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.
Zuccaro, L., Tesauro, C., Cerroni, B., Ottaviani, A., Knudsen, B., Balasubramanian, K., et al. (2014). Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads. ANALYTICAL BIOCHEMISTRY, 451, 42-44 [10.1016/j.ab.2014.02.003].
Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads.
DESIDERI, ALESSANDRO
2014-01-01
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 0.3pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
