|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
35. Long-Read DNA Sequencing by Capillary Array Electrophoresis
Oscar Salas-Solano, Lev Kotler, Zoran Sosic,
Arthur W. Miller, Yongwu Yang, Haihong Zhou, and Barry L. Karger
The increasing prominence of capillary array electrophoresis for DNA sequencing raises the importance of being able to obtain long reads on a regular basis with such instruments. We have recently reported the use of capillary electrophoresis (CE) for routine DNA sequencing of 1000 bases in less than one hour using replaceable linear polyacrylamide solutions (Salas-Solano et al., Anal. Chem. 1998, 70, 3996-4003). These results have now been extended to a multiple-capillary array. Cycle-sequencing reactions, and most steps of subsequent sample purification, are performed in 96-well microtiter plates with a system built around a Biomek 2000 robot. Before each run, the array of polyvinyl alcohol-coated capillaries is refilled with linear polyacrylamide solution. The 488 nm output of an argon laser is directed into an optic fiber, and then into a line generator, which focuses the beam into a 35-micrometer wide line extending across the entire bank of capillaries. The emitted dye fluorescence passes through notch filters, and through a transmission grating for spectral dispersion, and is imaged onto a CCD that is read at 3 Hz. This design has no moving parts and is easy to align. Base-calling is done by an expert system (see separate abstract of A. W. Miller and B. L. Karger), and read lengths of 1000 bases and above at 98-99% accuracy are routinely obtained. We will also report on our latest results for achieving long read length sequencing using capillary electrophoresis with replaceable polymer solutions.
This work is being supported by DOE grant DE-FG02-98ER 69895.
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