Comparison of fluorescence energy transfer primers with different donor-acceptor dye combinations

Fluorescence energy transfer (ET) primers are far superior to single dye-labeled primers as labels for DNA sequencing and polymerase chain reaction amplification. We compare here ET primers with different donor and acceptor dye combinations with respect to the relative acceptor fluorescence emission intensity and the amount of residual donor fluorescence emission. Primers with the following donor/acceptor pairs were synthesized: 6-carboxyfluorescein/6-carboxy-X-rhodamine (FAM-ROX), 3-(epsilon-carboxypentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine/ 6-carboxy-X-rhodamine (CYA-ROX), and the 4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene-3-propionic acid (BODIPY) derivatives, 5,7-dimethyl-BODIPY/5-(4-phenyl-1,3-butadienyl) BODIPY (BODIPY503/512-BODIPY581/591). Variables examined included the length of the 5'-amino linker arm, the number of base pairs between the donor and acceptor, and the excitation wavelength (488 or 514 nm). Of the primers examined, CYA-ROX primers offer the best combination of acceptor fluorescence emission intensity and spectral purity.