YAC CLONING ANALYSIS OF REGIONS OF THE HUMAN GENOME

酵母人工染色体（YAC）应用的基本方案 克隆到分析人类基因组的特定区域 将得到优化。 将作出系统的努力， YAC文库的平均插入片段大小并最大化多样性 的插入。 这些努力将包括使用插入物 由多种限制性内切酶以及内源性 核酸酶和流体动力学剪切。 速度沉积和 脉冲场凝胶电泳将用于预选 在克隆实验期间插入物和连接产物的大小。 基于与有序阵列杂交的筛选方案 单独挑选的克隆，以及随机接种的初级 转化，将被探索。 遗传筛选技术 基于YAC和探针序列之间的体内重组 A.这样测试。 最后，将实施技术， 通过低分辨率技术对YAC克隆的有效表征 测绘，以及高分辨率测绘和系统 亚克隆。

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.