One-step generation of recombineering constructs by asymmetric-end ligation and negative selection

Recombineering [1] or Red/ET recombination [2], a recently developed technique based on homologous recombination in Escherichia coli, is useful for the efficient production of a gene-targeting vector (TV) by modification of bacterial artificial chromosome (BAC) clones. This method requires the preparation of a linear DNA recombineering fragment, containing a selectable gene flanked by homologous regions (HRs) for recombination. The PCR method using primers containing HR sequences is the simplest way to produce the recombineering fragment with HRs placed at both ends. This procedure is not advantageous, however, when complex components such as reporter genes are included in the amplified region due to the high degree of mutations possibly generated by PCR. To avoid this problem, Liu and coworkers [3] and Valenzuela and coworkers [4] employed a ligation-based cloning method to introduce HRs into a vector used for recombineering. They used HRs provided as PCR products and oligonucleotides, respectively. In this strategy, however, several restriction enzymes (REs), including six-base cutters, are employed for cloning of HRs. Suitable enzyme sites or HRs, therefore, should be chosen according to the construct introduced. These properties sometimes necessitate screening to identify and select the correct clones. In addition, background clones derived from uncut vector could appear after recombination [3], although most of them can be eliminated by DpnI digestion when PCR-amplified recombineering fragments are used [2].