NEW ENERGY-TRANSFER DYES FOR DNA-SEQUENCING

We have synthesized a set ob four energy transfer dyes and demonstrate d their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes a re a novel set of four 4,7-dichloro-substituted rhodamine dyes which h ave narrower emission spectra than the standard, unsubstituted rhodami nes. A rigid amino acid linker; 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequenc ing instrument equipped with a dual line argon ion laser (488 and 514 nan excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvem ent in signal-to-noise was 4- to 5-fold. The utility of the new dye se t was demonstrated by sequencing of a BAC DNA with an 80 kb insert, Me asurement of the extinction coefficients and the relative quantum yiel ds of the dichlororhodamine components of the energy transfer dyes sho wed their values were reduced by 20-25% compared with the dichlororhod amine dyes alone.