YAC CLONING ANALYSIS OF REGIONS OF THE HUMAN GENOME

点击翻译按钮获取中文摘要

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.