Use of λ Exonuclease for Efficient Oligonucleotide-Mediated Site-Directed Deletion and Point Mutation of Double-Stranded DNA

ABSTRACT A novel approach to oligonucleotide-mediated, site-directed in vitro mutagenesis is described that allows for the efficient generation of sequence modifications on double-stranded substrates without the need for subcloning into special vectors. Site-directed deletions as well as point mutations were introduced into the genes encoding human tissue plasminogen activator (tPA) and the Bacillus amyloliquefaciens α-amylase gene using λ exonuclease to enzymatically degrade DNA 5′ to 3′ in order to generate a single-stranded template in the immediate vicinity of the oligonucleotide annealing site. The mutagenizing oligonucleotide, used both to redefine the 5′ end of the molecule and to introduce base changes, was annealed to the single-stranded target sequence producing substrates for both the exonucleolytic and polymerizing activities of DNA polymerase Klenow fragment. Resolution of the resultant heteroduplex by Escherichia coil resulted in the generation of the desired deletion point mutation in the t...