of Work: Efforts to pursue transformation-associated recombination cloning (TAR cloning) of large chromosomal fragments has been transferred to other groups formerly trained in this lab. The ability to clone and modify sequences has now taken on a major new direction and is providing a novel opportunity to conduct in vivo site directed mutagenesis. We have developed an in vivo site-directed mutagenesis system based on direct transformation with unpurified oligonucleotides and recombination processes that provides for the rapid creation of site-specific mutations in DNA within yeast. The two-step, cloning-free process, referred to as delitto perfetto, generates products in vivo having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion. The delitto perfetto system has been demonstrated in the chromosomes of S. cerevisiae and a cassette to aid in its application has been developed. The system also enables repeated rounds of specific or random changes in a window of up to 200 bp. A variety of individual and complementary oligonucleotides have been examined in order to establish the requirements for the site-directed mutagenesis and to develop several applications. This process, which is dependent on the RAD52 pathway, is not constrained by the distribution of naturally occurring restriction sites and requires minimal DNA sequencing. Given the simplicity of the two steps involved, this strategy will greatly facilitate functional genetic studies. The delitto perfetto approach has been used for modification of p53 responsive elements, to insert unique restriction sites in specific locations in the yeast genome, and to create changes in MRE11. The use of this procedure to achieve in vivo site-directed mutagenesis provides a powerful and straightforward way to modify DNA in yeast.Since yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes as YACs, the delitto perfetto strategy also provides an efficient way to create precise changes within mammalian DNA sequences.
