The research plan will develop novel engineering systems for genetic (DNA) analysis. The work will use silicon microfabrication technology to assemble complex, integrated, DNA biochemical analysis devices. The silicon fabrication resources created for the microprocessor industry have the potential to make DNA analysis systems that are inexpensive, robust, and miniaturized. Examples of a few of the scientific fields that may benefit from these devices include: (i) multigenic trait analysis, (ii) infectious disease diagnostics, (iii) agricultural genetics, and (iv) the Human Diversity Program. Specific c Aims The biochemical and electrophoretic manipulations for DNA genotyping and sequencing are well characterized, but have not been assembled into a simple automated processing system. On the basis of on established methods, the proposed effort will develop the electrophoretic, detection, and control components of a fully integrated technology for DNA sequencing. The use of silicon photolithographic fabrication techniques will allow components to be compatible, readily assembled as a single device, and inexpensive to mass produce. The planned work will complement and extend existing efforts in the lab to construct silicon devices for DNA genotyping. Microfabricated sequencing devices will be developed with the following experimental plan: 1. Construction of a miniature high resolution electrophoresis system. Existing electrophoresis technology is able to sizefractionatesequencingreactions800basepairsandgreaterusing gels 50-100 m thick and 50 cm long. The lab will duplicate these results using silicon micromachined 5-100 m diameter channels. Beginning with short (I cm length) channels, the channel length and detector resolution will gradually be increased. For longer channels (> 5 cm), folded columns will be used. The necessary gel channels, DNA detectors, and controllers will be designed, fabricated, and tested. 2. Fluidic and electronic integration of t he sequencing system. Template preparation, biochemical reactions, and electrophoresis systems will be integrated on a single device using silicon microfabricated components. Integration of electronic components (detector, heaters, liquid detectors, and temperature sensors) should allow the construction of a self-contained sequencing system. Interconnections with external fluidics and circuitry will be developed. 3. Elimination of sequencing bottlenecks using intelligent systems. The proposed system of integrated fluid-handling, electrophoresis, detector, and circuitry components allows feedback and decision-making directly within the device. Information-based processing will be used to reduce both the systemic and random errors for each sequencing sample. Sequencing strategies will be tested for improved reproducibility, error-detection, length of readable data, and compatibility with existing sequencing; protocols. Research description. The initial proposed device will contain several components: liquid injection ports, self-pumping channels based upon surface-force gradient phenomena, thermally-isolated reaction (PCR) chambers, decision split points, and gel electrophoresis channels. Next to, and underneath, these components are the system's electronic detectors and controlling circuitry. Within the system a purified DNA sample is injected, moved to a specific location, and the enzymatic sequencing reactions are performed. A portion of the sequencing product is isolated and sent to a preliminary electrophoresis gel for screening. Using the preliminary information, sequence data acquisition can be optimized by dividing the remaining product between electrophoresis gels with different resolution characteristics. Increasingly complex devices can be assembled from the individual components and basic functional modules described above. The initial components do not perform any preliminary template handling, and consequently well characterized template must be used as starting material. The proposed work will start from the basic DNA sequencing reaction and gel electrophoresis system and work "back" through the sample processing stream. Since the production of high-quality template is one of the current difficulties in "on-site" DNA sequencing, development of integral template synthesis will be essential. Eventually, it is anticipated that the entire processing stream will be incorporated onto the device, with all steps included within the silicon-fabricated environment. In the limit, this will eliminate process bottlenecks, since each sample will have its own dedicated series of instruments. Using silicon micromachining techniques, it may be possible to design and build reaction and separation units that are impractical to build by any other techniques. For example, silicon fabrication can construct an electrophoresis chamber with hundreds of DNA detectors along its length for the same cost as constructing a chamber with only one sensor. The same technology that makes transistors in integrated circuits inexpensive will allow these complicated, integrated systems to be produced for a fraction of larger-scale equivalents. This work will integrate proven biochemical technology with the existing technology for micromachining and microelectronics to produce a sequencing system that is fast, accurate, and cheap. A broad range of disciplinary knowledge, including nucleic acid biochemistry, silicon fabrication, circuit and sensor design bioseparations, and surface chemistry will be essential to completing the project. Therefore, research will continue to take advantage of established faculty collaborations in Chemical Engineering (Dr. Mark Burns) and Electrical Engineering (Dr. Carlos Mastrangelo), but will occur primarily within my laboratory group.
