General FRET-based coding for application in multiplexing methods.
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.
Giestas
Letícia
L
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
Petrov
Vesselin
V
Baptista
Pedro V
PV
Lima
João Carlos
JC
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D015345:Oligonucleotide Probes / Q000032:analysis* / Q000737:chemistry; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009841:Oligonucleotides