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

Laura Zuccaro, Cinzia Tesauro, Barbara Cerroni, Alessio Ottaviani, Birgitta Ruth Knudsen, Kannan Balasubramanian, Alessandro Desideri

    Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

    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 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

    Keywords

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

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