In vivo site-directed mutagenesis using oligonucleotides

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Abstract
Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae (Resnick and Cox in Mutat. Res. 451:1, 2000). Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning (Scherer and Davis in Proc. Natl. Acad. Sci. USA 76:4951, 1979, Barton et al. in Nucleic Acids Res. 18:7349, 1990). PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations (Langle-Rouault and Jacobs in Nucleic Acids Res. 23:3079, 1995, Erdeniz et al. in Genome Res. 7:1174, 1997). An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide (Moerschell et al. in Proc. Natl. Acad. Sci. USA 85:524, 1988), but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast (Duno et al. in Nucleic Acids Res. 27:e1, 1999); 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-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of 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 (Larionov et al. in Proc. Natl. Acad. Sci. USA 94:7384, 1997) such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.

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