DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

21. High-Performance DNA Sequencing and Analysis

Richard A. Mathies

Department of Chemistry, University of California, Berkeley, CA 94720

rich@zinc.cchem.berkeley.edu

Capillary array electrophoresis (CAE) systems¹ coupled with high sensitivity detection provided by energy-transfer labeling reagents² is now the accepted standard for high-throughput DNA sequencing facilities. Further advances are focused on the development of capillary array systems capable of running more than 96 capillaries as illustrated by Scherer et al.³ and the development of microfabricated CAE systems that provide higher throughput as well as the important ability to integrate microfluidic chemistries. Toward this end we have recently shown that microfabricated CE channels only 7-cm long can produce >500 bp 4-color sequencing reads in under 30 minutes⁴.

Microfabrication also permits the production of very high density electrophoretic analysis devices that provide unprecedented analysis throughput. Radial microplates coupled with a novel rotary confocal scanning system have been developed that can rapidly analyze 96 genotyping samples in parallel in seconds on 5-cm long channels⁵. To integrate arrays of 10-15 cm long channels on a radial microplate, it is necessary to devise ways to fold channels without reducing resolution. We have determined that "pinched turns", where the channel width is reduced before the turn and widened after the turn, enable folded designs that minimize turn-induced broadening while maintaining facile matrix introduction⁶.

Sequencing results on radial microplates with 96 15-cm long channels fabricated on a 6"-diameter wafer will be presented. We have also developed microfabrication methods for the integration of nanoliter volume PCR sample preparation directly with microfabricated CE analysis systems⁷.

Genotyping results will be presented along with plans for integrated thermal cycling devices. The implementation of microfabricated separation systems with integrated chemistries will be the next paradigm shift in DNA sequencing and genomic analysis.

• Kheterpal, I. and Mathies, R. A. Capillary Array Electrophoresis DNA Sequencing, Analytical Chemistry, 71, 31A-37A (1999).
• Xie, J., Hung, S.-C., Glazer, A. N. and Mathies, R. A. Energy Transfer Fluorescent Labels for DNA Sequencing and Analysis, in Topics in Fluorescence Spectroscopy, Volume 7: ANA Technology, in press (1999).
• Scherer, J. R., Kheterpal, I., Radhakrishnan, A., Ja, W. W. and Mathies, R. A. Ultra-High Throughput Rotary Capillary Array Electrophoresis Scanner for Fluorescent DNA Sequencing and Analysis, Electrophoresis 20, 1508-1517 (1999).
• Liu, S., Shi, Y., Ja, W. W. and Mathies, R. A. Optimization of High-Speed DNA Sequencing on Microfabricated Capillary Electrophoresis Channels, Anal. Chem. 71, 566-573 (1999).
• Shi, Y., Simpson, P., Scherer, J. R., Wexler, D., Skibola, C., Smith, M. T. and Mathies, R. A. Radial Capillary Array Electrophoresis Microplate and Scanner for High-Performance Nucleic Acid Analysis, Anal. Chem. 71, 5354-5361 (1999).
• Paegel, B. M., Hutt, L. D., Simpson, P. C. and Mathies, R. A. Turn Geometries for Minimizing Band Broadening in Microfabricated Capillary Electrophoresis Channels, Analytical Chemistry, submitted.
• Lagally, E., Simpson, P. C. and Mathies, R. A. Monolithic Integrated Microfluidic DNA Amplification and Capillary Electrophoresis System, Sensors and Actuators B, submitted (1999).

(LBNL) and DE-AC05-96OR22464 (ORNL).