EFFICIENT DNA-SEQUENCING WITH A PULSED SEMICONDUCTOR-LASER AND A NEW FLUORESCENT DYE SET

A new method is presented for automated one-lane four-dye DNA sequenci ng in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. T he primer sequencing reaction was applied in a confocal optical system . Detection and identification of the differently 5'-labeled primers w as done by time-correlated single-photon counting and a specially deve loped pattern-recognition technique based on the characteristic fluore scence lifetimes of the fluorescent dyes used as labels. Efficient exc itation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM) . With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential fo r high-throughput DNA sequencing in parallel capillaries or microfabri cated DNA sequencing chips. (C) 1997 Elsevier Science B.V.