INTEGRATED DNA GENOTYPING AND SEQUENCING MICROSYSTEMS

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Analysis methods for DNA genotyping and sequencing are linked into multiple-step processing system that begin with DNA source material and extract genetic information. In conventional methods, the processing system operates on batches of microliter-volume reaction samples, together with matched-liquid handling devices and electrophoretic analysis equipment. Project 1 will provide a systems-level approach to evaluate the integrated nano-liter volume components and fabrication technologies developed in the Program Project. The success of microfabricated components will be measured by their ability to work as an autonomous multi-step processing system. Project 1 has four Specific Aims: 1) Develop the microfluidics necessary for integrated nanoliter-scale DNA analysis. Microfluidic components will be used to construct an integrated system that can perform and monitor multiple parallel reactions. An interface with microliter-volume source samples will also be addressed. 2) Demonstrate sequencing and genotyping reactions in an integrated system. Microfluidic devices will be optimized for use with Sanger sequencing or genomic amplification reactions and denaturing gel electrophoresis. The system-level device will perform and monitor polymerase reactions, post reaction treatment, and electrophoretic band migration, with minimal operator intervention. 3) Demonstrate high-quality genomic DNA sequencing in an integrated systems. The resolution of the integrated nanoliter-volume system will be improved to match the level of existing microliter-scale integrated nanoliter-volume system will be improved to match the level of existing microliter-scale technologies. This will include improved fluorescence detectors (with Project 3) and higher resolution gels (with Project 2). Successful completion of this aim will result in a device that can obtain 500-1000 bp of sequence from a sample of template DNA. 4) Demonstrate complex sequencing strategies in an integrated device. Complex sequencing strategies that obtain maximal information from a single DNA sample, such as bi-directional sequencing or simultaneous restriction digestion, will be added to the integrated sequencing device. Issues of quality control, accuracy, and data reproducibility will also be examined.