse 1, D-70569 Stuttgart, Germany. Electronic address: b.kannan@fkf.mpg.de.
  • ,
  • Alessandro Desideri, Denmark
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 at 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 of enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.
Original languageEnglish
JournalAnalytical Biochemistry
Volume451
Pages (from-to)42-44
Number of pages3
ISSN0003-2697
DOIs
Publication statusPublished - 15 Apr 2014

    Research areas

  • Topoisomerase, Activity assay, Rolling circle amplification, Magnetic beads, Confocal microscope

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