IMPLEMENTATION OF A 384 CAPILLARY SEQUENCING SYSTEM

提出了一种用于开发和实施毛细管阵列DNA测序系统的加速方案。该系统将包括一个384毛细管仪器，与分离基质、试剂和客户支持结构相结合，以在不到或等于2小时内实现一致的DNA测序读数超过1200个碱基。该系统将有助于将现有的最先进的长阅读测序技术整合到生产设施中。该系统产生的数据吞吐量将比现有的基于毛细管的系统快约10倍，并将试剂使用量减少约20倍。DNA测序仪将由4-PMT共聚焦扫描仪设计组成，4个96毛细管阵列在固态加热元件中恒温，工作温度高达85摄氏度。分离矩阵将基于东北大学开发的高、低分子量线性聚丙烯酰胺的混合物，已被证明可为1200个碱基提供高质量的序列数据。样品制备将在384个毛细管盒中进行，该盒可制备500nl规模的样品。这些组件利用了东北大学、分子动力学和amersham Pharmacia的现有技术，但配合起来将使系统的开发具有显著增强的性能。该项目的目标是：1)设计和组装仪器，2)制造优化试剂，3)改进现有软件，4)提供样品制备设备和协议，5)提供现场支持。这一新系统的加速开发将极大地促进人类基因组计划的DNA序列获取阶段和其他序列密集型工作。

An accelerated program for the development and implementation of a capillary-array DNA sequencing system is proposed. The system will comprise a 384-capillary instrument coupled with the separation matrix, reagents, and customer support structure necessary to achieve consistent DNA sequencing reads greater than 1200 bases in less than or equal to 2 hours. The system will facilitate integration of extant state-of-the-art technologies for long-read sequencing into production facilities. The system will produce data with a throughput approximately 10x faster than existing capillary-based systems, and reduce reagent usage by approximately 20x. The DNA sequencing instrument will consist of a 4-PMT confocal scanner design with 4 96-capillary arrays thermostated in a solid-state heating element, operating as high as 85 degree C. The separation matrix will be based on mixtures of high- and low-molecular weight linear polyacrylamide developed at Northeastern University that have already been shown to give high-quality sequence data to 1200 bases. Sample preparation will be done in a 384-capillary cassette that prepares samples on a 500nl scale. These components leverage existing technologies at Northeastern University, Molecular Dynamics, and Amersham Pharmacia, but in concert will enable development of a system with dramatically enhanced performance. The goals of the project are to: 1) Design and assemble the instrumentation, 2) manufacture optimized reagents, 3) improve on available software, 4) deliver sample preparation devices and protocols, 5) deliver field support. Accelerated development of this new system will greatly facilitate the DNA sequence acquisition phases of the Human Genome Project and other sequence-intensive efforts.