Site-Directed Mutagenesis on Plasmid Using Polymerase Chain Reaction

  • Abdul Aziz Ahmad
  • Hamzah Mohd SallehEmail author
  • Ibrahim Ali Noorbatcha


Site-directed mutagenesis (SDM) is a very useful technique to study changes in protein function that may occur as a result of the DNA manipulation. A detail procedure to employ SDM on whole plasmid using polymerase chain reaction (PCR) with successful application of the method on a pET28a plasmid harboring an endoglucanase I gene from Fusarium oxysporum is described here.


Site directed mutagenesis Endoglucanase Fusarium oxysporum 



The authors would like to acknowledge the Ministry of Higher Education, (MoHE) Malaysia for awarding fund via Fundamental Research Grant Scheme: Grant No: FRGS-13-070-0311 and Professor S.G. Withers (UBC, Vancouver) for providing the original wild-type plasmid construct.


  1. Akcapinar GB, Alessandro VA, Pier LM, Casadio R, Ugur OS (2015) Modulating the thermostability of endoglucanase I from Trichodermareesei using computational approaches. Protein Eng Des Sel 28(5):127–135CrossRefGoogle Scholar
  2. Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82(2):488–492CrossRefGoogle Scholar
  3. Papworth C, Bauer JC, Braman J, Wright DA (1996) Site-directed mutagenesis in one day with >80% efficiency. Strateg Mol Biol 9:3–4Google Scholar
  4. Salleh HM (2011) In: Noorbatcha IA, Karim MIA, Salleh HM (eds) Experimental methods in modern biotechnology, vol 1. IIUM Press, Kuala LumpurGoogle Scholar
  5. Sugimoto M, Esaki N, Tanaka H, Soda K (1989) A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide. Anal Biochem 179(2):309–311CrossRefGoogle Scholar
  6. Taylor JW, Ott J, Eckstein F (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res 13(24):8765–8785CrossRefGoogle Scholar
  7. Vandeyar MA, Weiner MP, Hutton CJ, Batt CA (1988) A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene 65(1):129–133CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Abdul Aziz Ahmad
    • 1
  • Hamzah Mohd Salleh
    • 1
    • 2
    Email author
  • Ibrahim Ali Noorbatcha
    • 1
  1. 1.BioProcess and Molecular Engineering Research Unit, Kulliyyah of EngineeringInternational Islamic University MalaysiaKuala LumpurMalaysia
  2. 2.International Institute for Halal Research and Training, International Islamic University MalaysiaKuala LumpurMalaysia

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