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

Abstract

A procedure for selection of specific cosmid clones by homologous recombination between cosmid clones from a library and sequences cloned into a plasmid was developed. Cosmid libraries constructed in a rec- host strain are packaged in vivo into .lambda. phage particles. Appropriate aliquots are then introduded 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.