title: Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering.
abstract: DNA cloning and protein engineering are basic methodologies employed for various applications in all life-science disciplines. Manipulations of DNA however, could be a lengthy process that slows down subsequent experiments. To facilitate both DNA cloning and protein engineering, we present Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector. TPCR can be applied for incorporation of DNA fragments into any desired position within a circular plasmid without the need for purification of the intermediate PCR product and without the use of any commercial kit. Using several examples, we demonstrate the applicability of the TPCR platform for both DNA cloning and for multiple-site targeted mutagenesis. In both cases, we show that the TPCR reaction is most efficient within a narrow range of primer concentrations. In mutagenesis, TPCR is primarily advantageous for generation of combinatorial libraries of targeted mutants but could be also applied to generation of variants with specific multiple mutations throughout the target gene. Adaptation of the TPCR platform should facilitate, simplify and significantly reduce time and costs for diverse protein structure and functional studies.
authors.affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Ariel
authors.initials: A
authors.lastname: Erijman
authors.forename: Ada
authors.initials: A
authors.lastname: Dantes
authors.forename: Reut
authors.initials: R
authors.lastname: Bernheim
authors.forename: Julia M
authors.initials: JM
authors.lastname: Shifman
authors.forename: Yoav
authors.initials: Y
authors.lastname: Peleg
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002147:Calmodulin / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D002147:Calmodulin; C034284:IgG Fc-binding protein, Streptococcus; D011994:Recombinant Proteins