DNA SEQUENCING BY CAPILLARY GEL ELECTROPHORESES AND LASER-INDUCED FLUORESCENCE DETECTION

Detection limits of one to two thousand analyte molecules is produced by laser-induced fluorescence with the sheath flow cuvette for DNA sequencing. Two different detector systems are developed for the sequencing of real DNA samples: a single laser and single emission spectral channel system for the sequencing technique of Richardson and Tabor and a single laser excitation and dual emission spectral channel detector for the sequencing technique of DuPont. The best detection limits (1,400 molecules) are obtained with a 750-mu w helium-neon laser (lambda = 544 nm), a cooled PMT, and tetramethylrhodamine labeled primer used with the Richardson/Tabor sequencing technique. Although better detection limits have been reported for the determination of highly dilute neat dye solution, these results for DNA sequencing are unprecedented for the analysis of real samples and are made possible by the extreme specificity of the Sanger chain-terminating reaction, the extreme resolution of capillary gel electrophoresis, and the extreme detection performance of laser induced fluorescence with the sheath flow cuvette.