Abstract
The polymerase chain reaction (PCR) is the technique of choice used to obtain DNA for cloning, because it rapidly provides high amounts of desired DNA fragments and allows the easy introduction of extremities adequate for enzyme restriction or homologous recombination, and of artificial, native, or modified sequence elements for specific applications. In this context, the use of megaprimer-based PCR strategies allows the versatile and fast assembly and amplification of tailor-made DNA sequences readily available for cloning.
In this chapter, we describe the design and use of a megaprimer-based PCR protocol to construct customized fusion genes ready for cloning into commercial expression plasmids by restriction digestion and ligation.
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Acknowledgments
This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte, Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), Project GlycoCBMs PTDC/AGR-FOR/3090/2012 (FCOMP-01-0124-FEDER-027948), and grant SFRH/BDP/63831/2009 to Carla Oliveira.
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Aguiar, T.Q., Oliveira, C., Domingues, L. (2017). Synthesis of Fusion Genes for Cloning by Megaprimer-Based PCR. In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 1620. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-7060-5_6
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DOI: https://doi.org/10.1007/978-1-4939-7060-5_6
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