Ex Cyto DNA Sequencing from Single Bacterial Colonies

单菌落的前细胞 DNA 测序

• 批准号: 7156326
• 负责人: DAVID Alan MEAD
• 金额: $ 13.72万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-21 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7156326
• 关键词: DNA directed DNA polymerase; culture; enzymes; genes; genome; nucleic acid sequence; pyrophosphatase; reading

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.

描述（由申请人提供）：项目摘要/摘要：DNA聚合酶是驱动DNA序列分析的传统方法以及许多正在开发的较新测序方法的引擎。迄今为止，Taq DNA聚合酶的变体已用于几乎所有的基因组测序。然而，Taq DNA聚合酶具有许多固有的生物化学缺陷，这些缺陷限制了序列分析的速率和可靠性并增加了其成本。在这个项目中，将通过将DNA结合结构域融合到酶的N-末端来创建高度进行性的Taq聚合酶。这种经过修饰的聚合酶，采用优化的方案，将提高从痕量DNA测序的能力，从而能够从单个细菌菌落进行直接序列分析（“细胞外”测序）。类似于菌落PCR，细胞外测序将消除细菌培养物的过夜生长、昂贵的模板纯化和购买纯化的DNA聚合酶的需要，从而显著降低DNA序列分析的成本。具有高持续合成能力的Taq DNA聚合酶还将改善测序过程的技术方面，例如通过“困难”模板进行阅读并提供增加的读取长度，从而改善所调用的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来自于更快和更便宜地获得许多生物体的更高质量的基因组序列数据。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理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，但该过程仍然是时间、劳动力和成本密集型的。该提案旨在大幅提高速度，同时降低DNA测序的成本。