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RSS FeedsIJMS, Vol. 20, Pages 3908: PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries (International Journal of Molecular Sciences)

 
 

11 august 2019 20:00:06

 
IJMS, Vol. 20, Pages 3908: PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries (International Journal of Molecular Sciences)
 




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.


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138 viewsCategory: Biochemistry, Biophysics, Molecular Biology
 
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IJMS, Vol. 20, Pages 3906: Structural Comparison of a Promiscuous and a Highly Specific Sucrose 6F-Phosphate Phosphorylase (International Journal of Molecular Sciences)
 
 
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