Mark A. Quesada, Shiping Zhang, Janine Graves and F. William Studier
Biology Department, Brookhaven National Laboratory, Upton, New York 11973
An 8-capillary prototype electrophoresis system for DNA sequencing has been constructed. The sequence of more than 400 bases can be obtained from each capillary in less than an hour, from sequencing reactions generated with ABI 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 re-usable coated capillaries generously provided by Karger's group.[1]
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 8 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 micro-lenses. The OH stretch Raman line was convenient for aligning the fibers. In the prototype version, the fibers are fixed to the electrophoresis capillary with optical cement, but more convenient configurations are being explored. The 8 collection fibers are passed in parallel through holographic filters for Rayleigh rejection and into an imaging spectrograph, which simultaneously displays the full fluorescence spectrum (500-670 nm) from the 8 capillaries in parallel on the surface of an intensified CCD. The CCD is read out at a rate of 3.4 complete images per second. We are developing base-calling software for this system and intend to generate confidence levels as an intrinsic part of the base-calling process.
Improvements of this prototype system will be aimed at developing a reliable and fully automated system capable of sustained production of tens of kb of sequence per hour.
Supported by the Office of Health and Environmental Research of the U. S. Department of Energy.
[1] Ruiz-Martinez, M. C., Berka, J., Belenkii, A., Foret, F., Miller, A. W., and Karger, B. L. DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser-induced fluorescence detection. Anal. Chem. 65, 2851-2858 (1993).