Edward S. Yeung, Huan-Tsung Chang, Qingbo Li, Xiandan Lu, Eliza Fung
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011.
We have developed novel separation, detection, and imaging techniques for real-time monitoring in capillary electrophoresis. These techniques will be used to substantially increase the speed, throughput, reliability, and sensitivity in DNA sequencing applications in highly multiplexed capillary arrays. We estimate that it should be possible to eventually achieve a raw sequencing rate of 40 million bases per day in one instrument based on the standard Sanger protocol. We have reached a stage where an actual sequencing instrument with 100 capillaries can be built to replace the Applied Biosystems 373 or 377 instruments, with a net gain in speed and throughput of 100-fold and 24-fold, respectively.
The substantial increase in sequencing rate is a result of several technical advances in our laboratory. (1) The use of commercial linear polymers for sieving allows replaceable yet reproducible matrices to be prepared that have lower viscosity (thus faster migration rates) compared to polyacrylamide. (2) The use of a charge-injection device camera allows random data acquisition to decrease data storage and data transfer time. (3) The use of distinct excitation wavelengths and cut-off emission filters allows maximum light throughput for efficient excitation and sensitive detection employing the standard 4-dye coding. (4) The use of indexmatching and 1:1 imaging reduces stray light without sacrificing the convenience of on-column detection.
Continuing efforts include further optimization of the separation matrix, development of new column conditioning protocols, refinement of the excitation/emission optics, design of a pressure injection system for 96-well titer plates, validation of a new 2-color base-calling scheme, simplification of software to allow essentially real-time data processing, implementation of voltage programming to shorten the total run times, and scale up of the technology to allow parallel sequencing in up to 1,000 capillaries.
References
K. Ueno and E. S. Yeung, "Simultaneous Monitoring of DNA Fragments Separated by Capillary Electrophoresis in a Multiplexed Array of 100 Channels", Anal. Chem. 66, 1424-1431 (1994).
X. Lu and E. S. Yeung, "Optimization of Excitation and Detection Geometry for Multiplexed Capillary Array Electrophoresis of DNA Fragments", Appl. Spectrosc. 49, 605-609 (1995).
Q. Li and E. S. Yeung, "Evaluation of the Potential of a Charge Injection Device for DNA Sequencing by Multiplexed Capillary Electrophoresis", Appl. Spectrosc. 49, 825-833 (1995).
E. N. Fung and E. S. Yeung, "High-Speed DNA Sequencing by Using Mixed Poly(ethyleneoxide) Solutions in Uncoated Capillary Columns," Anal. Chem. 67, 1913-1919(1995).
Q. Li and E. S. Yeung, "Simple Two-Color Base-Calling Schemes for DNA Sequencing Based on Standard 4-Label Sanger Chemistry", Appl. Spectrosc. 49, 1528-1533 (1995).