A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides.

J M Prober, G L Trainor, R J Dam, F W Hobbs, C W Robertson, R J Zagursky, A J Cocuzza, M A Jensen, K Baumeister

Index: Science 238(4825) , 336-41, (1987)

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Abstract

A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleotides, each carrying a different chemically tuned succinylfluorescein dye distinguished by its fluorescent emission is described. Avian myeloblastosis virus reverse transcriptase is used in a modified dideoxy DNA sequencing protocol to produce a complete set of fluorescence-tagged fragments in one reaction mixture. These DNA fragments are resolved by polyacrylamide gel electrophoresis in one sequencing lane and are identified by a fluorescence detection system specifically matched to the emission characteristics of this dye set. A scanning system allows multiple samples to be run simultaneously and computer-based automatic base sequence identifications to be made. The sequence analysis of M13 phage DNA made with this system is described.