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.

Gene therapy studies require techniques that allow alteradapted an inducible homologous recombination system ation of human genomic DNA sequences. Bacterial artifor use in E. coli DH10B cells, the host strain for BACs ficial chromosome cloning systems (BACs/PACs) bridge and PACs. Using this system, we have introduced PCR the gap between vectors with small inserts and yeast artifragments carrying a selectable marker and a reporter ficial chromosomes (YACs). We report the use of a second gene downstream of the IVS I-110 splicing mutation into a generation BAC vector, pEBAC, containing eukaryotic selspecific site within the ␤-globin gene sequence. The use ectable markers and combining some of the best features of this inducible system minimises the risk of unwanted of the BAC, PAC and HAEC systems, into which a 185 kb rearrangements by recombination between repetitive sequence containing the human ␤-globin gene cluster was elements and allows the introduction of relevant modifiretrofitted. To permit the introduction of mutations correcations or reporters at any specific sequence within sponding to those causing human pathology, we have BACs/PACs in E. coli DH10B cells.Keywords: homologous recombination; ␤-globin; PAC; BAC; recE; recT PACs 1 and BACs 2 are used increasingly for long-range physical mapping, 3,4 positional cloning of disease genes, 5 whole genome sequencing projects 6 and functional studies. 7,8 However, the lack of convenient techniques for performing genetic manipulations on BACs/PACs in the host E. coli DH10B strain imposes serious limitations for functional analysis. Homologous recombination in an F plasmid-based vector in E. coli was first used in 1989 to join overlapping Drosophila cosmid fragments to form a 125 kb fragment. 9 Homologous recombination has also been used recently to introduce targeted modifications in BACs, 10-12 but the available techniques either require the construction of shuttle plasmids or are not directly applicable in DH10B cells.The bacteriophage Red-Gam and the RecE systems promote homologous recombination between linear DNA fragments and circular plasmid molecules or the host chromosome. 13,14 The reaction catalysed by the RecE pathway, termed ET cloning, 14 could be transferred to other recBC strains by cloning part of the recET operon into the l-arabinose-inducible expression plasmid pBAD24 to give plasmid pBAD24-trecET (YZ and AFS, 14 with the gam gene of bacteriophage being used as a natural inhibitor of recBCD nuclease, to allow ET cloning in recBC+ strains of E. coli. This plasmid enabled targeted modification of a 76 kb Drosophila P1 clone in its recBC+ host strain (NS3145) with PCR fragments having about 50 bp of homology in each arm.A second generation BAC/PAC cloning vector, pEBAC140, (Figure 1a, PAI and J-M Vos), combining features of the first generation PAC 1 and BAC 2 cloning systems and the HAEC system 15 was used in the initial efforts to optimize homologous recombination. EBAC/148␤ (Figure 1b) contains the entire ␤-globin locus (about 73 kb) in a 185 ...