Abstract
The restriction free (RF) cloning has emerged as one of the highly efficient techniques in the area of genetic engineering. RF cloning has wide range of applications in plasmid DNA manipulation including cloning of a single gene, simultaneous assembly of multiple DNA fragments, and mutagenesis from single to multiple simultaneous alterations of a target DNA. Recently, we have developed a new technique of circular permutation using RF cloning. Circular permutation is widely used to investigate the mechanisms of protein folding and function. Previously, restriction enzyme based cloning was used to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.
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Acknowledgments
We thank Prof. Amnon Horovitz for his support. The SPU is supported by the Dana and Yossie Holander Center for Structural Proteomics.
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Bandyopadhyay, B., Peleg, Y. (2022). Application of Restriction Free (RF) Cloning in Circular Permutation. In: Currin, A., Swainston, N. (eds) Directed Evolution. Methods in Molecular Biology, vol 2461. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2152-3_10
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DOI: https://doi.org/10.1007/978-1-0716-2152-3_10
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