Abstract
Bacterial artificial chromosomes (BACs) offer many advantages for functional studies of large eukaryotic genes. To utilize the potential applications of BACs optimally, new approaches that allow rapid and precise engineering of these large molecules are required. Here, we describe a simple and flexible two-step approach based on ET recombination, which permits point mutations to be introduced into BACs without leaving any other residual change in the recombinant product. Introduction of other modifications, such as small insertions or deletions, is equally feasible. The use of ET recombination to achieve site-directed mutagenesis opens access to a powerful use of BACs and is extensible to DNA molecules of any size in Escherichia coli, including the E. coli chromosome.