General FRET-based coding for application in multiplexing methods

Photochem Photobiol Sci. 2009 Aug;8(8):1130-8. doi: 10.1039/b906033b. Epub 2009 May 26.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / analysis*
  • Fluorescent Dyes / chemistry
  • Oligonucleotide Probes / analysis*
  • Oligonucleotide Probes / chemistry
  • Oligonucleotides / analysis*
  • Oligonucleotides / chemistry

Substances

  • Fluorescent Dyes
  • Oligonucleotide Probes
  • Oligonucleotides