Our goal is to develop Miniaturized Integrated DNA Analysis Systems that provide high-speed, high-throughput DNA sequencing and genotyping at reduced cost. These capabilities will facilitate sequencing of the human and other genomes, the analysis of genetic variation, and genetic and infectious disease diagnosis. Capillary electrophoresis channels and microfluidic sample preparation components will be fabricated on glass substrates using photolithography and wet ch4emical etching. High-speed capillary gel electrophoresis sensations will be achieved by applying high-fields to very small 10-100 mum diameter channels and by exploiting microfluidic sample loading. High- throughput and sensitivity will be achieved by producing high density radial arrays of capillary electrophoresis channels and by using a rotary confocal fluorescence scanner. Low-cost and robust processes will be achieved by integrating the DNA sample preparation, sample transport, sample injection, and electrophoretic analysis onto the chips. These long-term goals will be approached by completing the following specific aims: (1) 96-Channel radial sequencing microplates and methods will be optimized for DNA sequencing with the goal of >500 Phred 20 bases per lane in <30 min separations. (2) PCR-CAE microplates will e developed and tested that can PCR amplify and analyze 96 genomic DNA samples on a single microplate. (3) Microfluidic solid-phase methods will be developed for performing on-chip thermal cycling (TC) to produce DNA extension reactions in 100-300 nL reactors followed by on-chip extension fragment purification and loading onto microfabricated CE channels for sequencing. (4) Chip designs and a thermal cycling platform for PC-CAE microplates will then be developed and tested that can prepare, purify and analyze 96 sequencing samples on a single microplate. (5) Finally, a BC-chip will be developed that can into E. coli along with marker like GFP. After brief growth to achieve selection, the E. coli cell library will be introduced into the chip and run through a cell sorter to identify and route individual transformed cells into single cell PCR reactors. After PCR amplification of the insert, solid phase capture chemistries will be used to immobilize the template and to purify the subsequent extension products for sequencing analysis. A single run of this chip to analyze 96 clones will produce approximately IX coverage.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
2R01HG001399-07
Application #
6437105
Study Section
Genome Study Section (GNM)
Program Officer
Schloss, Jeffery
Project Start
1995-09-30
Project End
2004-11-30
Budget Start
2001-12-01
Budget End
2002-11-30
Support Year
7
Fiscal Year
2002
Total Cost
$369,546
Indirect Cost
Name
University of California Berkeley
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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