title: Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis.
abstract: The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.
authors.affiliation: Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
authors.forename: Hasnain
authors.identifier: 0000-0002-2630-450X
authors.initials: H
authors.lastname: Hussain
authors.affiliation: Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
authors.forename: Nikson Fatt-Ming
authors.initials: NF
authors.lastname: Chong
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics*; D003062:Codon / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics*
chemical_list: D003062:Codon; D004247:DNA