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Article Dans Une Revue Journal of Physical Chemistry B Année : 2010

Measuring number of fluorophores labelling cDNA strands, in solution, with Fluorescence Correlation Spectroscopy and photobleaching

Résumé

We present different approaches that aim at determining, in solution, the brightness and the number of Alexa Fluor 647 molecules labelling the C bases of two sequences of cDNA, corresponding to two transcripts of different sizes, a short and a long transcript (123 and 306 base long, with 45 and 74 dCTP residues, respectively). In each case, the Alexa labeled bases have been incorporated during reverse-transcription. Two kinds of experiments have been performed and combined: photobleaching and Fluorescence Correlation Spectroscopy (together with the factorial cumulant analysis method). As a result, we show that the photobleaching cross-section of labelled cDNA strands is about half that of free Alexa in aqueous solution, while their brightness is about twice. The factorial cumulant analysis put into evidence the fact that the brightness of cDNA strands varies from molecule to molecule, due to the statistical distribution of the number of Alexa fluorophores labelling cDNA. Our measurements are consistent with a poissonian distribution of the number of fluorescent labels per cDNA, with a mean value of about 2 for the long cDNA strands. This indicates that only a few percent of the dCTP incorporated during the reverse transcription hold a fluorescent label, which is consistent with the fact that these Alexa Fluor 647 labels do not interact with each other. Finally, the analysis of experiments also reveals that the photobleaching decay rate of the excited state of Alexa Fluor 647 decreases by about 30% when incorporated into cDNA, while its non radiative decay rate is increased such that the brightness of individual Alexa labels is decreased by 25%.
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Dates et versions

hal-00411842 , version 1 (30-08-2009)
hal-00411842 , version 2 (01-03-2010)

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Antoine Delon, Irène Wang, Emeline Lambert, Silva Lerbs-Mache, Régis Mache, et al.. Measuring number of fluorophores labelling cDNA strands, in solution, with Fluorescence Correlation Spectroscopy and photobleaching. Journal of Physical Chemistry B, 2010, 114 (8), pp.2988. ⟨10.1021/jp910082h⟩. ⟨hal-00411842v1⟩
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