Rapid cloning by homologous recombination in vivo.

Abstract

Cloning techniques are fundamental to molecular biology. Classically, recombinant plasmid and/or phage vectors are prepared in vitro, the transformation step only serving for amplification purposes. A different approach would exploit the natural intracellular enzymatic machinery to produce recombinant DNA molecules. Such techniques are well-known to mutagenize chromosomal DNA by e.g. transposon mutagenesis (1) but uncommon yet in the production of recombinant plasmid molecules. Here, we describe a novel cloning method which seems to be mediated by homologous recombination. To add DNA sequence stretches homologous to the plasmid vector to both ends of a linear DNA fragment, we amplified a 1.1 kbp DNA insert cloned into the EcoRI/XhoI sites of pBluescript SK (-) (Stratagene) by PCR using the plasmid primers P1 and P2. The resulting DNA fragment contains an additional 33 bp plasmid-derived DNA sequence stretch at the EcoRI, and a 42 bp stretch at the XhoI-end of its sequence (see Figure la). Cotransformation of this insert with EcoRI/XhoI linearized pBluescript SK (-) into E. coli DH5ca (competency > 106 CFU/lAg) yielded transformants harbouring recombinant pBluescript with the insert in correct orientation. DNA sequencing indicated a homologous recombinatorial reaction mechanism since