Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

• Main Authors: Hasnain, Hussain, Chong, Nikson Fatt Ming
• Format: E-Article
• Language: English
• Published: Hindawi Publishing Corporation 2016
• Subjects: Q Science (General); QR Microbiology
• Online Access: http://ir.unimas.my/id/eprint/14294/1/Combined%20Overlap%20Extension%20PCR%20Method%20for%20Improved%20Site%20Directed%20Mutagenesis%20%28abstract%29.pdf; http://ir.unimas.my/id/eprint/14294/; https://www.hindawi.com/journals/bmri/

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.