Simplified DNA manupulations based on in vitro mutagenesis

Abstract

A modification of the procedure of oligonucleotide-directed mutagenesis of DNA fragments cloned in M13 bacteriophage vectors [1] is described. The production efficiency of closed, circular, double-stranded DNA molecules is greatly improved when the oligonucleotide extensions are performed at 30°C, enzymes and substrates replenished after 1 hr, and the total reaction time limited to 4 hr. The reaction mixture is then used, with no further treatment, to transform competent Escherichia coli cells. Using this simplified procedure, we have obtained specific single-base substitutions in the IFN-α1 gene at high frequency (5–30% of all plaques). More significantly, the procedure also allows the insertion of relatively long nucleotide sequences at predetermined positions. As an illustration of this technique, we have modified the IFN-α14 gene for subsequent cloning into a yeast expression vector. Using a single 37-base oligonucleotide, we have inserted 12 bases immediately 5′ to the cysteine 1 codon of IFN-α14. These 12 new bases created an ATG translation initiation codon, the appropriate yeast translation initiation nucleotide environment, and a 5′ BamHI site for direct insertion of the tailored gene into the yeast vector. The production of such insertion mutants is generally applicable in both structure/function studies and in simplyfing complex DNA manipulations, particularly where suitable restriction endonuclease sites are lacking.