PTO-QuickStep: A fast and efficient method for cloning random mutagenesis libraries

Jajesniak, P., Tee, K. L. and Wong, T. S. (2019) PTO-QuickStep: A fast and efficient method for cloning random mutagenesis libraries. International Journal of Molecular Sciences, 20(16), 3908. (doi: 10.3390/ijms20163908) (PMID:31405219) (PMCID:PMC6720219)

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.

• Item Type: Articles
• Additional Information: Funding: We thank the Department of Chemical & Biological Engineering, ChELSI, EPSRC (EP/E036252/1), BBSRC (BB/R020183/1), the RAEng/Leverhulme Trust Senior Research Fellowship (to T.S.W.), the University of Sheffield GCRF Fellowship (to K.L.T.), and the University of Sheffield PhD Scholarship (to P.J.) for financial support.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Jajesniak, Dr Pawel
• Authors: Jajesniak, P., Tee, K. L., and Wong, T. S.
• College/School: College of Science and Engineering > School of Engineering > Biomedical Engineering
• Journal Name: International Journal of Molecular Sciences
• Publisher: MDPI
• ISSN: 1661-6596
• ISSN (Online): 1422-0067
• Published Online: 11 August 2019
• Copyright Holders: Copyright © 2019 by the authors
• First Published: First published in International Journal of Molecular Sciences 20(16): 3908
• Publisher Policy: Reproduced under a Creative Commons license