Yonemoto IT, Weyman PD
Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.
Methods in Molecular Biology (Clifton, N.J.). 2017 Oct 01; 1498: 359-366.
Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (>~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation.