Jajesniak, P. and Wong, T.S. (2015) QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids. Journal of Biological Engineering, 9 (15). ISSN 1754-1611
Abstract
Background
Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with performing point insertion of DNA. These limitations result in low product yield and reduced flexibility in the design of a genetic construct.
Result
Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3′-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. QuickStep-Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. It allows DNA fragment integration into any plasmid at any position, in an efficient, time- and cost-effective manner, without tedious intermediate DNA gel purification, modified oligonucleotides, specialty enzymes and ultra-competent cells. The method is compatible with competent E. coli cells prepared using the conventional calcium chloride method.
Conclusion
QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2015 Jajesniak and Wong. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
Keywords: | Molecular cloning; Gene cloning; Megaprimer; Recombinant DNA; Recombinant plasmid; Protein engineering; Directed evolution; Synthetic biology; Metabolic engineering |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 26 Jul 2017 13:59 |
Last Modified: | 26 Jul 2017 13:59 |
Published Version: | https://doi.org/10.1186/s13036-015-0010-3 |
Status: | Published |
Publisher: | BioMed Central |
Refereed: | Yes |
Identification Number: | 10.1186/s13036-015-0010-3 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:119406 |