Rapid cloning by homologous recombination in vivo.

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 EcdRUXhol sites of pBluescript SK ( ) (Stratagene) by PCR using the plasmid primers PI and P2. The resulting DNA fragment contains an additional 33 bp plasmid-derived DNA sequence stretch at the EcoN, and a 42 bp stretch at the X7iol-end of its sequence (see Figure la). Cotransformation of this insert with EcoKUXhol linearized pBluescript SK ( ) into E.coli DH5a (competency > 10 CFU/l/ig) yielded transformants harbouring recombinant pBluescript with the insert in correct orientation. DNA sequencing indicated a homologous recombinatorial reaction mechanism since the overlapping homologous DNA regions were not duplicated in the recombinant plasmids. Maximum yields were obtained with about equimolar concentrations of plasmid and insert in submicrogram quantities. However, in contrast to E.coli made competent by CaCl2 treatment, we were unable to detect any true recombinants when electroporation was used for transformation. To define the influence of the length of the homologous DNA sequence overlap on transformant yield, the overlapping regions between plasmid and insert were successively shortened on one side of the cloning site by digesting pBluescript with a panel of several different restriction endonucleases, leading to a series of linearized vectors with 0—33 bp overlap on one side of the cloning site. Transformant yields steadily increased with increasing length of the sequence overlap, though about 10 bp overlap represented a practical minimum length to obtain sufficiently high numbers of transformants in DH5a (Table 1). This corresponds to the length of a typical PCR primer containing a 5' add-on restriction endonuclease site plus about 5 b overhang sequence which must, however, be chosen identical to the vector sequence extending 5' to the cloning site to allow recombinatorial cloning.