Cyanine dyes with high absorption cross section as donor chromophores in energy transfer primers

Energy transfer (ET) fluorescent primers are significantly superior to single dye-labeled primers for DNA sequencing and multiplex genetic analyses (Ju, J., Glazer, A. N., and Mathies, R. A. (1996) Nature Med. 2, 246-249). We describe here ET primers in which a donor chromophore with a large absorption cross section but a low fluorescence quantum yield is exploited to increase the Stokes-shifted fluorescence emission of acceptor dyes. The new ET primers have 3-(epsilon-carboxypentyl)-3' -ethyl-5,5' -dimethyloxacarbocyanine (CYA; epsilon(M)(488)nm 142,000 M(-1) cm(-1)) at the 5'-end as a common energy donor, and fluorescein or rhodamine derivatives (FAM, R6G, TAMRA, and ROX), attached to a modified thymidine 10 bases away within the primer sequence, as acceptors. With 488-nm excitation, the fluorescence emission intensity of these four ET primers is 1.4- to 24-fold stronger than that of the corresponding primers labeled only with the single acceptor dye. When compared with the corresponding ET primers with a fluorescein derivative (FAM; epsilon(M)(488) 60,000 M(-1) cm(-1)) as donor, the fluorescence emissions of primers with CYA as donor and FAM, R6G, TAMRA, and ROX as accepters are respectively 0.8-, 1.0-, 1.7-, and 1.7-fold as intense. The low fluorescence quantum yield of the CYA donor resulted in distinct fluorescence signals for the DNA-sequencing fragments with much lower crosstalk between the four detection channels than that seen with ET primers based on a FAM donor. With single stranded M13mp18 DNA as the template, the CYA ET primers provided DNA sequences on a four-color capillary sequencer with 100% accuracy in the first 500 bases. (C) 1996 Academic Press, Inc.