The obtainment of a high-quality sequence of the very first human genome has provided us with incredibly important information. However, in order to better understand the origins of genetic diseases such as cancer, sequencing data must be obtained from multiple human genomes and tumor genomes, as well as from the genomes of other complex organisms. To enable this important work, the high costs associated with sequencing the DMA of complex organisms must be greatly reduced. The current workhorse technology used in Genome Sequencing Centers, capillary array electrophoresis, has reached a plateau in terms of the cost per sequenced base, and we request NIH/NHGRI funds to complete our work in the development of novel polymer networks and wall coatings to enable a lower-cost, more efficient sequencing technology to take its place: microfluidic chip electrophoresis. DNA separation is much faster and cheaper in microfluidic chips, because much narrower DNA sample zones are injected. (We demonstrate here the sequencing of 550 bases, with 98.5% base-calling accuracy, in just 5.5 minutes - a new speed record by a factor of three! Capillaries require at ~ 70 minutes to give the same result). Chip-based technology also promises to enable the total integration of the sequencing process: one will merely load sheared BAG DNA on the chip, and the Sanger reaction, sample cleanup, and separation of DNA to give called sequence will be done automatically. We will create and test polymer matrices and wall coatings that enable read lengths of 700 to 800 bases in < 10 minutes on glass chips with human genomic samples. Other matrices and coatings will be developed to deliver, at a minimum, 500-base reads on plastic chips (a lower-cost chip substrate) in < 15 minutes. Our approach is to carefully control polymer and copolymer synthesis conditions and accurately characterize the physical and chemical properties of the polymers, so that we can better understand and design the next generation of materials for improved performance. The polymer networks we create will be of sufficiently low viscosity to enable automated, low-pressure loading (< 150 psi) of matrix into chips and will be 'self-coating' to remove extra steps in the chip preparation process, increasing throughput and lowering costs. We will carefully study the molecular mechanisms responsible for the ultrafast DNA sequencing we have recently observed in our polymer matrices, a critical aspect of optimizing microchip-based sequencing systems. ? ? ?
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