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.
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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).
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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
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DOI: https://doi.org/10.1007/978-1-62703-764-8_7
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-764-8
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