CLONING SYSTEM BASED ON THE E COLI F FACTOR

基于大肠杆菌 F 因子的克隆系统

• 批准号: 2209228
• 负责人: John M Sedivy
• 金额: $ 24.22万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-01 至 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2209228
• 关键词: Escherichia coli; electroporation; genetic library; genetic manipulation; genetic mapping; genetic techniques; human genetic material tag; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; oligonucleotides; pulsed field gel electrophoresis; technology /technique development; tissue /cell culture; transfection /expression vector

The sheer size of the human genome is the major impediment to the construction of a comprehensive physical map. The need to rapidly clone, map, and manipulate large DNA molecules has become acute in conjunction with the Human Genome Project. Considerable successes have already been achieved with Yeast Artificial Chromosomes (YACs), and they are currently the major technology being employed in the mapping of the human genome. The main objective of this proposal is to investigate whether a new cloning system based on the E. coli F factor would be a valuable addition and/or alternative to the existing YAC cloning system. Preliminary results suggest that the F factor cloning system may offer advantages over the YAC cloning system in three important areas. First, it should be possible to prepare F factor libraries that contain very few chimeric clones. Second, it should be possible to stably propagate in F factor vectors certain DNA sequences that cause instability and rearrangements in YAC vectors. Third, the isolation and analysis of F factor clones should be substantially easier and faster. Adoption of the F factor cloning system would thus be expected to achieve significant savings of time and money associated with the mapping and analysis of complex genomes. Experiments are proposed to explicitly address these issues. To achieve these goals it will be necessary to develop specific protocols for the construction of large F factor libraries. In this respect it is significant that transformation frequencies of constructs in the 100-200 kbp range are some 100-fold higher in E. coli than in yeast. It should thus be possible to construct large F factor libraries in a single experiment. In addition, it should be possible to significantly improve the construction of libraries from very rare sources of DNA. Depending on the success of these investigations, the long-term objective of this proposal is to construct a representative human library in the F factor cloning system with 500 kbp average inserts and a 4- to 5-fold coverage of the human genome.

人类基因组的庞大规模是人类进化的主要障碍。 绘制一幅全面的自然地图。 需立即 克隆、绘制和操纵大的DNA分子已成为一个紧迫的问题， 与人类基因组计划合作。了可观的成绩 已经用酵母人工染色体 （YAC），它们是目前采用的主要技术 在人类基因组的绘制中。 的主要目标 建议是调查是否有一个新的克隆系统， 急诊大肠杆菌F因子将是有价值的添加和/或 替代现有的YAC克隆系统。 初步 结果表明，F因子克隆系统可能提供 与YAC克隆系统相比，该系统在三个重要领域具有优势。 首先，应该可以制备F因子文库， 含有很少的嵌合克隆。 第二，应该能够 在F因子载体中稳定繁殖某些DNA序列， 导致YAC载体的不稳定和重排。 三是 F因子克隆的分离和分析应基本上 更容易更快。 采用F因子克隆系统将 从而有望实现时间和金钱的显著节省 与复杂基因组的绘制和分析相关。 实验提出明确解决这些问题。 到 为了实现这些目标，必须制定具体的 用于构建大型F因子文库的方案。 在 在这方面， 100-200 kbp范围内的构建体在E. 大肠杆菌比酵母菌。 因此，应该可以建造大型的 F因子库在单个实验中。 此外还应 可以大大提高图书馆的建设 非常罕见的DNA来源。 取决于这些项目的成功 这项建议的长远目标是 在F因子克隆中构建具有代表性的人文库 具有500 kbp平均插入片段和4- 5倍覆盖率的 人类基因组