CASSETTE LABELING FOR FACILE CONSTRUCTION OF ENERGY-TRANSFER FLUORESCENT PRIMERS

DNA primer sets, labeled with two fluorescent dyes to exploit fluoresc ence energy transfer (ET), can be efficiently excited with a single la ser line and emit strong fluorescence at distinctive wavelengths, Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple colorbased analyses [J, Ju, C, Ruan, C, W, Fuller, A, N, Glazer and R, A, Mathies (1995) Proc, Natl. Acad. Sc i. USA 92, 4347-4351], We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorpo rated by conventional synthesis at the 5'-end of an oligonucleotide pr imer of any sequence, In this cassette, the donor and acceptor fluorop hores are separated by a polymer spacer (S-6) formed by six 1',2'-dide oxyribose phosphate monomers (S), The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attache d to the 3' end of the ribose spacer in the ET cassette, The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fl uorescein and rhodamine dyes as accepters, display well-separated acce ptor emission spectra with 2-12-fold enhanced fluorescence intensity r elative to that of the corresponding single dye-labeled primers, With single-stranded M13mp18 DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% ac curacy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequenc er.