Oligonucleotide-Directed Random Mutagenesis Using the Phosphorothioate Method

Oligonucleotide-directed mutagenesis has become the primary method for testing theories of protein structure/function and control of gene expression by specifically altering DNA sequences. Random oligonucleotides can be used to extend this approach to genes where less structural information is available. These can be prepared by spiking each of the phosphoramidites used in oligonucleotide synthesis with a mixture of the other three bases. The spiked oligonucleotides are then used for in vitro mutagenesis to produce a library of random mutants (Fig. 1 ).