title: Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.
abstract: 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.
authors.affiliation: J. Craig Venter Institute, Synthetic Biology and Bioenergy Group, 4120 Capricorn Lane, La Jolla, CA, 92137, USA.
authors.forename: Isaac T
authors.initials: IT
authors.lastname: Yonemoto
authors.affiliation: J. Craig Venter Institute, Synthetic Biology and Bioenergy Group, 4120 Capricorn Lane, La Jolla, CA, 92137, USA. pweyman@jcvi.org.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Weyman
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D004247:DNA; D004262:DNA Restriction Enzymes
keywords: DNA assembly; Escherichia coli; Gibson assembly; Polymerase chain reaction; Site-directed mutagenesis