THE CROSSOVER LINKER. MECHANISMS AND APPLICATIONS IN GENE MODIFICATION

Abstract

We have developed a novel method for mutating DNA sequences, based on site-specific, in vivo, recombination, known as the crossover linker method. A typical crossover linker contains; (i) a single-stranded overhang for an initial cohesive-end ligation with one terminus of a linearized plasmid, (ii) a mid-section carrying modified sequence information, and (iii) a "homology-searching" sequence at the other end, that is similar to a specific region in the opposite terminus of the plasmid. Following transformation of an E. coli host with a plasmid/linker complex, intramolecular recombination between the homologous regions of the resultant intermediate completes the circularization of the plasmid, with concomitant integration of the linker. Crossover linking is performed on double-stranded DNA and can be used to create deletions andinsertions, as well as to perform site-specific mutagenesis. Both single- and double-stranded linkers with "homology searching" region as short as 5 nucleotides can be used for gene modification. Deletions of over 1000 bp have been achieved using "homology searching" regions of approx. 20 nucleotides in length. In this article, the effectiveness, limitations and mechanism of this process are discussed with emphasis on the application of the crossoverlinker to the manipulation of protein-encoding sequences.