BioMed Research International · 2016 · 40 citations · 10 references
Life SciencesGeneticsMolecular BiologyDna RepairGenetic EngineeringGenome EditingError-prone MutagenesisMutagenesis StrategiesPolymerase Chain ReactionSite-directed MutagenesisCodon MutagenesisCodon OptimizationCodon Usage OptimizationOverlap Extension PcrOverlap-extension Polymerase Chain ReactionPcr-based Mutagenesis
The study introduces the COE-PCR method, combining the strengths of overlap extension PCR with the speed and ease of asymmetrical overlap extension PCR. COE-PCR integrates OE-PCR and AOE-PCR protocols into a single workflow, leveraging OE-PCR’s robustness and AOE-PCR’s rapidity. The method allows mutating up to six base pairs per experiment, enabling changes of up to four adjacent codons, and was successfully applied in eight experiments, each altering two to six base pairs, making it especially useful for codon optimization with a single primer set.
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.
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Michael D. Abràmoff, Paulo Magalhães, Sunanda J. Ram · Data Archiving and Networked Services (DANS) · 2004 · 11.9K citations · Full text