The mapping and sequencing of the approximately 3 X 10exp9 base pairs of DNA that constitute the human genome is the colossal task undertaken in the Human Genome Initiative (HGI). While advances in molecular biological techniques make this possible, a methodological """"""""bottleneck"""""""" in the sequencing protocol has been created by the necessity to separate sequencing fragments via conventional slab gel electrophoresis. Consequently, there has been intense interest in augmenting the conventional slab gel-based methodology with new technologies that can enhance sequencing throughput. Two relatively new electrophoretic formats, capillary electrophoresis and electrophoresis in microfabricated """"""""chips,"""""""" have shown promise for providing this technology. Despite some inherent technical challenges, the ability to execute ultrafast separations in multiple channels etched into the surface of a planar glass or plastic substrate could enhance sequencing speed significantly. However, the true power of the chip platform stems, not only from the capability for rapid electrophoretic separations, but from the ability to seamlessly integrate sample preparation into the chip. This would allow for direct and automated interfacing of PCR and electrophoresis in a single device. The object of this proposal is to develop a novel thermocycling approach that, unlike other approaches, can easily be integrated into the chip platform. PCR will be carried out using an infrared-based thermocycling technology that exploits a tungsten lamp as a simple but effective heat delivery system, coupled with infrared detection/control of solution temperature. Combined, this will allow for PCR to be executed on the chip with nanoliter volumes without contacting the chip. Consequently, the cost- and time-efficiency of PCR will be improved dramatically over conventional methodology. This development will set the stage for interfacing rapid cycle sequencing reactions with the prerequisite sample clean-up process and the post-PCR electrophoretic separation, all in a single, disposable microanalysis device.
