SIEVING MEDIA FOR DNA SEQUENCING WITH LONG READ LENGTHS

第二阶段拨款的目标是开发一个DNA测序矩阵，用于 毛细管电泳法可实现非常长的读取长度(1000 基地)在半自动、高吞吐量的环境中。我们将使用 在第一阶段开发的分析工具，用于进行高级调查 线性聚合物作为DNA筛分基质的行为 测序。我们将集中精力识别和消除 导致峰值展宽的因素。我们将调查 筛分聚合物的分子质量分布对其性能的影响 Ogston和Reputation模式下的选择性以及效果 聚合物的化学组成对选择性和 分离效率。此外，我们还将优化 提高选择性和分离性的背景电解液 效率。这一努力将导致筛选矩阵公式 支持半自动、高通量的长测序读数长度 测序系统。 建议的商业应用： 目前商业化的毛细管测序仪产生高精度的测序 阅读长度平均约为450个碱基，很少超过600个碱基。 这项工作有可能使平均阅读长度翻一番。这将会 将EST测序的成本降低一半，这将是更重要的 对于基因组测序，由于长的读数长度极大地促进了 序列组装和闭合。由此产生的矩阵可能会很快 在MegaBACE 1000 DNA测序系统上提供，用于商业用途。

The goal of the Phase II grant is to develop a DNA sequencing matrix for capillary electrophoresis that achieves very long read lengths (1000 bases) in a semi-automatic, high-throughput environment. We will use the analysis tools developed in Phase I to conduct an advanced investigation of the behavior of linear polymers as sieving matrices for DNA sequencing. We will concentrate on identification and elimination of the factors that contribute to peak broadening. We will investigate the effect of the molecular mass distribution of the sieving polymers on selectivity in both Ogston and reputation modes, as well as the effect of chemical composition of the polymers on both selectivity and separation efficiency. Further, we will optimize the composition of the background electrolyte to improve both selectivity and separation efficiency. This effort will lead to a sieving matrix formulation that supports long sequencing readlengths in a semi-automatic, high-throughput sequencing system. PROPOSED COMMERCIAL APPLICATIONS: Current commercial capillary sequencers produce high-accuracy sequencing read lengths averaging about 450 bases and rarely exceeding 600 bases. This work has the potential to double the average read length. This would reduce the cost of EST sequencing by half, and would be more significant for genome sequencing, since long read lengths greatly facilitate sequence assembly and closure. The resulting matrix could be quickly supplied for commercial use on the MegaBACE 1000 DNA Sequencing System.