Exonuclease III action on microarrays: Observation of DNA degradation by fluorescence correlation spectroscopy

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...

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Bibliographic Details
Published in:Analytical biochemistry 2010-04, Vol.399 (2), p.251-256
Main Authors: Ehrlich, Nicky, Anhalt, Katrin, Hübner, Christian, Brakmann, Susanne
Format: Article
Language:English
Subjects:
DNA
DNA - chemistry
DNA - metabolism
Exodeoxyribonucleases - metabolism
Exonuclease
Fluorescence labeling
Fluorescence spectroscopy
Fluorescent Dyes - chemistry
Hydrolysis
Microscopy, Confocal
Oligonucleotide Array Sequence Analysis - methods
Single-molecule studies
Spectrometry, Fluorescence
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2009.12.004