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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Resnick, M.A. & Cox, B.S. Yeast as an honorary mammal. Mutat. Res. 451, 1–11 (2000).
Scherer, S. & Davis, R.W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc. Natl. Acad. Sci. USA 76, 4951–4955 (1979).
Barton, M.C., Hoekstra, M.F. & Emerson, B.M. Site-directed, recombination-mediated mutagenesis of a complex gene locus. Nucleic Acids Res. 18, 7349–7355 (1990).
Langle-Rouault, F. & Jacobs, E. A method for performing precise alterations in the yeast genome using a recycable selectable marker. Nucleic Acids Res. 23, 3079–3081 (1995).
Erdeniz, N., Mortensen, U.H. & Rothstein, R. Cloning-free PCR-based allele replacement methods. Genome Res. 7, 1174–1183 (1997).
Moerschell, R.P., Tsunasawa, S. & Sherman, F. Transformation of yeast with synthetic oligonucleotides. Proc. Natl. Acad. Sci. USA 85, 524–528 (1988).
Duno, M., Bendixen, C., Krejci, L. & Thomsen, B. Targeted deletions created in yeast vectors by recombinational excision. Nucleic Acids Res. 27, e1 (1999).
Larionov, V., Kouprina, N., Solomon, G., Barrett, J.C. & Resnick, M.A. Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast. Proc. Natl. Acad. Sci. USA 94, 7384–7387 (1997).
Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808 (1994).
Lewis, L.K. & Resnick, M.A. Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae. Mutat. Res. 451, 71–89 (2000).
Sung, P., Trujillo, K.M. & Van Komen, S. Recombination factors of Saccharomyces cerevisiae. Mutat. Res. 451, 257–275 (2000).
Yamamoto, T., Moerschell, R.P., Wakem, L.P., Ferguson, D. & Sherman, F. Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast. Yeast 8, 935–948 (1992).
Peterson, K.R., Clegg, C.H., Li, Q. & Stamatoyannopoulos, G. Production of transgenic mice with yeast artificial chromosomes. Trends Genet. 13, 61–66 (1997).
Boren, J., Lee, I., Callow, M.J., Rubin, E.M. & Innerarity, T.L. A simple and efficient method for making site-directed mutants, deletions and fusions of large DNA such as P1 and BAC clones. Genome Res. 6, 1123–1130 (1996).
Tucker, R.M. & Burke, D.T. Directed mutagenesis of YAC-cloned DNA using a rapid, PCR-based screening protocol. Nucleic Acids Res. 24, 3467–3468 (1996).
Muyrers, J.P. et al. Point mutation of bacterial artificial chromosomes by ET recombination. EMBO Rep. 1, 239–243. (2000).
Schaefer, D.G. & Zryd, J.P. Efficient gene targeting in the moss Physcomitrella patens. Plant J. 11, 1195–1206 (1997).
Dieken, E.S., Epner, E.M., Fiering, S., Fournier, R.E. & Groudine, M. Efficient modification of human chromosomal alleles using recombination-proficient chicken/human microcell hybrids. Nat. Genet. 12, 174–182 (1996).
Brachmann, C.B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132 (1998).
Lewis, L.K., Kirchner, J.M. & Resnick, M.A. Requirement for end-joining and checkpoint functions, but not RAD52-mediated recombination, after EcoRI endonuclease cleavage of Saccharomyces cerevisiae DNA. Mol. Cell. Biol. 18, 1891–1902 (1998).
Delneri, D., Gardner, D.C., Bruschi, C.V. & Oliver, S.G. Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain. Yeast 15, 1681–1689 (1999).
Ayyagari, R., Impellizzeri, K.J., Yoder, B.L., Gary, S.L. & Burgers, P.M. A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair. Mol. Cell. Biol. 15, 4420–4429 (1995).
Acknowledgements
We thank Dmitry Gordenin, Elaine Yeh, Alberto Inga, Craig Bennett and Kirill Lobachev for advice, stimulating discussions and comments on the manuscript. L.K.L. was supported by the US Department of Energy Grant DE-A102-99ER62749.
Author information
Authors and Affiliations
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Storici, F., Lewis, L. & Resnick, M. In vivo site-directed mutagenesis using oligonucleotides. Nat Biotechnol 19, 773–776 (2001). https://doi.org/10.1038/90837
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/90837
This article is cited by
-
Dicentric chromosomes are resolved through breakage and repair at their centromeres
Chromosoma (2024)
-
Chance and necessity in the pleiotropic consequences of adaptation for budding yeast
Nature Ecology & Evolution (2020)
-
Immediate, multiplexed and sequential genome engineering facilitated by CRISPR/Cas9 in Saccharomyces cerevisiae
Journal of Industrial Microbiology and Biotechnology (2020)
-
Transcription-dependent targeting of Hda1C to hyperactive genes mediates H4-specific deacetylation in yeast
Nature Communications (2019)
-
Chaperone-mediated ordered assembly of the SAGA and NuA4 transcription co-activator complexes in yeast
Nature Communications (2019)