Abstract
We developed a simple method (simple cloning) for subcloning DNA fragments into any location of a targeted vector without the need of restriction enzyme, ligase, exonuclease, or recombinase in Escherichia coli. This technology can be applied to common E. coli hosts (e.g., DH5α, JM109, TOP10, BL21(DE3)). The protocol includes three steps: (1) generate DNA insert and linear vector backbone by regular high-fidelity PCR, where these two DNA fragments contain 3′ and 5′ overlap** termini; (2) generate DNA multimers based on these two DNA fragments by using prolonged overlap extension-PCR (POE-PCR) without primers added; and (3) transform POE-PCR product to competent Escherichia coli cells directly, yielding the desired plasmid. Simple cloning provides a new cloning method with great simplicity and flexibility. Furthermore, this new method can be modified for the preparation of a large-size mutant library for directed evolution in E. coli. Using this method, it is very easy to generate a mutant library with a size of more than 107 per 50 μL of the POE-PCR product within 1 day.
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Acknowledgement
This work could not be finished without the support of the Nan**g Tech University and Virginia Tech. In addition, funding to YPZ for this work was provided in part by DOE EERE award (DE-EE0006968) and by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, US Department of Agriculture.
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Zhong, C., You, C., Wei, P., Zhang, YH.P. (2017). Simple Cloning by Prolonged Overlap Extension-PCR with Application to the Preparation of Large-Size Random Gene Mutagenesis Library in Escherichia coli . In: Hughes, R. (eds) Synthetic DNA. Methods in Molecular Biology, vol 1472. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6343-0_4
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DOI: https://doi.org/10.1007/978-1-4939-6343-0_4
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