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 over-hangs 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. © Springer Science+Business Media New York 2014.
CITATION STYLE
Schmid-Burgk, J. L., Xie, Z., & Benenson, Y. (2014). Hierarchical ligation-independent assembly of PCR fragments. Methods in Molecular Biology, 1116, 49–58. https://doi.org/10.1007/978-1-62703-764-8_4
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