Ex Cyto DNA Sequencing from Single Bacterial Colonies

描述（由申请人提供）：这项研究的长期目标是开发改进的DNA聚合酶，这将大大推进Sanger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和廉价的方法，直接从单个细菌菌落进行桑格测序。该方法绕过了传统DNA测序所需的模板纯化的时间和费用。它是基于改进的Taq融合DNA聚合酶（DNAP）的发展，具有改进的DNA亲和力和加工能力。将这种聚合酶整合并表达到用于克隆的宿主细胞中，应该会产生一个新的概念，称为“前细胞测序”。与集落PCR类似，Ex细胞测序将消除细菌培养物的过夜生长，昂贵的模板纯化和纯化DNA聚合酶的购买。在I期研究中，我们开发了一种新的融合聚合酶，具有新的酶特性，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的痕量DNA进行测序外，新的Taq融合聚合酶还实现了以下程序：对其他酶无法解析的困难模板进行测序，直接从液体培养物中使用少至5 ul的培养基进行测序，在各种缓冲条件下紧密结合DNA，以及长PCR （10 kb）。II期申请的具体目标是完成Ex细胞测序的开发和优化，并修饰下一代测序中使用的其他DNA聚合酶，如Bst、Klenow和T4 DNAPs。这些改良的酶应该提供更好的读取长度和通过复杂结构进行测序的能力，从而提高碱基调用的准确性和随后的基因组组装。这些酶将改善许多核酸合成应用，如乳液PCR，用于模板的体外克隆扩增，全基因组扩增，长PCR扩增，以及许多其他受现有DNAP能力限制的方法。

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.