COMPARISON OF FLUORESCENCE ENERGY-TRANSFER PRIMERS WITH DIFFERENT DONOR-ACCEPTOR DYE COMBINATIONS

Fluorescence energy transfer (ET) primers are far superior to single d ye-labeled primers as labels for DNA sequencing and polymerase chain r eaction amplification. We compare here ET primers with different donor and acceptor dye combinations with respect to the relative acceptor f luorescence emission intensity and the amount of residual donor fluore scence emission. Primers with the following donor/acceptor pairs were synthesized: 6-carboxyfluorescein/6-carboxy-X-rhodamine (FAM-ROX), 5,5 '-dimethyloxacarbocyamine/6-carboxy-X-rhodamine (CYA-ROX), and the 4,4 -difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene-3-propionic acid (BO DIPY) derivatives, 5,7-dimethyl-BODIPY/5-(4-phenyl-1,3-butadienyl) (BO DIPY503/512-BODIPY581/591). Variables examined included the length of the 5'-amino linker arm, the number of base pairs between the donor an d acceptor, and the excitation wavelength (488 or 514 nm). Of the prim ers examined, CYA-ROX primers offer the best combination of acceptor f luorescence emission intensity and spectral purity. (C) 1998 Academic Press.