Energy transfer primers with 5- or 6-carboxyrhodamine-6G as acceptor chromophores

Energy-transfer (ET) fluorescent primers for DNA sequencing and multiplex genetic analysis (Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351) are named according to the convention D-N-A, where D is the donor, N is the number of bases between the donor and the acceptor, and A is the acceptor. Thus, a primer that carries 6-carboxyfluorescein (FAM) at the 5'-end and 6-carboxy-4', 5'-dichloro-2',7'-dimethoxyfluorescein (JOE) attached to a modified thymidine 10 bases away is designated F10J. We describe here new ET primers, with 5- or 6-carboxyrhodamine-6G (G5 or G6) as acceptors (with FAM as the donor) in place of JOE, with improved match in the electrophoretic mobilities of the DNA fragments extended from the ET dye-labeled primers, and less overlap in the fluorescence emission of the various labeled DNA fragments. This reduced spectral overlap is most likely due to the narrower emission from G5 or G6 in F10G compared to that from JOE in F10J. With single-stranded M13mp18 DNA as the template, a typical run with F10G6 and three other ET primers on a capillary sequencer provided DNA sequences with 99% accuracy in the first 620 bases.