Existing methods for site-directed plasmid mutagenesis are restrained by the 
small spectrum of modifications that can be introduced by mutagenic primers and 
the amplicon size limitations of in vitro DNA synthesis. As demonstrated here, 
the combined use of Red/ET recombination and unique restriction site elimination 
enables extensive manipulation regardless of plasmid size and DNA sequence 
elements. First, a selectable marker is PCR-amplified with synthetic primers 
attaching 50-bp homology target flanks for Red/ET recombination and an arbitrary 
restriction site absent in the substrate plasmid. The resulting cassette is 
co-electroporated with substrate plasmids in Red/ET-proficient Escherichia coli 
cells. Following isolation of recombinant plasmids, linear nonselectable DNA 
replaces the cassette and introduces the desired mutation(s) in a second Red/ET 
recombination step. Upon selective digestion of parental plasmids and 
retransformation, a 38% mutation efficiency was achieved using a synthetic 
97-nucleotide oligonucleotide to cure a 17-bp deletion within lacZalpha of pUC19 
(2,686 bp). A PCR fragment was used with similar efficiency to co-replace mouse 
Cdkn1b codons 9 and 76 in gene-targeting vector pGTC (13,083 bp).