The long term objective of this proposal is to automate and improve a key area and current bottleneck of genomic research: the automated handling of submicroliter fluid samples. Specifically, the research proposed in this application is the development of a novel, high-throughput, high-yield, fully automated system capable of performing restriction enzyme digests, PCPs, and sample preparation for DNA sequencing. The immediate goals of this system are to process 5,000 1 mu l samples per day, at a cost per finished sequence base pair of less than 20 cents. This will be accomplished through a 10 fold reduction in reagents and sample volumes, and a 10 fold increase in throughput derived from automation, parallelization, and integration of: (I) prior technology developed by the proposing team, and (2) new technology that will be developed during the course of this project. The system will interface in an automated fashion with microtiter plates, Eppendorf tubes, or capillaries at the front end, and with electrophoretic capillaries, electrophoretic gel loading combs, or microtiter plates at the back end. Other input/output media are also possible. The proposed system uses glass capillary tubes and several novel methods to reduce sample size, automate the handling of small fluid samples, reduce thermal cycling and incubation times, and minimize the amount of disposables used to perform DNA sequencing. All processing steps are performed within these capillaries. This use of capillaries also helps facilitate sample containment and minimize evaporation losses, which are particularly important for the high surface area to volume ratios of small samples. An important feature of the proposed system is that it is consistent with current methods of sample handling. Furthermore, it will reduce costs, increase yield, and increase throughput in deriving DNA sequence. The system described in this proposal will greatly aid the Human Genome Project in meeting its sequencing goals. It will also have a synergistic effect outside of the Human Genome Project, benefiting efforts in clinical testing, medical diagnostics, pharmaceutical development, environmental testing, DNA fingerprinting, and agricultural research.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG001497-04
Application #
2889666
Study Section
Special Emphasis Panel (SRC (03))
Program Officer
Schloss, Jeffery
Project Start
1996-05-01
Project End
2001-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Washington
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Shi, Xu; Lin, Liang-I; Chen, Szu-yu et al. (2011) Real-time PCR of single bacterial cells on an array of adhering droplets. Lab Chip 11:2276-81
Chao, Shih-Hui; Meldrum, Deirdre R (2009) Spontaneous, oscillatory liquid transport in surface tension-confined microfluidics. Lab Chip 9:867-9
Lin, Liang-I; Chao, Shih-Hui; Meldrum, Deirdre R (2009) Practical, microfabrication-free device for single-cell isolation. PLoS One 4:e6710
Koschwanez, John H; Carlson, Robert H; Meldrum, Deirdre R (2009) Thin PDMS films using long spin times or tert-butyl alcohol as a solvent. PLoS One 4:e4572
Koschwanez, John H; Carlson, Robert H; Meldrum, Deirdre R (2007) Easily fabricated magnetic traps for single-cell applications. Rev Sci Instrum 78:044301
Meldrum, D R; Evensen, H T; Pence, W H et al. (2000) ACAPELLA-1K, a capillary-based submicroliter automated fluid handling system for genome analysis. Genome Res 10:95-104
Evensen, H T; Meldrum, D R; Saenphimmachak, C et al. (1999) High-density small-volume gel loading directly from capillary tubes. Biotechniques 27:974-8
Arutunian, E B; Meldrum, D R; Friedman, N A et al. (1998) Flexible software architecture for user-interface and machine control in laboratory automation. Biotechniques 25:698-702, 704-5
Friedman, N A; Meldrum, D R (1998) Capillary tube resistive thermal cycling. Anal Chem 70:2997-3002