Mutagenesis: Site‐Directed

Abstract

Site‐directed mutagenesis (SDM) aims 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 hundred; 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 must 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 and 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. In recent years, technologies employing in vivo gene as well genome modifications (editing) has been significantly improved. CRISPR/Cas9 system for genome editing has already been used in different systems.

Key Concepts

  • A selection system to distinguish and select the molecules with the desired site‐directed mutations from the rest of the molecules has to be planned.
  • DNA sequence analysis to verify the presence of the desired alteration with no other changes in the rest of the molecules is essential component of the technique.
  • All site‐directed alterations requiring site‐directed mutagenesis technique are done at the DNA level.
  • Engineered site‐directed mutations are 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 parent molecules is essential.
  • DNA sequence to verify the DNA change is a fundamental part of the technique.
  • Biological and biochemical ramifications because of SDM are usually the purpose that SDM is done in the first place.
  • In addition to the in vitro modification of genes, in vivo methods for gene and whole genome modifications are being developed. In such methods, no transformation of the modified DNA is transformed back to cells, but the tools to bring the changes have to.

Keywords: codon optimisation; mutation efficiency; site‐saturation mutagenesis; random mutagenesis; N‐end rule; synthetic mutations; protein structure–function; SLiP site‐directed mutagenesis; genome editing; CRISPER/Cas9

Figure 1. In vitro mutagenesis using dutung genetic selection method. Based on Su and El‐Gewely 1988, 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 performs thermal cycling to denature DNA (deoxyribonucleic acid) template, anneal mutagenic primers containing desired mutation and extend and incorporate primers with high‐fidelity DNA polymerase (a) or QuikChange 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. ©
Figure 3. Q5® Site‐Directed Mutagenesis Kit. Reprinted from www.neb.com (2013) with permission of New England Biolabs. 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 employs 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 2012. © Elsevier.
Figure 5. PCR‐based site‐directed mutagenesis using ‘SLiCE’ prepared from laboratory E. coli strain. Arrows represent PCR primers. (a) Overlap extension method for site‐directed mutagenesis. (b) SLiCE‐mediated PCR‐based site‐directed mutagenesis (SLiP site‐directed mutagenesis). Reprinted from Motohashi 2015 © Biomed Central.
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Walquist, Mari J, and El‐Gewely, M Raafat(Jan 2018) Mutagenesis: Site‐Directed. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001000.pub4]