PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries
AbstractQuickStep 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
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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.
Jajesniak P, Tee KL, Wong TS. PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries. International Journal of Molecular Sciences. 2019; 20(16):3908.Chicago/Turabian Style
Jajesniak, Pawel; Tee, Kang L.; Wong, Tuck S. 2019. "PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries." Int. J. Mol. Sci. 20, no. 16: 3908.
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