Abstract
DNA cloning is a basic methodology employed for multiple applications in all life-science disciplines. In order to facilitate DNA cloning we developed 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. TPCR reaction is most efficient within a narrow range of primer concentrations. Adaptation of the TPCR should facilitate, simplify, and significantly reduce time and costs for DNA assembly, as well as protein engineering studies. In the current publication we describe a detailed protocol for implementation of the TPCR method for DNA assembly.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Graslund S, Nordlund P, Weigelt J et al (2008) Protein production and purification. Nat Methods 5:135–146
Benoit RM, Wilhelm RN, Scherer-Becker D et al (2006) An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids. Protein Expr Purif 45:66–71
Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256
Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074
Gileadi O, Burgess-Brown NA, Colebrook SM et al (2008) High throughput production of recombinant human proteins for crystallography. Methods Mol Biol 426:221–246
Neilan BA, Tillett D (2002) Enzyme-free cloning of PCR products and fusion protein expression. Methods Mol Biol 192:125–132
Chen GJ, Qiu N, Karrer C et al (2000) Restriction site-free insertion of PCR products directionally into vectors. BioTechniques 28:498–500, 504–495
Geiser M, Cebe R, Drewello D et al (2001) Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase. BioTechniques 31:88–90, 92
Miyazaki K, Takenouchi M (2002) Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. BioTechniques 33:1033–1034, 1036–1038
Unger T, Jacobovitch Y, Dantes A et al (2010) Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172:34–44
van den Ent F, Lowe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67:67–74
Erijman A, Dantes A, Bernheim R et al (2011) Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J Struct Biol 175:171–177
Peleg Y, Unger T (2008) Application of high-throughput methodologies to the expression of recombinant proteins in E. coli. Methods Mol Biol 426:197–208
Peleg Y, Unger T (2012) Resolving bottlenecks for recombinant protein expression in E. coli. Methods Mol Biol 800:173–186
Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28
Acknowledgments
We thank Prof. J. Sussman, Prof. I. Silman, Prof. G. Schreiber, and Prof. Yigal Burstein for their continuous support throughout the study. The ISPC is supported by the Divadol Foundation. This research was supported by the ISF grant 1372/10 (J.M.S.), Deutsche Forschungsgemeinschaft grant EI 423/2-1 (J.M.S.), and the Abisch Frenkel foundation (J.M.S).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Erijman, A., Shifman, J.M., Peleg, Y. (2014). A Single-Tube Assembly of DNA Using the Transfer-PCR (TPCR) Platform. In: Valla, S., Lale, R. (eds) DNA Cloning and Assembly Methods. Methods in Molecular Biology, vol 1116. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-764-8_7
Download citation
DOI: https://doi.org/10.1007/978-1-62703-764-8_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-763-1
Online ISBN: 978-1-62703-764-8
eBook Packages: Springer Protocols