Accelerated BAC Library Construction And Analysis

描述(由申请人提供)：大插入DNA文库对于人类基因组和其他对我们的福祉至关重要的基因组的分子分析是必不可少的。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。这也大大增加了对额外基因组BAC文库的需求。这些图书馆的建设既耗时又费钱，而且具有挑战性。这项提议的长期目标是促进克隆目前人类基因组中的空白，并将构建大型BAC文库所需的时间从几个月减少到几周。这些图书馆的质量将超过目前的偏差、随机性、保真度和低污染标准。具体目标包括开发新的BAC克隆工具、菌株和方法，以绕过传统的瓶颈，显著提高高分子量基因组DNA的分子克隆和转化效率。一种新的大肠杆菌转化系统可将大DNA摄取率提高100倍，并使平均插入片段大小增加到300kb以上。其他技术创新包括一种随机剪切高分子量DNA的简单方法，一种零背景克隆系统，以及一种消除几种形式克隆偏见的无转录克隆系统。 建议的商业应用： 商业产品和服务包括：具有极高DNA摄取效率的新的大肠杆菌转化系统、用于无偏向克隆顽固基因和片组的零背景、无转录载体、BAC文库构建kt、BAC DNA亲和纯化试剂盒、减少质粒骨架重测序的载体弹出，以及经济的BAC文库构建服务。

DESCRIPTION (provided by applicant): Large insert DNA libraries are essential for the molecular analysis of the human genome and other genomes important to our well-being. 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 greatly increased the demand for additional genomic BAC libraries. Construction of these libraries is time consuming, costly and challenging. The long-term objectives of this proposal are to facilitate cloning the current gaps in the human genome and reduce the time required for constructing large BAC libraries to a few weeks from several months. The quality of these libraries will surpass current standards for bias, randomness, fidelity, and low contamination. Specific aims include the development of new BAC cloning tools, strains, and methods that circumvent traditional bottlenecks and substantially improve the molecular cloning and transformation efficiency of high molecular weight genomic DNA. A new E. coli transformation system could improve large DNA uptake 100-fold and increase average insert size to above 300 kb. Other technical innovations include a simple method for randomly shearing high molecular weight DNA, a zero background cloning system, and a transcription-free cloning system that eliminates several forms of cloning bias. PROPOSED COMMERCIAL APPLICATIONS: Commercial products and services include: a new E. coli transformation system with exceptionally high DNA uptake efficiency, a zero background, transcription-free vector for unbiased cloning of recalcitrant genes and gneomes, a BAC library construction kt, a BAC DNA affinity purification kit, a vector pop-out to reduce re-sequencing of the plasmid backbone, and an economical BAC library construction service.