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SIEVING MEDIA FOR DNA SEQUENCING WITH LONG READ LENGTHS

用于长读长 DNA 测序的筛选介质

基本信息

批准号：
6125637
负责人：
Vladislav Dolnik
金额：
$ 22.52万
依托单位：
MOLECULAR DYNAMICS, INC.
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-08-22 至 2001-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6125637
关键词：
biomaterial development /preparation biomaterial evaluation

项目摘要

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.

第二阶段资助的目标是开发 DNA 测序矩阵毛细管电泳可实现非常长的读长（1000 碱基）在半自动、高通量环境中。我们将使用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影响峰展宽的因素。我们将调查筛分聚合物分子量分布的影响奥格斯顿和声誉模式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。进一步优化结构背景电解质可提高选择性和分离度效率。这项工作将导致筛分矩阵配方支持半自动、高通量的长测序读长测序系统。拟议的商业应用：当前的商业毛细管测序仪可产生高精度测序读长平均约为 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增加一倍。这会 EST测序成本降低一半，效果更显着对于基因组测序，因为长读长极大地促进了顺序组装和闭合。由此产生的矩阵可以很快供 MegaBACE 1000 DNA 测序系统商业使用。