Analysis methods for DNA genotyping and sequencing are linked into multi-step processing systems 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 3 will provide component-level design and testing of improved nanoliter-volume processing components for DNA sequencing and genotyping. To fulfill the goal of high-quality, continuous DNA sequencing significant improvements in fluorescence detection and gel electrophoresis will be required. New or improved fabrication technologies will also be examined for biochemical compatibility, ease of fabrication, cost, or reproducibility. All of the components developed in the Project retain the target of eventual integration into the complex system of Project 1. Project 3 has three Specific Aims: 1) Development of polymer-silicon micromachined components. Both thin film polymer channels and molding/embossing patterns on polymer substrates will be used for integrated device construction. Thin film methods will be extended to thicker channel walls, thereby reducing evaporative liquid loss. Bonding of thermoplastic and silicon surfaces to generate hermetic sealed channels will also be developed using two types of polymer handling methods. 2) Development of enhanced sensitivity fluorescence detectors. The initial diode detector we have constructed will be improved to (a) maximize its response to the DNA fluorescent signal, (b) minimize its dark current noise, and (c) electrically isolate the detector from the electrophoresis stage. We anticipate at 10- to 100-fold improved in the signal to noise ratio for the detector. 3) Development of non-collinear fluorescence excitation. In the current device arrangement, the excitation illumination is collinear with the detector. We will develop non-collinear illumination schemes relying on either micromachined reflecting surfaces on polymer wave-guides.
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