1. PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries
- Author
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Pawel Jajesniak, Tuck Seng Wong, and Kang Lan Tee
- Subjects
0106 biological sciences ,0301 basic medicine ,Computer science ,cloning ,random mutagenesis ,Mutagenesis (molecular biology technique) ,DNA-Directed DNA Polymerase ,Computational biology ,Polymerase Chain Reaction ,01 natural sciences ,Article ,Catalysis ,Inorganic Chemistry ,lcsh:Chemistry ,03 medical and health sciences ,Synthetic biology ,Plasmid ,010608 biotechnology ,Escherichia coli ,Cloning, Molecular ,Physical and Theoretical Chemistry ,directed evolution ,Deoxyribonucleases, Type II Site-Specific ,Molecular Biology ,lcsh:QH301-705.5 ,Spectroscopy ,Gene Library ,Cloning ,Expression vector ,Oligonucleotide ,Organic Chemistry ,protein engineering ,DNA ,General Medicine ,Protein engineering ,Directed evolution ,Computer Science Applications ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,Mutagenesis ,synthetic biology ,Directed Molecular Evolution ,QuickStep ,PTO-QuickStep ,Plasmids - 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&prime, 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.
- Published
- 2019
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