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 sequencing 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. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5′-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation 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 for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.