DNA sequencing by ELFSE on microfluidic devices

描述(申请人提供)：当前最先进的DNA测序技术依赖于大阵列熔融二氧化硅毛细管中的电泳，这些阵列填充了提供DNA分离的缠绕聚合物基质。如果下一代测序仪能够基于玻璃或塑料微流控设备，将实现重大的成本节约和产量的大幅增加。然而，目前正在开发的微流控设备，如今天的毛细管阵列仪器，需要用于DNA分离的粘性聚合物基质，这固有地限制了读取长度，并且难以加载到芯片微通道中。开发一种新的、实用的无溶液微通道DNA测序模式，将极大地增加微流控设备在基因组中心实现高通量DNA测序的可能性。在这里，我们提出的结果清楚地展示了末端标记的自由溶液电泳(ELFSE)的潜力，以测序微通道中的DNA，而不需要筛选矩阵。ELFSE的基本原理是在测序混合物中的每个DNA分子上附加一个单分散的扰动实体，或“拖拽标签”，允许通过自由溶液微通道电泳法根据DNA大小进行分离。我们实验室发明的新型阻力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。我们刚刚建成了一套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、长阅读长度的DNA测序技术。结合基因工程和有机化学技术，我们将合成和提纯基于重复的非天然多肽(“蛋白质聚合物”)的非天然、单分散的拖拽标签。将开发用于将拖拽标签连接到DNA引物、循环测序和样品清理的优化方案。ELFSE的DNA测序方案将使用四色毛细管阵列仪器和微流控芯片进行开发和优化。将探索基于先前报道的化学成分的各种壁涂层，以最小化阻力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，将进一步努力进行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.