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Updated: Jul 6, 2025

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
Published on: February 5, 2019
Site-Directed Mutagenesis to Mutate Multiple Residues in a Single Reaction
Saima Usman1, Antesar Bushaala1, Muy Teck Teh1
1Centre for Oral Immunobiology and Regenerative Medicine, Institute of Dentistry, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
Abstract:
Site-directed mutagenesis (SDM) is a technique that allows mutation of specific nucleotide(s) in a codon to study its functional implications in a protein. Commercial kits are available, which require high-performance liquid chromatography purified oligos for this purpose. These kits are expensive, and they are not very efficient, so one has to sequence several clones to get a desired one. We present here a simple method that requires only crude oligos, commercially available high-fidelity enzymes, and the success rate is close to 100%. In addition, up to 6 different mutations can be introduced in one reaction without causing any fortuitous change in the vector backbone. Using this strategy, we have introduced 32 S/T➔A substitutions in the N-terminus head and 13 changes in the C-terminus tail domain of vimentin.
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