A PCR amplification strategy for unrestricted generation of chimeric genes.
For analyzing protein function, protein dynamics, or protein-protein interactions, the use of chimeric proteins has become an indispensable tool. The generation of DNA constructs coding for such fused proteins can, however, be a tedious process. Currently used strategies often make use of available endonuclease sites, leading to limitations in the choice of the site of fusion between two genes and problems in maintaining protein secondary structure. We have developed a cloning strategy to get around these disadvantages that is based on a single round of PCR amplification followed by antibiotic-resistant gene complementation.
Department of Cell Biology, Section of Radiation & Stress Cell Biology, University Medical Center Groningen, University of Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Michel J
MJ
Vos
Harm H
HH
Kampinga
D019677:Artificial Gene Fusion; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D050939:Gene Fusion; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D050888:Heat-Shock Proteins, Small / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection
C485182:Cyan Fluorescent Protein; D050888:Heat-Shock Proteins, Small; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins