Cassette labeling for facile construction of energy transfer fluorescent primers

Abstract

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347–4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5′-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S₆) formed by six 1′,2′-dideoxyribose phosphate monomers (S). The donor is attached to the 5′ side of the ribose spacer and the acceptor to a modified thymidine attached to the 3′ end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxyfluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2–12-fold enhanced fluorescence intensity relative 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% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.