In vivo site-directed mutagenesis using oligonucleotides

Functional characterization of the genes of higher eukaryotes has been aide d by their expression in model organisms and by analyzing site-specific cha nges in homologous genes in model systems such as the yeast Saccharomyces c erevisiae(1), Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together wi th subcloning(2,3). PCR-based procedures that do not involve cloning are in efficient or require multistep reactions that increase the risk of addition al mutations(4,5). An alternative approach, demonstrated in yeast, relies o n transformation with an oligonucleotide(6), but the method is restricted t o the generation of mutants with a selectable phenotype. Oligonucleoticles, when combined with gap repair, have also been used to modify plasmids in y east(7); however, this approach is limited by restriction-site availability . We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-spec ific DNA mutations in vivo. A two-step, cloning-free process, referred to a s delitto perfetto, generates products having only the desired mutation, su ch as a single or multiple base change, an insertion, a small or a large de letion, or even random mutations. The system provides for multiple rounds o f mutation in a window up to 200 base pairs. The process is RAD52 dependent , is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes(8) such as yeast artificial chromosomes, the delitto perfetto strategy also pr ovides an efficient way to create precise changes in mammalian or other DNA sequences.