ENERGY-TRANSFER PRIMERS WITH 5- OR 6-CARBOXYRHODAMINE-6G AS ACCEPTOR CHROMOPHORES

Energy-transfer (ET) fluorescent primers for DNA sequencing and multip lex 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) ar e 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 a cceptor. Thus, a primer that carries 6-carboxyfluorescein (FAM) at the 5'-end and carboxy-4',5'-dichloro-2',7'-dimethoxy-fluorescein (JOE) a ttached to a modified thymidine 10 bases away is designated F10J. We d escribe here new ET primers, with 5- or 6-carboxyrhodamine-6G (G(5) or G(6)) as accepters (with FAM as the donor) in place of JOE, with impr oved match in the electrophoretic mobilities of the DNA fragments exte nded from the ET dye-labeled primers, and less overlap in the fluoresc ence emission of the various labeled DNA fragments. This reduced spect ral overlap is most likely due to the narrower emission from G(5) or G (6) in F10G compared to that from JOE in F10J. With single-stranded M1 3mp18 DNA as the template, a typical run with F10G(6) and three other ET primers on a capillary sequencer provided DNA sequences with 99% ac curacy in the first 620 bases. (C) 1996 Academic Press, Inc.