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PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries

Department of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, UK
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Int. J. Mol. Sci. 2019, 20(16), 3908; https://doi.org/10.3390/ijms20163908
Received: 13 July 2019 / Revised: 7 August 2019 / Accepted: 9 August 2019 / Published: 11 August 2019
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Abstract

QuickStep is a cloning method that allows seamless point integration of a DNA sequence at any position within a target plasmid using only Q5 High-Fidelity DNA Polymerase and DpnI endonuclease. This efficient and cost-effective method consists of two steps: two parallel asymmetric PCRs, followed by a megaprimer-based whole-plasmid amplification. To further simplify the workflow, enhance the efficiency, and increase the uptake of QuickStep, we replaced the asymmetric PCRs with a conventional PCR that uses phosphorothioate (PTO) oligos to generate megaprimers with 3′ overhangs. The ease and speed of PTO-QuickStep were demonstrated through (1) right-first-time cloning of a 1.8 kb gene fragment into a pET vector and (2) creating a random mutagenesis library for directed evolution. Unlike most ligation-free random mutagenesis library creation methods (e.g., megaprimer PCR of whole plasmid [MEGAWHOP]), PTO-QuickStep does not require the gene of interest to be precloned into an expression vector to prepare a random mutagenesis library. Therefore, PTO-QuickStep is a simple, reliable, and robust technique, adding to the ever-expanding molecular toolbox of synthetic biology and expediting protein engineering via directed evolution. View Full-Text
Keywords: cloning; QuickStep; PTO-QuickStep; random mutagenesis; directed evolution; protein engineering; synthetic biology cloning; QuickStep; PTO-QuickStep; random mutagenesis; directed evolution; protein engineering; synthetic biology
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Jajesniak, P.; Tee, K.L.; Wong, T.S. PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries. Int. J. Mol. Sci. 2019, 20, 3908.

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