Rapid engineering of bacterial artificial chromosomes using oligonucleotides

A rapid method obviating the use of selectable markers to genetically manip ulate large DNA inserts cloned into bacterial artificial chromosomes is des cribed. Mutations such as single-base changes, deletions, and insertions ca n be recombined into a BAC by using synthetic single-stranded oligonucleoti des as targeting vectors, The oligonucleotides include the mutated sequence flanked by short homology arms of 35-70 bases on either side that recombin e with the BAG. In the absence of any selectable marker, modified BACs are identified by specific PCR amplification of the mutated BAC from cultures o f pooled bacterial cells. Each pool represents about 10 electroporated cell s from the original recombination mixture. Subsequently, individual clones containing the desired alteration are identified from the positive pools. U sing this BAC modification method, we have observed a frequency of one reco mbinant clone per 90-260 electroporated cells. The combination of high targ eting frequency and the sensitive yet selective PCR-based screening method makes BAC manipulation using oligonucleotides both rapid and simple. genesi s 29:14-21, 2001. Published 2001 Wiley-Liss, Inc.(dagger).