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

Ariel Erijman, Ada Dantes, Reut Bernheim, Julia M. Shifman*, Yoav Peleg

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

104 Scopus citations

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.

Original languageAmerican English
Pages (from-to)171-177
Number of pages7
JournalJournal of Structural Biology
Volume175
Issue number2
DOIs
StatePublished - Aug 2011

Bibliographical note

Funding Information:
We thank Prof. J. Sussman, Prof. I. Silman, Prof. G. Schreiber and Dr. Tamar Unger for moral support and their comments on the manuscript. This research was supported by the European Commission Sixth Framework Research and Technological Development Program ‘SPINE2-COMPLEXES’ Project, under contract No. 031220 ; a grant of the Israel Ministry of Science, Culture, and Sport to the Israel Structural Proteomics Center; the Divadol Foundation; the Neuman Foundation; the Israel Science Foundation ( 1372/10 to J.M.S.) and DFG ( El 423/2-1 to J.M.S.).

Keywords

  • Combinatorial libraries
  • DNA cloning
  • Ligation-independent cloning (LIC)
  • Multiple-site mutagenesis
  • Protein design
  • Targeted protein engineering
  • Transfer-PCR (TPCR)

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