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Issue 8, 2009
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General FRET-based coding for application in multiplexing methods

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Abstract

FRET can be used as a strategy to assign different simultaneous events in the same sample but “cross-talk” problems are a limitation. Here we present a contribution for the better understanding of the “cross-talk” in FRET experiments that include several pairs in the same sample. Using oligonucleotide probes labeled with fluorescent dyes which can be selectively excited at a specific wavelength, and using target oligonucleotides tagged with a fluorescent dye with specific characteristics that allow only it to emit light upon selective excitation of a specific probe by energy transfer (FRET), we aim to identify the exact probe–target hybridized pair. When using three donors to probe the presence of complementary targets, only 20% of possible donor/acceptor combinations give straightforward signals readily identifiable with the sample composition, while in the remaining cases severe cross-excitation prevents the direct identification of the sample composition. To correctly resolve the samples identity, we developed a theoretical model that enables the unequivocal attribution of a sample composition to a given set of fluorescence signals, in the presence of three donors.

Graphical abstract: General FRET-based coding for application in multiplexing methods

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Supplementary files

Article information


Submitted
26 Mar 2009
Accepted
05 May 2009
First published
26 May 2009

Photochem. Photobiol. Sci., 2009,8, 1130-1138
Article type
Paper

General FRET-based coding for application in multiplexing methods

L. Giestas, V. Petrov, P. V. Baptista and J. C. Lima, Photochem. Photobiol. Sci., 2009, 8, 1130
DOI: 10.1039/B906033B

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