Restoration of gene function by homologous recombination: from PCR to gene expression in one step.
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 lambdaPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage lambda 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 beta-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.
Yosef
Ido
I
Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel.
Bloushtain
Noga
N
Shapira
Michal
M
Qimron
Udi
U
D003001:Cloning, Molecular / Q000379:methods*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005819:Genetic Markers; D005822:Genetic Vectors; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D005819:Genetic Markers; D019868:Kanamycin Kinase; D001616:beta-Galactosidase