Ex Cyto DNA Sequencing from Single Bacterial Colonies

项目概述/摘要：DNA聚合酶是驱动传统DNA序列分析方法以及许多正在开发的新测序方法的引擎。迄今为止，Taq DNA聚合酶的变体已被用于几乎所有的基因组测序。然而，Taq DNA聚合酶具有许多内在的生化缺陷，限制了序列分析的速度和可靠性，并增加了其成本。在这个项目中，将通过将DNA结合域融合到酶的n端来创建一个高度程序化的Taq聚合酶。这种经过优化的聚合酶将提高对痕量DNA进行测序的能力，从而实现对单个细菌菌落的直接测序（“前细胞”测序）。与集落PCR类似，前细胞测序将消除对细菌培养物过夜生长的需要，昂贵的模板纯化和纯化DNA聚合酶的购买，大大降低了DNA序列分析的成本。具有高处理能力的Taq DNA聚合酶还将改善测序过程的技术方面，例如读取“困难”的模板并提供更长的读取长度，从而提高被叫碱基及其后续组装的准确性。医学科学和基础研究的重要进展将源于更快、更便宜地获得众多生物体的高质量基因组序列数据。项目简介：人类基因组计划的成功催生了对DNA序列信息需求的爆炸性增长。从各种物种中发现新基因将对了解人类健康和疾病产生重大影响。尽管DNA序列分析的速度和成本有所提高，但这一过程仍然是时间、劳动力和成本密集型的。这一提议旨在大幅提高速度，同时降低DNA测序的成本。

DESCRIPTION (provided by applicant): Project Summary/Abstract: DNA polymerases are the engines that drive conventional methods of DNA sequence analysis, as well as many of the newer sequencing methods being developed. Variants of Taq DNA polymerase have been used for nearly all genomic sequencing to date. However, Taq DNA polymerase has a number of inherent biochemical deficiencies that limit the rate and reliability of sequence analysis and increase its cost. In this project, a highly processive Taq polymerase will be created by fusing a DNA binding domain to the N-terminus of the enzyme. This modified polymerase, with optimized protocols, will improve the ability to sequence from trace amounts of DNA, enabling direct sequence analysis from single bacterial colonies ("ex cyto" sequencing). Analogous to colony PCR, ex cyto sequencing will eliminate the need for overnight growth of bacterial cultures, expensive template purification, and purchase of purified DNA polymerase, dramatically decreasing the cost of DNA sequence analysis. A Taq DNA polymerase with high processivity will also improve technical aspects of the sequencing process, such as reading through "difficult" templates and providing increased read lengths, thereby improving the accuracy of called bases and their subsequent assembly. Important advances in medical science and basic research will result from faster and cheaper access to higher quality genomic sequence data of numerous organisms. Project Narrative: The success of the human genome project has spawned explosive growth in the demand for DNA sequence information. The discovery of new genes from a variety of species will have a large impact on understanding human health and disease. Despite improvements in speed and reduction in costs of DNA sequence analysis, the process is still time, labor, and cost-intensive. This proposal seeks to dramatically improve the speed while decreasing the costs of DNA sequencing.