Mutagenesis: Site‐Specific

Abstract

Site‐specific mutagenesis techniques, also known as site‐directed mutagenesis (SDM), aim to introduce precise alterations in any coding or noncoding deoxyribonucleic acid (DNA) sequence, usually in vitro. These modifications could be as small as a nucleotide or several hundreds; in one site or in multisite in the same DNA sequence. Recently, these alterations have been also developed in vivo. SDM success depends on how changes are introduced and mutant selection is done. DNA sequence analysis has to be made to verify change(s) before any biochemical or biological experiments are done. Recent methods for SDM and most used commercial kits are discussed. A list of companies offering SDM service is included. The authors have also listed software used for mutagenic oligonucleotide primer‐design. These techniques are revolutionising our understanding of the genetic and molecular mechanisms, protein structure–function relationship, protein–protein interaction, binding sites in any biological system. In addition to the academic benefits of SDM, SDM techniques have impacted biotechnology and the applied field such as engineering new enzymes, drug development, optimisation of heterologous gene expression and secretion.

Key Concepts:

  • All site‐specific alterations requiring site‐directed mutagenesis technique are done at the DNA level making it heritable modifications. Modifications done at the protein levels are not heritable.

  • The results of these alterations are reflected in the encoded amino acids sequence of the proteins or in any targeted binding site in the DNA sequence.

  • Several simplified Techniques are now available.

  • Selection of the altered DNA molecules from the pool of nonmodified parental molecules is essential.

  • DNA sequence to verify the DNA change is fundamental part of the technique.

  • Biological and biochemical ramifications as a result of SDM are usually the purpose that SDM is done in the first place.

Keywords: codon optimisation; mutation efficiency; site‐directed mutagenesis; site‐saturation mutagenesis; N‐end rule; synthetic mutations

Figure 1.

In vitro mutagenesis using dutung genetic selection method. Based on Su and El‐Gewely (), utilising the genetic selection system and the dutungE. coli strain (Kunkel, ).

Figure 2.

QuikChange (Agilent Technologies) One‐Day Method (a) and the Lightning Fast Method (b). 1. Mutant strand synthesis that perform thermal cycling to: Denature DNA template, anneal mutagenic primers containing desired mutation and extend and incorporate primers with high‐fidelity DNA polymerase (a) or QuickChange Lightning fusion enzyme (b). 2. I Digestion of Template: Digest parental methylated and hemimethylated DNA with DpnI (a) or NEW DpnI enzyme (b). 3. DpnI Transformation: Transform mutated molecule into competent cells for nick repair. Reproduced with Permission, Courtesy of Agilent Technologies, Inc. © Agilent Technologies, Inc. Nov 2013. QuikChange kit, for more details see: http://www.genomics.agilent.com/en/product.jsp?cid=AG‐PT‐175&tabId=AG‐PR‐1162&_requestid=371895

Figure 3.

Reprinted from www.neb.com (2013) with permission of New England Biolabs. © New England Biolabs, Inc. For more details see: https://www.neb.com/products/e0554‐q5‐site‐directed‐mutagenesis‐kit

Figure 4.

Overview of the TA cloning from Adachi and Fukuhara, . This method has engineered 5′‐phosphrylated double stranded primers with 3′T‐overhang sticky ends that will be ligated to the plasmids A‐overhang. Reproduced with permission from Adachi and Fukuhara, . © Elsevier.

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Walquist, Mari, Kjeldsen Buvang, Elisabeth, and El‐Gewely, M Raafat(Jun 2014) Mutagenesis: Site‐Specific. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001000.pub3]