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.
Highlights
Site directed mutagenesis is a technique used for substitution, addition, and deletion of specific base sequences in DNA [1]
Site directed mutagenesis is used in codon optimization to remedy codon bias during heterologous expression of proteins [7, 8]
The mutagenic primers (MP) were complementary to each other, with the mutagenic bases in the center of each primer, and were between 17 and 32 bp in length depending on the number of base pairs to be changed
Summary
Site directed mutagenesis is a technique used for substitution, addition, and deletion of specific base sequences in DNA [1]. It is an important tool to generate mutants with altered amino acid sequences for enzyme studies, investigation of the relationship between structure and functions of proteins, and functional analysis of genes or their regulatory sequences [2,3,4]. Mutations are introduced through mutagenic primers which contain one or more mismatched bases [1, 2]. These mutagenic primers are incorporated during PCR and the mutant DNA is amplified exponentially [1, 2]
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