SIEVING MEDIA FOR DNA SEQUENCING WITH LONG READ LENGTHS

第二阶段赠款的目标是开发一个DNA测序矩阵， 毛细管电泳实现非常长的读取长度（1000 在半自动、高通量的环境中。我们将使用 在第一阶段开发的分析工具， 线性聚合物作为DNA筛选基质的行为 测序我们将集中精力查明和消除 导致峰加宽的因素。我们将调查 筛分聚合物的分子量分布对 Ogston和声誉模式的选择性，以及 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 提高选择性和分离的背景电解质 效率这一努力将导致筛选基质配方， 支持半自动、高通量的长测序读长 定序系统 拟议的商业应用： 目前的商业毛细管测序仪产生高精度测序 读段长度平均约450个碱基，很少超过600个碱基。 这项工作有可能使平均读取长度加倍。这将 将EST测序的成本降低一半， 对于基因组测序，因为长的读取长度极大地促进了 序列组装和闭合。由此产生的矩阵可以很快 提供用于MegaBACE 1000 DNA测序系统的商业用途。

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.