DOE Human Genome Program Contractor-Grantee
22. Radial Capillary Array Electrophoresis Microplate and Scanner for High-Performance DNA Sequencing and Analysis
Yining Shi1, Brian M. Paegel1, James R. Scherer1, Peter C. Simpson1, David Wexler1, Christine Skibola2, Martyn T. Smith2, and Richard A. Mathies1
1Department of Chemistry and 2School of Public Health, University of California, Berkeley, CA 94720
The design, fabrication and operation of a radial capillary array electrophoresis (CAE) microplate and scanner for high-throughput DNA analysis are presented1. The microplate consists of a central common anode reservoir coupled to 96 microfabricated separation channels connected to sample injectors on the perimeter of the wafer. Detection is accomplished by a laser-excited rotary confocal scanner with four-color detection. Loading of 96 samples in parallel is achieved using a pressurized capillary array system. High-quality separations of 96 pBR322 restriction digest samples are achieved in <120 s using a 4"-diameter microplate. The practical utility and multicolor detection capability of this system is demonstrated by analyzing 96 methylenetetrahydrofolate reductase (MTHFR) alleles in parallel using a non-covalent 2-color staining method. This work establishes the feasibility of high-performance genotyping with capillary array electrophoresis microplates.
To explore the capabilities of our radial CAE microplate and scanner for high-speed and high-throughput DNA sequencing, we have designed and fabricated a CAE microplate containing 96 folded 12-cm-long separation channels on a 6"-diameter wafer. While high-quality four-color sequencing separations can be achieved on 7-cm-long straight microchannels2, integration of an array of such straight channels into a 6"-diameter wafer in the radial format is difficult due to the dimensional restrictions of the wafer. To address this issue, Paegel et al. introduced an optimized channel design which allows the fabrication of 96 folded separation channels on a 6"-diameter wafer. Each of the 96 folded 12-cm-long channels has two complementary tapered turns that minimize turn-induced band broadening during electrophoresis separations3,4. Four-color sequencing separations and automatic base-calling analyses of 96 single stranded M13mp18 DNA sequencing samples with our 6"-diameter radial CAE microplate and scanner will be presented.
(LBNL) and DE-AC05-96OR22464 (ORNL).
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