Ex Cyto DNA Sequencing from Single Bacterial Colonies

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、廉价的方法，直接从单个细菌菌落进行桑格测序。该方法避免了常规DNA测序所需的模板纯化的时间和费用。它是基于改进的DNA亲和力和持续合成能力的修饰的Taq融合DNA聚合酶（DNAP）的开发。将这种聚合酶整合到用于克隆的宿主细胞中的染色体整合和表达应该能够实现称为“细胞外测序”的新概念。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。在I期研究中，我们开发了一种具有新型酶属性的新型融合聚合酶，该聚合酶已被证明可改善核酸测序和扩增的多个方面。除了对来自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，使用少至5 μ l的生长培养基直接从液体培养物进行测序，在各种缓冲液条件下紧密结合DNA，以及长PCR（10 kb）。II期提案的具体目标是完成Ex cyto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，如Bst、Klenow和T4 DNAP。这些改进的酶应该提供上级读长和通过困难结构测序的能力，从而提高碱基识别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，例如用于模板的体外克隆扩增的乳液PCR、全基因组扩增、长PCR扩增以及受现有DNAP能力限制的许多其他方法。 公共卫生相关性：人类基因组计划的成功引发了对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。该提案旨在通过开发下一代DNA聚合酶来显著提高DNA测序的速度，同时降低DNA测序的成本。

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.