Exonuclease III action on microarrays: Observation of DNA degradation by fluorescence correlation spectroscopy |
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| Authors: | Nicky Ehrlich Christian Hübner |
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| Institution: | a Physics Institute, University of Lübeck, D-23538 Lübeck, Germany
b Department of Chemical Biology/BCMT, Faculty of Chemistry, Technische Universität Dortmund, D-44227 Dortmund, Germany |
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| Abstract: | DNA with all cytosines, thymines, or all pyrimidines of one strand substituted by fluorescently labeled analogs shows diminished solubility in aqueous media and a strong tendency to aggregation that hampers enzymatic downstream processing. In this study, immobilization of fully fluorescently labeled DNA on microarrays was shown to resolve the named problems and to enable successive DNA degradation by exonuclease III. Fluorescence correlation spectroscopy and single-molecule counting for monitoring the course of DNA hydrolysis in real time revealed the virtually processive degradation of labeled DNA that occurred at an average rate of approximately 4 nt/s. |
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| Keywords: | DNA Fluorescence labeling Fluorescence spectroscopy Exonuclease Single-molecule studies |
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