Selective isolation of cosmid clones by homologous recombination in Escherichia coli.

作者：Annemarie Poustka, Hans‐Richard Rackwitz, Anna‐Maria Frischauf, Barbara Höhn, Hans R. Lehrach · 发表于：Proceedings of the National Academy of Sciences · 年份：1984 · DOI：10.1073/pnas.81.13.4129 · 被引用次数：150 · 研究领域：Protein purification and stability、Viral Infectious Diseases and Gene Expression in Insects、Microbial Metabolic Engineering and Bioproduction

A procedure for selection of specific cosmid clones by homologous recombination between cosmid clones from a library and sequences cloned into a plasmid has been developed. Cosmid libraries constructed in a rec- host strain are packaged in vivo into lambda particles. Appropriate aliquots are then introduced into a rec+ host containing the sequence used for selection cloned into a plasmid vector without sequence homology to the cosmid vector. After a short time for recombination, the cosmids are packaged in vivo. Cosmids that have taken up the plasmid by homologous recombination are isolated by plating under conditions selecting for the antibiotic resistance markers carried by both vectors. The recombined cosmids can lose the inserted sequence by another homologous recombination event and, after packaging in vivo, these revertants can be identified on appropriate indicator plates.