DOE Human Genome Program Contractor-Grantee
21. High-Performance DNA Sequencing and Analysis
Richard A. Mathies
Department of Chemistry, University of California, Berkeley, CA 94720
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.
(LBNL) and DE-AC05-96OR22464 (ORNL).
|The online presentation of this publication is a special feature of the Human Genome Project Information Web site.|