A System for Rapid DNA Sequencing with Fluorescent Chain-Terminating Dideoxynucleotides

• Published in: Science (American Association for the Advancement of Science) Vol. 238; no. 4825; pp. 336 - 341
• Main Authors: Prober, James M., Trainor, George L., Dam, Rudy J., Hobbs, Frank W., Robertson, Charles W., Zagursky, Robert J., Cocuzza, Anthony J., Jensen, Mark A., Baumeister, Kirk
• Format: Journal Article
• Language: English
• Published: Washington, DC The American Association for the Advancement of Science 16.10.1987; American Association for the Advancement of Science
• Subjects: Analytical, structural and metabolic biochemistry; Automation; Avian Myeloblastosis Virus - enzymology; Bacteriophages - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Chemical bases; deoxyribonucleotides; Deoxyribonucleotides - adverse effects; Deoxyribonucleotides - therapeutic use; DNA; DNA - genetics; DNA, Viral - genetics; Dyes; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Fluoresceins; Fluorescence; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Gels; General aspects, investigation methods; Genetic engineering; Genetic technics; Laser beams; Methods. Procedures. Technologies; Nucleic acids; nucleotide sequence; Optical filters; RNA-Directed DNA Polymerase - metabolism; Sequencing; Spectrometry, Fluorescence; Succinates; Synthetic digonucleotides and genes. Sequencing; Wavelengths; Fluorescence; Method; Nucleotide sequence; Process improvement
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.2443975

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.