|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
38. Ultra-High Throughput DNA Genotyping and Sequencing on Radial Capillary Array Electrophoresis Microplates
Peter C. Simpson, James R. Scherer,
Yining Shi, and Richard A. Mathies
The microfabrication of DNA sample preparation, electrophoretic separation and detection devices is making possible a new generation of high-speed, high-throughput DNA analysis systems. Our research is focused on the ultra-high throughput analysis of PCR products for genotyping applications as well as DNA sequencing on microfabricated capillary array electrophoresis (CAE) microplates. These CAE microplates perform high speed analysis of multiple samples in parallel increasing the throughput by several orders of magnitude over conventional slab or capillary array systems1. Several generations of CAE microplates have been developed to optimize layout and performance. Our current design uses a circular scanning confocal fluorescence detection system together with radially symmetric channel layouts. The design consists of a common anode reservoir in the center of a circular 4" or 6" diameter wafer and an array of 96 channels extending radially outward towards injector units at the perimeter of the wafer. This radial design gives quality high speed separations by eliminating resolution reducing turns and allows the analysis of 96 samples in parallel on a single microplate. The confocal rotary scanner can measure fluorescence from DNA fragments in all channels at a rate of up to 15 samples/sec. The major advantage of a rotary scanner over linear scanners is that the motion of the scanner is continuous, making it easier to control the velocity at high sample rates. High sample rates are necessary to ensure good resolution of electrophoretic bands. The scanner is capable of collecting 2880 data points/revolution at 23.15 ms intervals. The scanner utilizes four independent ADCs to simultaneously acquire data from four color electrophoresis runs.
The operation and capabilities of the radial CAE microplates with the rotary scanning system were first demonstrated by performing high speed electrophoretic separations of 96 pBR322 MspI DNA samples in 40 seconds. Genotyping of methylenetetrahydrofolate reductase (MTHFR), a candidate gene for vascular disease and neutral tube defects, was also performed on 4'' diameter radial CAE microplates to demonstrate the rapid analysis of biologically relevant samples (in collaboration with Prof. M. Smith and C. Skibola in the School of Public Health, UCB). Two-color multiplexed fluorescence detection of the MTHFR genotypes was accomplished by prelabeling standard pBR322 MspI DNA ladder with a red emitting bisintercalation dye (butyl TOTIN) and prelabeling of the MTHFR DNA with a green emitting bisintercalation dye (TOTO)2. Using this two-color multiplexing method, 96 MTHFR DNA samples were genotyped in less than 2 minutes with 4 bp resolution. Radial CAE microplates fabricated on 6" wafers are currently being developed for ultra-high throughput DNA sequencing applications.
1P.C. Simpson, D. Roach, A.T. Woolley, T. Thorsen, R. Johnston, G. F. Sensabaugh, and R.A. Mathies, Proc. Nat. Acad. Sci., USA, 95, 2256-2261 (1998)
2S.M. Clark, and R.A. Mathies, Anal. Chem., 69, 13354-1363 (1997)
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