Current state-of-the-art DNA sequencing technologies rely on electrophoresis in large arrays of fused silica capillaries, which are filled with entangled polymer matrixes that provide DNA separation. Major cost savings, and a large increase in throughput, would be realized if next-generation sequencers could be based on glass or plastic microfluidic devices. However, microfluidic devices currently under development, like today's capillary array instruments, require viscous polymer matrixes for DNA separation, which inherently limit read length and are difficult to load into chip rnicrochannels. The development of a new and practical paradigm for microchannel DNA sequencing in free solution would greatly increase the chances that microfluidic devices could be made practical for high-throughput DNA sequencing in Genome Centers. Here, we present results that clearly demonstrate the potential of End-Labeled Free Solution Electrophoresis (ELFSE) to sequence DNA in microchannels without a sieving matrix. The basic principle of ELFSE is to attach a monodisperse perturbing entity, or """"""""drag-tag"""""""", to each DNA molecule in a sequencing mixture, allowing separation based on DNA size by free-solution rnicrochannel electrophoresis. Novel drag-tags and microchannel wall coatings invented in our laboratory show highly promising properties for DNA sequencing by ELFSE. We have just finished building an advanced chip electrophoresis system. With further development, ELFSE on chips could be the next generation of easily automated, high-throughput, long-read length DNA sequencing technologies. Using a combination of genetic engineering and organic chemistry techniques we will synthesize and purify non-natural, monodisperse drag-tags based on repetitive, nonnatural polypeptides (""""""""protein polymers""""""""). Optimized protocols for conjugation of drag-tags to DNA primers, cycle sequencing, and sample cleanup will be developed. Protocols for DNA sequencing by ELFSE will be developed and optimized using both a 4-color capillary array instrument and microfluidic chips. A variety of wall coatings based on previously reported chemistries would be explored for minimization of interactions between drag-tags and capillary walls to maintain high-efficiency DNA peaks. The theory of ELFSE is under development, and further efforts will be geared to theoretical predictions and molecular dynamics simulations of ELFSE. Theoretical predictions will guide every aspect of ELFSE experimentation. ? ?
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