Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae
Article first published online: 20 MAY 2013
Copyright © 2013 John Wiley & Sons, Ltd.
Volume 30, Issue 6, pages 243–253, June 2013
How to Cite
Fukunaga, T., Cha-aim, K., Hirakawa, Y., Sakai, R., Kitagawa, T., Nakamura, M., Nonklang, S., Hoshida, H. and Akada, R. (2013), Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae. Yeast, 30: 243–253. doi: 10.1002/yea.2957
- Issue published online: 4 JUN 2013
- Article first published online: 20 MAY 2013
- Accepted manuscript online: 19 APR 2013 04:20AM EST
- Manuscript Accepted: 15 APR 2013
- Manuscript Revised: 8 APR 2013
- Manuscript Received: 8 MAR 2013
- recombinant DNA;
- homologous recombination;
- overlap extension PCR
Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre-existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C-rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.