YAC CLONING ANALYSIS OF REGIONS OF THE HUMAN GENOME

人类基因组区域的 YAC 克隆分析

• 批准号: 3878841
• 负责人: MAYNARD V OLSON
• 金额: --
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3878841
• 关键词: DNA; Saccharomyces; endonuclease; gel electrophoresis; genetic library; genetic manipulation; genetic mapping; genetic markers; genetic recombination; molecular cloning; nucleic acid hybridization; nucleic acid probes; nucleic acid repetitive sequence; plasmids; transposon /insertion element

The basic protocols for applying yeast artificial-chromosome (YAC) cloning to the analysis of specific regions of the human genome will be optimized. Systematic efforts will be made to increase the average insert size of YAC libraries and to maximize the diversity of the inserts. These efforts will include the use of inserts produced by a variety of restriction enzymes as well as endogenous nucleases and hydrodynamic shear. Velocity sedimentation and pulsed-field gel electrophoresis will be used to pre-select the sizes of inserts and ligation products during cloning experiments. Screening protocols based on hybridization to ordered arrays of individually picked clones, as well as to randomly plated primary transformants, will be explored. Genetic screening techniques based on in vivo recombination between YACs and probe sequences will a)so be tested. Finally, techniques will be implemented for the efficient characterization of YAC clones by low-resolution mapping, as well as by high-resolution mapping and systematic subcloning.

应用酵母人工染色体(YAC)的基本方案 克隆用于分析人类基因组的特定区域 将会得到优化。将有系统地努力增加 YAC文库的平均插入大小和最大化多样性 插入物的数量。这些努力将包括使用镶件 由各种限制性内切酶以及内源 核酸酶和流体动力剪切。速度沉积和 将使用脉冲场凝胶电泳法预选 克隆实验中插入和连接产物的大小。 基于对有序阵列的杂交的筛选方案 单独挑选的克隆，以及随机分配的主克隆 变形体，将会被探索。遗传筛选技术 基于YAC与探针序列的体内重组 A)是否会接受测试。最后，将针对以下方面实施技术 YAC克隆的低分辨率高效表征 测绘以及通过高分辨率测绘和系统化 亚克隆。