A straightforward way to engineer DNA in E. coli using homologous recombination is described. The homologous recombination reaction uses RecE and RecT and is transferable between E. coli strains. Several target molecules were manipulated, including high copy plasmids, a large episome and the E. coli chromosome. Sequential steps of homologous or site-specific recombination were used to demonstrate a new logic for engineering DNA, unlimited by the disposition of restriction endonuclease cleavage sites or the size of the target DNA.

We present a method to modify bacterial artificial chromosomes (BACs) resident in their host strain. The method is based on homologous recombination by ET-cloning. We have successfully modified BACs at two distinct loci by recombination with a PCR product containing homology arms of 50 nt. The procedure we describe here is rapid, was found to work with high efficiency and should be applicable to any BAC modification desired.

The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination, for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.

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