Site-Specific Recombination Directed by Single-Stranded Crossover Linkers: Specific Deletion of the Amino-Terminal Region of the β-Galactosidase Gene in pUC Plasmids

Abstract

The "duplex crossover linker" technique was simplified and used to delete the β-galactosidase (β-Gal)-coding sequence upstream from the multiple restriction sites in pUC plasmids. A single-stranded crossover linker, with a homology-searching sequence as short as 5 bases, was initially ligated to a linearized plasmid. Inside Escherichia coli, the plasmid was circularized by intramolecular, homologous recombination between the (5′-or 3′-) protruding homology-searching sequence and a targeted region in the opposite terminus. As a consequence, sequences beyond the point of integration were deleted. Specific deletion of sequences up to 1472 bp was demonstrated. The single-stranded linkers apparently avoided generation of undesirable mutants associated with the usage of duplex linkers. A mechanism has been proposed for the intramolecular recombination directed by the crossover linkers. It principally involves either 3′- or 5′-exonucleolytic breakdown of the homologous terminus of the plasmid, circularization by spontaneous pairing of the exposed complementary strands, and subsequent degradation of any redundant sequence.