Multiple capillary DNA sequencer that uses fiber-optic 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 generated with four-color fluorescent terminators. Illumination of each capillary and collection of fluorescence is through individual optical fibers. Resolution of the DNA ladder is through a replaceable sieving matrix of linear polyacrylamide in reusable coated capillaries. Light from an argon ion laser is introduced into a fused biconically tapered fiber-optic splitter, and individual fibers deliver approximately 10 mW of 514 nm light to each of the eight electrophoresis capillaries. Illumination and collection are by fibers normal to the surface of the electrophoresis capillary and at right angle to each other. Illumination by a fiber with low numerical aperture and collection by a fiber with high numerical aperture provides good sensitivity and signal-to-noise ratios without the need for microlenses (limit of detection: 1.5 x 10(-11)M fluorescein analog dye with a signal-to-noise ratio of 2). The eight collection fibers are passed in parallel through holographic filters for Rayleigh rejection and into an imaging spectrograph, which simultaneously displays the full fluorescence spectrum (475-648 nm) from the eight capillaries in parallel on the surface of an intensified charge-coupled device (CCD). The CCD is read out at a rate of 3.4 complete images per second.
Quesada
M A
MA
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Zhang
S
S
D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D005336:Fiber Optic Technology; D055100:Optical Fibers; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods; D013050:Spectrometry, Fluorescence
D004247:DNA