Ex Cyto DNA Sequencing from Single Bacterial Colonies

单菌落的前细胞 DNA 测序

• 批准号: 7870519
• 负责人: DAVID Alan MEAD
• 金额: $ 37.5万
• 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 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7870519
• 关键词: Affinity; Base Sequence; Biological Sciences; Buffers; Bypass; Cells; Cloning; DNA; DNA Binding; DNA Sequence; DNA Sequence Analysis; DNA amplification; DNA-Directed DNA Polymerase; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Emerging Technologies; Emulsions; Enzyme Tests; Enzymes; Escherichia coli; Genes; Genome; Goals; Growth; Health; Human; Human Genome Project; In Vitro; Individual; Ions; Length; Liquid substance; Methods; Nucleic Acids; Nucleic acid sequencing; Nucleotides; Phase; Polymerase; Polymerase Gene; Procedures; Process; Proteins; Reaction; Reading; Recombinant DNA; Research; Research Personnel; Sampling; Speed; Structure; Taq Polymerase; Technology; Testing; Time; Work; base; cost; genome sequencing; improved; instrument; next generation; novel; public health relevance; pyrophosphatase; success; tool

DESCRIPTION (provided by applicant): The long term objective of this research is to develop improved DNA polymerases that will substantially advance Sanger and "next generation" sequencing technologies. The primary goal of the Phase I research was to develop a rapid and inexpensive method to perform Sanger sequencing directly from single bacterial colonies. This method bypasses the time and expense of template purification needed for conventional DNA sequencing. It is based on the development of a modified Taq fusion DNA polymerase (DNAP) with improved DNA affinity and processivity. Chromosomal integration and expression of such a polymerase into host cells used for cloning should enable a new concept, termed "Ex cyto sequencing". Analogous to colony PCR, Ex cyto sequencing will eliminate the overnight growth of bacterial cultures, expensive template purification, and the purchase of purified DNA polymerase. In the Phase I research we developed a new fusion polymerase with novel enzymatic attributes, which was shown to improve multiple aspects of nucleic acid sequencing and amplification. In addition to sequencing trace amounts of DNA from a single bacterial colony, the new Taq fusion polymerase enabled the following procedures: sequencing difficult templates unresolved by other enzymes, sequencing directly from liquid cultures using as little as 5 ul of outgrowth media, tight DNA binding in a variety of buffer conditions, and long PCR (10 kb). The specific aims of the Phase II proposal are to complete the development and optimization of Ex cyto sequencing and to modify other DNA polymerases used in next generation sequencing, such as Bst, Klenow, and T4 DNAPs. These improved enzymes should provide superior read lengths and the ability to sequence through difficult structures, thereby improving the accuracy of base calling and the subsequent assembly of genomes. These enzymes will improve numerous nucleic acid synthesis applications, such as emulsion PCR, used for the in vitro clonal amplification of templates, whole genome amplification, long PCR amplification, and a host of other methods that are constrained by existing DNAP capabilities. PUBLIC HEALTH RELEVANCE: 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 by developing a next generation DNA polymerase.

描述（由申请人提供）：本研究的长期目标是开发改进的 DNA 聚合酶，这将大大推进桑格和“下一代”测序技术。第一阶段研究的主要目标是开发一种快速且廉价的方法，直接对单个细菌菌落进行桑格测序。该方法省去了传统 DNA 测序所需的模板纯化时间和费用。它基于改进的 Taq 融合 DNA 聚合酶 (DNAP) 的开发，具有改进的 DNA 亲和力和持续合成能力。这种聚合酶的染色体整合和表达到用于克隆的宿主细胞中应该会产生一个新概念，称为“细胞外测序”。与菌落 PCR 类似，Ex 细胞测序将消除细菌培养物的过夜生长、昂贵的模板纯化以及购买纯化的 DNA 聚合酶。在第一阶段研究中，我们开发了一种具有新颖酶属性的新型融合聚合酶，它被证明可以改善核酸测序和扩增的多个方面。除了对单个细菌菌落中的痕量 DNA 进行测序之外，新的 Taq 融合聚合酶还可以实现以下程序：对其他酶无法解析的困难模板进行测序、使用少至 5 ul 的生长培养基直接从液体培养物中进行测序、在各种缓冲液条件下紧密 DNA 结合以及长 PCR (10 kb)。 II期提案的具体目标是完成Ex细胞测序的开发和优化，并修改下一代测序中使用的其他DNA聚合酶，例如Bst、Klenow和T4 DNAP。这些改进的酶应提供卓越的读取长度和对困难结构进行测序的能力，从而提高碱基调用和随后的基因组组装的准确性。这些酶将改善众多核酸合成应用，例如用于模板体外克隆扩增的乳液 PCR、全基因组扩增、长 PCR 扩增以及许多受现有 DNAP 能力限制的其他方法。 公共健康相关性：人类基因组计划的成功引发了对 DNA 序列信息的需求的爆炸性增长。来自各种物种的新基因的发现将对了解人类健康和疾病产生重大影响。尽管 DNA 序列分析的速度有所提高并降低了成本，但该过程仍然是时间、劳动力和成本密集型的​​。该提案旨在通过开发下一代 DNA 聚合酶来显着提高 DNA 测序速度，同时降低 DNA 测序成本。