MULTIPLE CAPILLARY DNA SEQUENCER THAT USES FIBEROPTIC ILLUMINATION AND DETECTION

An 8-capillary prototype electrophoresis system for DNA sequencing has been constructed. The sequence of 400-450 bases can be obtained from each capillary in less than an hour from sequencing reactions generate d with four-color fluorescent terminators. Illumination of each capill ary and collection of fluorescence is through individual optical fiber s. Resolution of the DNA ladder is through a replaceable sieving matri x of linear polyacrylamide in reusable coated capillaries. Light from an argon ion laser is introduced into a fused biconically tapered fibe r-optic splitter, and individual fibers deliver approximately 10 mW of 514 nm light to each of the eight electrophoresis capillaries. Illumi nation and collection are by fibers normal to the surface of the elect rophoresis capillary and at right angle to each other. Illumination by a fiber with low numerical aperture and collection by a fiber with hi gh numerical aperture provides good sensitivity and signal-to-noise ra tios without the need for microlenses (limit of detection: 1.5 x 10(-1 1) M fluorescein analog dye with a signal-to-noise ratio of 2). The ei ght collection fibers are passed in parallel through holographic filte rs for Rayleigh rejection and into an imaging spectrograph, which simu ltaneously displays the full fluorescence spectrum (475-648 nm) from t he eight capillaries in parallel on the surface of an intensified char ge-coupled device (CCD). The CCD is read out at a rate of 3.4 complete images per second.