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

doi:10.1016/j.ab.2014.02.003

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 newspaper › Journal article › Research › peer-review

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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 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 language	English
Journal	Analytical Biochemistry
Volume	451
Pages (from-to)	42-44
Number of pages	3
ISSN	0003-2697
DOIs	https://doi.org/10.1016/j.ab.2014.02.003
Publication status	Published - 15 Apr 2014

Keywords

Topoisomerase
Activity assay
Rolling circle amplification
Magnetic beads
Confocal microscope

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@article{2aaf7bd44d12427a8781153a830b39e7,

title = "Rolling circle amplification based detection of human topoisomerase I activity on magnetic beads",

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.",

keywords = "Topoisomerase, Activity assay, Rolling circle amplification, Magnetic beads, Confocal microscope",

author = "Laura Zuccaro and Cinzia Tesauro and Barbara Cerroni and Alessio Ottaviani and Knudsen, {Birgitta Ruth} and Kannan Balasubramanian and Alessandro Desideri",

note = "Copyright {\textcopyright} 2014. Published by Elsevier Inc.",

year = "2014",

month = apr,

day = "15",

doi = "10.1016/j.ab.2014.02.003",

language = "English",

volume = "451",

pages = "42--44",

journal = "Analytical Biochemistry",

issn = "0003-2697",

publisher = "Academic Press Inc.",

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TY - JOUR

T1 - Rolling circle amplification based detection of human topoisomerase I activity on magnetic beads

AU - Zuccaro, Laura

AU - Tesauro, Cinzia

AU - Cerroni, Barbara

AU - Ottaviani, Alessio

AU - Knudsen, Birgitta Ruth

AU - Balasubramanian, Kannan

AU - Desideri, Alessandro

PY - 2014/4/15

Y1 - 2014/4/15

N2 - 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.

AB - 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.

KW - Topoisomerase

KW - Activity assay

KW - Rolling circle amplification

KW - Magnetic beads

KW - Confocal microscope

U2 - 10.1016/j.ab.2014.02.003

DO - 10.1016/j.ab.2014.02.003

M3 - Journal article

C2 - 24525043

SN - 0003-2697

VL - 451

SP - 42

EP - 44

JO - Analytical Biochemistry

JF - Analytical Biochemistry

ER -

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

Abstract

Keywords

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