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

Content Provider	PubMed Central
Author	Yosef, Ido Noga, Bloushtain Shapira, Michal Qimron, Udi
Copyright Year	2004
Abstract	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 λPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage λ 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 β-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.
Related Links	http://dx.doi.org/10.1128/aem.70.12.7156-7160.2004
Ending Page	7160
Page Count	5
Starting Page	7156
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	12
Volume Number	70
Language	English
Publisher	American Society for Microbiology
Publisher Date	2004-12-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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