Efficient Saturation Mutagenesis of a Pentapeptide Coding Sequence Using Mixed Oligonucleotides

Abstract

Site-directed mutagenesis using oligonucleotides that are degenerate at a specific codon was employed to construct a set of mutations in a pentapeptide sequence targeting cytosolic proteins to lysosomes during serum withdrawal. Low-temperature annealing of the mixed oligonucleotides to single-stranded phage DNA and a genetic selection for the DNA strand carrying the mutations were utilized. The use of mixed oligonucleotides by this technique provides an economical means of generating a large set of substitution mutations. A single codon can be changed to codons for most other amino acids in one step. This approach eliminates the need for restriction enzyme cleavage sites flanking the target for mutagenesis and, therefore, is useful for targeting mutations to any DNA fragment cloned into an appropriate single-stranded bacteriophage.