This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yosef, I. Articles by Qimron, U. Search for Related Content PubMed PubMed Citation Articles by Yosef, I. Articles by Qimron, U. Agricola Articles by Yosef, I. Articles by Qimron, U.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, December 2004, p. 7156-7160, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7156-7160.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Restoration of Gene Function by Homologous Recombination: from PCR to Gene Expression in One Step

Ido Yosef,^1, Noga Bloushtain,¹ Michal Shapira,² and Udi Qimron^1*

Department of Microbiology and Immunology, Faculty of Health Sciences,¹ Department of Life Sciences, Faculty of Natural Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel²

Received 15 June 2004/ Accepted 31 July 2004

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete P_R or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding ß-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.

Present address: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.