Bacterial artificial chromosomes (BACs) offer many advantages for functiona
l 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 flexi
ble two-step approach based on ET recombination, which permits point mutati
ons to be introduced into BACs without leaving any other residual change in
the recombinant product. Introduction of other modifications, such as smal
l insertions or deletions, is equally feasible. The use of ET recombination
to achieve site-directed mutagenesis opens access to a powerful use of BAC
s and is extensible to DNA molecules of any size in Escherichia coli, inclu
ding the E. coli chromosome.