Transfer-PCR (TPCR): A highway for DNA cloning and protein engineering

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-...

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Bibliographic Details
Published in:Journal of structural biology 2011-08, Vol.175 (2), p.171-177
Main Authors: Erijman, Ariel, Dantes, Ada, Bernheim, Reut, Shifman, Julia M., Peleg, Yoav
Format: Article
Language:English
Subjects:
Bacterial Proteins - genetics
Calmodulin - genetics
Cloning, Molecular - methods
Combinatorial libraries
Computer Simulation
DNA cloning
Escherichia coli - genetics
Genetic Vectors
Ligation-independent cloning (LIC)
Models, Molecular
Multiple-site mutagenesis
Mutagenesis, Site-Directed - methods
Polymerase Chain Reaction - methods
Protein design
Protein Engineering - methods
Recombinant Proteins - genetics
Targeted protein engineering
Transfer-PCR (TPCR)
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Summary: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.
ISSN:1047-8477
1095-8657
DOI:10.1016/j.jsb.2011.04.005