Abstract
DNA cloning is fundamental for modern cell research and biotechnology. Various restriction enzymes have been isolated, characterized, and purified to facilitate the digestion and ligation of DNA molecules of different origins. Nevertheless, the very small numbers of enzymes capable of digesting novel and long DNA sequences and the tedious and nearly impossible task of re-engineering existing enzymes with novel specificities greatly limit the use of restriction enzymes for the construction of complex and long DNA molecules. Zinc finger nucleases (ZFNs) – hybrid restriction enzymes that can be tailor made for the digestion of both native and artificial DNA sequences – offer a unique opportunity for expanding the repertoire of restriction enzymes useful for various DNA cloning tasks. Here we present protocols for the assembly, expression, and purification of cloning-grade ZFNs and their use for DNA cloning. We focus our discussion on the assembly of a dual-cassette plant transformation vector, as an example of a task that is nearly impossible to perform using the current collection of naturally occurring and recombinant 6–8 bp long restriction enzymes.
The authors Vardit Zeevi and Andriy Tovkach contributed equally to this work.
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Acknowledgments
We thank Dr. G.N. Drews for the gift of pHS::QQR-QEQ/2300. We also thank Dan Weinthal for his instrumental advice and support. This work in our lab is supported by grants from the Human Frontiers Science Program, the Biotechnology Research and Development Corporation (BRDC) and University of Michigan startup funds.
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Zeevi, V., Tovkach, A., Tzfira, T. (2010). Artificial Zinc Finger Nucleases for DNA Cloning. In: Mackay, J., Segal, D. (eds) Engineered Zinc Finger Proteins. Methods in Molecular Biology, vol 649. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-753-2_12
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DOI: https://doi.org/10.1007/978-1-60761-753-2_12
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