DNA sequencing by ELFSE on microfluidic devices

描述（由申请人提供）：目前最先进的DNA测序技术依赖于在熔融二氧化硅毛细管的大阵列中进行电泳，这些毛细管中充满了纠缠的聚合物基质，可以提供DNA分离。如果下一代测序仪可以基于玻璃或塑料微流体装置，将实现重大的成本节约和吞吐量的大幅增加。然而，目前正在开发的微流体装置，如今天的毛细管阵列仪器，需要粘性聚合物基质进行DNA分离，这固有地限制了读取长度，并且难以加载到芯片微通道中。自由溶液中微通道DNA测序新范例的发展将极大地增加微流控装置在基因组中心实现高通量DNA测序的机会。在这里，我们提出的结果清楚地证明了末端标记自由溶液电泳（ELFSE）在没有筛分基质的微通道中测序DNA的潜力。ELFSE的基本原理是将单分散的扰动实体或“拖拽标签”附着在测序混合物中的每个DNA分子上，允许通过自由溶液微通道电泳根据DNA大小进行分离。我们实验室发明的新型拖标签和微通道壁涂层在ELFSE DNA测序中显示出非常有前途的特性。我们刚刚建成了一个先进的芯片电泳系统。随着进一步的发展，芯片上的ELFSE可能成为下一代易于自动化，高通量，长读长度DNA测序技术。利用基因工程和有机化学技术的结合，我们将合成和纯化基于重复的、非天然的多肽（“蛋白质聚合物”）的非天然的、单分散的标签。将开发拖拽标签与DNA引物结合、循环测序和样品清理的优化方案。利用四色毛细管阵列仪器和微流控芯片开发和优化ELFSE DNA测序方案。将探索基于先前报道的化学物质的各种壁涂层，以最大限度地减少拖拽标签和毛细管壁之间的相互作用，以保持高效的DNA峰。ELFSE的理论正在发展中，进一步的工作将面向ELFSE的理论预测和分子动力学模拟。理论预测将指导ELFSE实验的各个方面。

DESCRIPTION (provided by applicant): 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.