A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides.
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.
D001331:Automation; D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D003854:Deoxyribonucleotides / Q000009:adverse effects / Q000627:therapeutic use; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins; D005456:Fluorescent Dyes; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013386:Succinates
D004279:DNA, Viral; D003854:Deoxyribonucleotides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013386:Succinates; D004247:DNA; D012194:RNA-Directed DNA Polymerase