Accelerated BAC Library Construction And Analysis

描述(由申请人提供)：大插入DNA文库是对人类基因组和其他对我们的福祉至关重要的基因组进行分子分析的基本资源。识别复杂疾病的易感基因至关重要地依赖于完整和准确的序列组装。比较基因组学揭示了代谢途径、癌症、水平基因转移、进化、蛋白质家族和众多物种的遗传谱系等方面的新信息。这也增加了对额外基因组袋库的需求。这种类型的遗传资源的构建既耗时又昂贵，而且具有挑战性。人类基因组中的许多缺口很难或不可能用当前的载体和宿主系统来弥合。这项工作的目标是开发新的BAG克隆工具、菌株和方法，以绕过传统的瓶颈，大幅提高分子克隆的保真度和高分子量DMA的转化效率。在第一阶段，开发了一种随机剪切高分子量DNA的简单方法和一种拷贝数可诱导的无转录载体，该载体消除了几种形式的克隆偏见。在新的无转录BAG载体中，多个难以克隆的基因组实例被证明是稳定的。拟议的第二阶段工作将扩大BAG载体的开发，包括功能和宿主范围的进一步改进，这将允许在许多革兰氏阴性微生物中表达抗菌素小分子。一种新的大肠杆菌转化系统的开发将对分子医学的许多方面产生影响。该系统可显著提高DNA摄取量并增加平均插入片段大小。这项提议的长期目标是获得人类基因组中的克隆缺口，并将构建大型BAG文库的速度从几个月压缩到几周。这些图书馆的质量将超过目前的偏差、随机性、保真度和污染标准。

DESCRIPTION (provided by applicant): Large insert DMA libraries are essential resources for the molecular analysis of the human genome and other genomes important to our well-being. The identification of susceptibility genes for complex disorders is critically dependent on a complete and accurate sequence assembly. Comparative genomics reveals new information about metabolic pathways, cancer, horizontal gene transfer, evolution, protein families, and the genetic repertoire of numerous species. It has also increased the demand for additional genomic BAG libraries. The construction of this type of genetic resource is time consuming, costly and challenging. Numerous gaps in the human genome are difficult or impossible to close with current vector, host systems. The goal of the proposed work is the development of new BAG cloning tools, strains and methods that circumvent traditional bottlenecks and substantially improve the fidelity of molecular cloning and transformation efficiency of high molecular weight DMA. Under Phase I, a simple method for randomly shearing high molecular weight DMA and a copy number inducible transcription-free vector that eliminates several forms of cloning bias was developed. Multiple examples of difficult to clone genomes are shown to be stable in the new transcription free BAG vector. The proposed Phase II work will extend the development of the BAG vector to include additional improvements in functionality and host range, which will allow for the expression of antimicrobial small molecules in numerous gram negative microorganisms. The development of a new E. coli transformation system to significantly improve large DNA uptake and increase average insert size will impact numerous aspects of molecular medicine. A long-term objective of this proposal is to access clone gaps in the human genome and compress the rate of constructing large BAG libraries from months to a few weeks. The quality of these libraries will surpass the current standards for bias, randomness, fidelity, and contamination.