Chinault A C, Sternberg N L
Baylor College of Medicine, Houston, Texas, USA.
Curr Protoc Hum Genet. 2001 May;Chapter 5:Unit 5.5. doi: 10.1002/0471142905.hg0505s00.
This unit describes procedures for screening large-insert libraries by multistep polymerase chain reaction (PCR) analysis of DNA samples from clone pools. In the basic protocol, PCR amplification and agarose gel electrophoresis are used to identify successively smaller pools of DNA or colonies that contain clones with the appropriate-sized amplification product. In the last screening step, individual clones are identified. The first and second support protocols describe the preparation of DNA and yeast-cell pools for screening, and the first alternate protocol describes the preparation of crude lysates suitable for PCR from individual clones or small-scale pools.
本单元介绍了通过对来自克隆池的DNA样本进行多步聚合酶链反应（PCR）分析来筛选大插入片段文库的方法。在基本方案中，使用PCR扩增和琼脂糖凝胶电泳来依次鉴定包含具有合适大小扩增产物的克隆的更小的DNA池或菌落。在最后的筛选步骤中，鉴定单个克隆。第一个和第二个支持方案描述了用于筛选的DNA和酵母细胞池的制备，第一个替代方案描述了适合从单个克隆或小规模池中进行PCR的粗裂解物的制备。
