Enzyme Engineering

Volume 978 of the series Methods in Molecular Biology pp 173-186


Protein Engineering: Single or Multiple Site-Directed Mutagenesis

  • Pei-Chung HsiehAffiliated withNew England Biolabs, Inc. Email author 
  • , Romualdas VaisvilaAffiliated withNew England Biolabs, Inc.

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Site-directed mutagenesis techniques are invaluable tools in molecular biology to study the structural and functional properties of a protein. To expedite the time required and simplify methods for mutagenesis, we recommend two protocols in this chapter. The first method for single site-directed mutagenesis, which includes point mutations, insertions, or deletions, can be achieved by an inverse PCR strategy with mutagenic primers and the high-fidelity Phusion® DNA Polymerase to introduce a site-directed mutation with exceptional efficiency. The second method is for engineering multiple mutations into a gene of interest. This can be completed in one step by PCR with mutagenic primers and by assembling all mutagenized PCR products using the Gibson Assembly™ Master Mix. This method allows multiple nucleotides to be changed simultaneously, which not only saves time but also reagents compared to traditional methods of mutagenesis.

Key words

Site-directed mutagenesis Inverse PCR mutagenesis Gibson assembly Synthetic biology