Mutagenesis: Site‐Specific


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:‐PT‐175&tabId=AG‐PR‐1162&_requestid=371895

Figure 3.

Reprinted from (2013) with permission of New England Biolabs. © New England Biolabs, Inc. For more details see:‐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.



Adachi Y and Fukuhara C (2012) TA strategy for rapid and efficient site‐directed mutagenesis. Analytic Biochemistry 431(1): 66–68.

Beaudoin J, Ioannoni R, Mailloux S, Plante S and Labbé S (2013) Transcriptional regulation of the copper transporter mfc1 in meiotic cells. Eukaryotic Cell 12(4): 575–590.

Beetham PR, Kipp PB, Sawycky XL, Arntzen CJ and May GD (1999) A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene‐specific mutations. Proceedings of the National Academy of Sciences of the USA 96: 8774–8778.

Bittova L, Sumandea M and Cho W (1999) A structure–function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues. Journal of Biological Chemistry 274: 9665–9672.

Bok JW and Keller NP (2012) Fast and easy method for construction of plasmid vectors using modified quick‐change mutagenesis. Methods in Molecular Biology 944: 163–174.

Bryksin AV and Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. BioTechniques 48(6): 463–465.

Chan J, Watson JN, Lu A et al. (2012) Bacterial and viral sialidases: contribution of the conserved active site glutamate to catalysis. Biochemistry 51(1): 433–441.

Dieffenbach CW, Lowe TMJ and Dveksler GS (1995) General concepts for PCR primer design. In: Dveksler GS (ed.) PCR Primer, A Laboratory Manual, pp. 133–155. New York: Cold Spring Harbor Laboratory Press.

Doan T and Aymerich S (2003) Regulation of the central glycolytic genes in Bacillus subtilis: binding of the repressor CggR to its single DNA target sequence is modulated by fructose‐1,6‐bisphosphate. Molecular Microbiology 47: 1709–1721.

Dymond JS (2013) PCR‐based random mutagenesis. Methods in Enzymology 529: 249–258.

El‐Gewely MR (1991) Oligonucleotide and multisite directed mutagenesis. In: El‐Gewely MR (ed.) Site Directed Mutagenesis and Protein Engineering, pp. 161–170. Amsterdam: Elsevier Science.

Guillier M, Allemand F, Raibaud S et al. (2002) Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: a possible case of ribosomal RNA–messenger RNA molecular mimicry. RNA 8: 878–889.

Hagen FK, Hazes B, Raffo R, DeSa D and Tabak LA (1999) Structure–function analysis of the UDP‐N‐acetyl‐d‐galactosamine:polypeptide N‐acetylgalactosaminyltransferase. Essential residues lie in a predicted active site cleft resembling a lactose repressor fold. Journal of Biological Chemistry 274: 6797–6803.

Hale R and Thompson G (1998) Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli . Protein Expression and Purification 12: 185–188.

Hammerle M, Bauer J, Rose M et al. (1998) Proteins of newly isolated mutants and the amino‐terminal proline are essential for ubiquitin‐proteasome‐catalyzed catabolite degradation of fructose‐1,6‐bisphosphatase of Saccharomyces cerevisiae . Journal of Biological Chemistry 273: 25000–25005.

Hogrefe HH, Cline J, Youngblood GL and Allen RM (2002) Creating randomized amino acid libraries with the QuikChange multi site‐directed mutagenesis kit. BioTechniques 33(5): 1158–1160, 1162, 1164–1165.

Hutchison CA III, Phillips S, Edgell MH et al. (1978) Mutagenesis at a specific position in a DNA sequence. Journal of Biological Chemistry 253(18): 6551–6560.

Innis MA and Gelfand DH (1990) Optimization of PCRs. In: Innis MA, Gelfand DH, Sninsky JJ and White TJ (eds) PCR Protocols: A Guide to Methods and Applications, pp. 5–11. San Diego, CA: Academic Press Inc.

Jonet MA, Mahadi NM, Murad AM et al. (2012) Optimization of a heterologous signal peptide by site‐directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli . Journal of Molecular Microbiology and Biotechnology 22(1): 48–58.

Koch B, Buchholz M, Wermann M et al. (2012) Probing secondary glutaminyl cyclase (QC) inhibitor interactions applying an in silico‐modeling/site‐directed mutagenesis approach: implications for drug development. Chemical Biology & Drug Design 80(6): 937–946.

Kochevenko A and Willmitzer L (2003) Chimeric RNA/DNA oligonucleotide‐based site‐specific modification of the tobacco acetolactate syntase gene. Plant Physiology 132: 174–184.

Komori K, Ichiyanagi K, Morikawa K and Ishino Y (1999) PI‐PfuI and PI‐PfuII, intein‐coded homing endonucleases from Pyrococcus furiosus. II. Characterization of the binding and cleavage abilities by site‐directed mutagenesis. Nucleic Acids Research 27: 4175–4182.

Kunkel TA (1985) Rapid and efficient site‐specific mutagenesis without phenotypic selection. Proceedings of the National Academy of Sciences of the USA 82: 488–492.

Kunkel TA, Bebenek K and McClary J (1991) Efficient site‐directed mutagenesis using uracil‐containing DNA. Methods in Enzymology 204: 125–139.

Kwok S, Kellogg DE, McKinney N et al. (1990) Effects of primer–template mismatches on the polymerase chain reaction: human immunodeficiency virus 1 model studies. Nucleic Acids Research 18: 999–1005.

Ma AC, Lee HB, Clark KJ and Ekker SC (2013) High efficiency in vivo genome engineering with a simplified 15‐RVD GoldyTALEN design. PLoS One 8(5): e65259.

Molloy EM, Field D, O'Connor PM et al. (2013) Saturation mutagenesis of lysine 12 leads to the identification of derivatives of nisin A with enhanced antimicrobial activity. PLoS One 8(3): e58530.

Munteanu B, Braun M and Boonrod K (2012) Improvement of PCR reaction conditions for site‐directed mutagenesis of big plasmids. Journal of Zhejiang University. Science. B 13(4): 244–247.

Olsen D, Sayers JR and Eckstein F (1993) Site‐directed mutagenesis of single‐stranded and double‐stranded DNA by phosphorothioate approach. Methods in Enzymology 217: 189–217.

Papp PP, Nagy T, Ferenczi S et al. (2002) Binding sites of different geometries for the 16‐3 phage repressor. Proceedings of the National Academy of Sciences of the USA 99: 8790–8795.

Parikh MR and Matsumura I (2005) Site‐saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta‐fucosidase from beta‐galactosidase. Journal of Molecular Biology 352(3): 621–628.

Phelan RM and Townsend CA (2013) Mechanistic insights into the bifunctional non‐heme iron oxygenase carbapenem synthase by active site saturation mutagenesis. Journal of the American Chemical Society 135(20): 7496–7502.

Rossant J and Nagy A (1995) Genome engineering: the new mouse genetics. Nature Medicine 1: 592–594.

Saleem RA, Murphy TC, Liebmann JM and Walter MA (2003) Identification and analysis of a novel mutation in the FOXC1 forkhead domain. Investigative Ophthalmology & Visual Science 44(11): 4608–4612.

Sharrocks AD (1994) The design of primers for PCR. In: Griffin HG and Griffin AM (eds) PCR Technology: Current Innovations, pp. 5–11. Boca Raton, FL: CRC Press.

Steffens DL and Williams JG (2007) Efficient site‐directed saturation mutagenesis using degenerate oligonucleotides. Journal of Biomolecular Techniques 18(3): 147–149.

Steffler F, Guterl JK and Sieber V (2013) Improvement of thermostable aldehyde dehydrogenase by directed evolution for application in Synthetic Cascade Biomanufacturing. Enzyme and Microbial Technology 53(5): 307–314.

Storbakk N, Fenton C, Riise HM, Nilsen IW and El‐Gewely MR (1996) In vivo interaction between mutated tryptophan repressors of Escherichia coli . Journal of Molecular Biology 256: 889–896.

Stuckey S and Storici F (2013) Gene knockouts, in vivo site‐directed mutagenesis and other modifications using the delitto perfetto system in Saccharomyces cerevisiae. Methods Enzymol 533: 103–131.

Su T‐Z and El‐Gewely MR (1988) A multisite‐directed mutagenesis using T7 DNA polymerase: application for reconstructing a mammalian gene. Gene 69: 81–89.

Swingle BM (2013) Oligonucleotide recombination enabled site‐specific mutagenesis in bacteria. Methods in Molecular Biology 978: 127–132.

Tang L, Jiang R, Zheng K and Zhu X (2011) Enhancing the recombinant protein expression of halohydrin dehalogenase HheA in Escherichia coli by applying a codon optimization strategy. Enzyme and Microbial Technology 49(4): 395–401.

Tee KL and Wong TS (2013) Polishing the craft of genetic diversity creation in directed evolution. Biotechnology Advances 31(8): 1707–1721.

Thein S and Wallace RB (1986) The use of synthetic oligonucleotides as specific hybridization probes in the diagnosis of genetic disorders. In: Davis KE (ed.) Human Genetic Diseases: A Practical Approach, pp. 33–35. Herndon: IRL Press.

Varshavsky A (1996) The N‐end rule: functions, mysteries, uses. Proceedings of the National Academy of Sciences of the USA 93: 12142–12149.

Vohra S and Biggin PC (2013) Mutationmapper: a tool to aid the mapping of protein mutation data. PLoS One 8(8): e71711.

Wäneskog M and Bjerling P (2014) Multi‐fragment site‐directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method. Analytical Biochemistry 444: 32–37.

Wang W and Malcolm BA (1999) Two‐stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site‐Directed Mutagenesis. BioTechniques 26(4): 680–682.

Wang X, Li X, Zhang Z, Shen X and Zhong F (2010) Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin‐A in E. coli . Protein Expression and Purification 72(1): 101–106.

Weiner MP, Costa GL, Schoettlin W et al. (1994) Site‐directed mutagenesis of double‐stranded DNA by the polymerase chain reaction. Gene 151: 119–123.

Winkler ML, Rodkey EA, Taracila MA et al. (2013) Design and exploration of novel boronic acid inhibitors reveals important interactions with a clavulanic acid‐resistant sulfhydryl‐variable (SHV) β-lactamase. Journal of Medicinal Chemistry 56(3): 1084–1097.

Xu H and El‐Gewely MR (2003) Differentially expressed downstream genes in cells with normal or mutated p53. Oncology Research 13: 429–436.

Xu H, Petersen EI, Petersen SB and El‐Gewely MR (1999) Random mutagenesis libraries: optimization and simplification by PCR. BioTechniques 27: 1102–1108.

Yao Y, Pattabiraman N, Michne WF, Huang XP and Hampson DR (2003) Molecular modeling and mutagenesis of the ligand‐binding pocket of the mGlu3 subtype of metabotropic glutamate receptor. Journal of Neurochemistry 86(4): 947–957.

Yedavalli P and Rao NM (2013) Engineering the loops in a lipase for stability in DMSO. Protein Engineering Design and Selection 26(4): 317–324.

Yoshikawa F, Uchiyama T, Iwasaki H et al. (1999) High efficient expression of the functional ligand binding site of the inositol 1,4,5‐triphosphate receptor in Escherichia coli . Biochemical and Biophysical Research Communications 257: 792–797.

Young L and Dong Q (2010) Targeted amplification of mutant strands for efficient site‐directed mutagenesis and mutant screening. Methods in Molecular Biology 634: 147–155.

Zhu L (1996) In vitro site‐directed mutagenesis using the unique restriction site elimination (USE) method. Methods in Molecular Biology 57: 13–29.

Zimmerman K, Scholten JD, Huang CC, Fierke CA and Hupe DJ (1998) High‐level expression of rat farnesyl: protein transferase in Escherichia coli as a translationally coupled heterodimer. Protein Expression and Purification 14: 395–402.

Further Reading

Andrews FH and McLeish MJ (2013) Using site‐saturation mutagenesis to explore mechanism and substrate specificity in thiamin diphosphate‐dependent enzymes. FEBS Journal 24: 6395–6411.

Barbosa O, Torres R, Ortiz C et al. (2013) Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules 14(8): 2433–2462.

Carey MF, Peterson CL and Smale ST (2013) PCR‐mediated site‐directed mutagenesis. Cold Spring Harbor Protocols 8: 738–742.

Heyn J, Hinske LC, Ledderose C, Limbeck E and Kreth S (2013) Experimental miRNA target validation. Methods in Molecular Biology 936: 83–90.

Liu Y, Wu T, Song J et al. (2013) A mutant screening method by critical annealing temperature‐PCR for site‐directed mutagenesis. BMC Biotechnology 13: 21.

Roulias A, Pichler U, Hauser M et al. (2013) Differences in the intrinsic immunogenicity and allergenicity of Bet v 1 and related food allergens revealed by site‐directed mutagenesis. Allergy. doi:10.1111/all.12306.

Wu D, Guo X, Lu J et al. (2013) A rapid and efficient one‐step site‐directed deletion, insertion, and substitution mutagenesis protocol. Analytical Biochemistry 434(2): 254–258.

Xie T, Song B, Yue Y, Chao Y and Qian S (2014) Site‐saturation mutagenesis of central tyrosine 195 leading to diverse product specificities of an α‐cyclodextrin glycosyltransferase from Paenibacillus sp. 602–1. Journal of Biotechnology 170: 10–16.

Yamamoto H and Shikanai T (2013) In planta mutagenesis of Src homology 3 domain‐like fold of Ndhs, a ferredoxin‐binding subunit of the chloroplast NADH dehydrogenase‐like complex in Arabidopsis: a conserved Arg193 plays a critical role in ferredoxin binding. Journal of Biological Chemistry. doi:10.1074/jbc.M113.511584.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

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