Abstract
The emerging field of synthetic biology requires novel cloning techniques that allow the rapid assembly of multiple expression units to build artificial genetic circuits. Here, we describe a rapid, flexible, and cost-efficient cloning method that requires only standard laboratory equipment and skills. Our technique relies on the 3′–5′ exonuclease activity of T4 DNA polymerase to generate 20 nt single-stranded DNA overhangs that allow annealing and ligation-independent cloning (LIC) of four DNA fragments in one tube. The resulting intermediate-size constructs can be reused to hierarchically assemble constructs of more than 24 kb by the same method.
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Acknowledgement
The authors thank Veit Hornung for his support. This work is financially supported by the Bauer Fellows Program, SFB 670, ETH Zurich, ERC starting grant, National Institutes of Health (1R01CA155320-01), National Institute of General Medical Sciences Grant for Centers of Systems Biology, and the German National Academic Foundation.
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Schmid-Burgk, J.L., Xie, Z., Benenson, Y. (2014). Hierarchical Ligation-Independent Assembly of PCR Fragments. 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_4
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DOI: https://doi.org/10.1007/978-1-62703-764-8_4
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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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