Site-Directed Mutagenesis (SDM)- Introduction, Principle, Steps, Protocol, Applications


Site-directed mutagenesis (SDM), also known as site-specific or directed mutagenesis, is a laboratory technique for modifying the nucleotide sequence of a gene at a specific position. It is a highly useful method for modifying genes and studying the structural and functional features of a protein based on the structure, function, catalytic mechanism, and catalytic residues of enzymes.


Site-directed mutagenesis is a laboratory technique that allows researchers to introduce particular modifications at specific sites in a DNA sequence of interest. This approach is widely utilized in research for protein engineering, drug discovery, and functional genomics. Researchers can explore the consequences of individual mutations in genes of interest on protein function, structure, and interactions, providing insight into disease causes and prospective treatment targets. There are various ways for executing site-directed mutagenesis, including PCR-based, oligonucleotide-directed, and CRISPR/Cas-based methods, and the method chosen depends on the specific application and the properties of the target gene or genome.


PCR-based site-directed mutagenesis: This method involves designing primers that contain the desired mutation and using them to amplify the entire gene or a specific region of interest. The resulting PCR product can then be cloned into a plasmid and verified through sequencing.

Oligonucleotide-directed mutagenesis

This method involves synthesizing short oligonucleotides that contain the desired mutation and using them to anneal to the template DNA. The DNA polymerase is then used to extend the oligonucleotide, creating a mutant DNA strand that can be cloned and verified through sequencing.

QuikChange site-directed mutagenesis

This method uses a modified PCR protocol in which two complementary primers, one of which contains the desired mutation, are used to amplify the target DNA. The resulting product is then treated with a restriction enzyme to digest the parental DNA, leaving only the mutated product. This method can be used to create multiple mutations simultaneously.

CRISPR/Cas-based site-directed mutagenesis

This method involves using the CRISPR/Cas system to introduce specific mutations at a desired location in the genome. Guide RNAs are designed to target the mutation site, and the Cas protein is used to introduce the desired changes.


Site-directed mutagenesis (SDM) is a technique used to introduce specific changes or mutations into a DNA sequence. Here are the general steps involved in SDM:

Design primers: Design primers that will introduce the desired mutation(s) into the target DNA sequence. These primers should be approximately 20-30 nucleotides in length, with the mutation(s) located at the centre of the primer.

PCR amplification: Use the designed primers to amplify the target DNA sequence using PCR. The PCR reaction mixture contains template DNA, primers, DNA polymerase, dNTPs, and PCR buffer. The cycling conditions for PCR are optimized to ensure specific amplification of the target DNA sequence.

Purify the PCR product: Purify the PCR product to remove any remaining primers, dNTPs, and PCR buffer. The purified PCR product can be used directly for downstream applications or further steps of SDM.

Mutagenesis reaction: Use the purified PCR product as a template for a mutagenesis reaction. The mutagenesis reaction contains mutagenic primers, DNA polymerase, dNTPs, and buffer. The mutagenic primers are designed to introduce the desired mutation(s) into the target DNA sequence.

Steps of Site-Directed Mutagenesis (SDM)

Fig: Steps of Site-Directed Mutagenesis (SDM)

Transformation: Transform the mutagenesis reaction into a suitable host strain, such as E. coli, to allow the DNA to replicate and produce colonies of cells containing the desired mutation.

Selection: Select for cells that contain the desired mutation(s) using appropriate selection methods, such as antibiotic resistance or screening assays.

Confirmation: Verify the presence of the desired mutation(s) by DNA sequencing or other molecular biology techniques.

These are the general steps involved in site-directed mutagenesis. Specific protocols may vary depending on the type of mutagenesis method used and the experimental requirements.


Site-directed mutagenesis (SDM) can be performed using various protocols, depending on the specific experimental requirements. Here are some general steps for SDM using the QuickChange method:

Materials Required

  • Template DNA
  • Primer pairs
  • DNA polymerase
  • dNTPs
  • PCR buffer
  • DpnI restriction enzyme
  • Agarose gel electrophoresis reagents


  • Design the mutagenic primers that will introduce the desired mutation(s) into the target DNA sequence. The primers should be approximately 20-30 nucleotides in length, with the mutation(s) located at the center of the primer.
  • Set up the PCR reaction mix in a PCR tube:
  • Add 1-10 ng of template DNA
  • Add 1 µM each of the mutagenic primers
  • Add the appropriate amount of PCR buffer, dNTPs, and DNA polymerase according to the manufacturer’s instructions.
  • Run PCR amplification using the following cycling conditions:
  • Denaturation at 95°C for 30 seconds
  • Annealing at 55-68°C for 30 seconds
  • Extension at 68°C for 1-3 minutes (depending on the length of the PCR product)
  • Run an agarose gel electrophoresis to confirm the amplification of the desired PCR product.
  • Digest the PCR product with DpnI restriction enzyme to remove the template DNA, which is methylated and resistant to DpnI digestion. Add 1-2 units of DpnI per 1 µg of PCR product and incubate at 37°C for 1 hour.
  • Transform the digested PCR product into a suitable host strain, such as E. coli, and select for transformants on antibiotic plates.
  • Verify the presence of the desired mutation(s) by DNA sequencing.

Other SDM protocols, such as overlap extension PCR or cassette mutagenesis, have different steps and requirements, but the basic principles are similar. It is important to carefully design primers and optimize PCR conditions to ensure successful mutagenesis.


  • To investigate variations in protein activity caused by DNA modification.
  • To find mutations (at the DNA, RNA, or protein levels) that have a desirable attribute.
  • It is used to change the structure and function of proteins, such as increasing enzymatic activity, changing substrate selectivity, or increasing binding affinity.
  • It can be used to generate animal models of human diseases by introducing disease-associated mutations, allowing researchers to investigate disease mechanisms and discover new remedies.
  • SDM can be used to research the effects of mutations on protein stability and structure, such as studying the protein folding route or identifying residues that contribute to protein stability.
  • SDM can be used to explore at the function of individual amino acid residues in protein function, such as those involved in binding, catalysis, or protein-protein interactions.


  • Bachman J. Site-directed mutagenesis. Methods Enzymol. 2013; 529:241-8.
  • Papworth, C., Bauer, J. C., Braman, J. and Wright, D. A. (1996) Strategies 9(3):3–4.
  • Nelson, M. and McClelland, M. (1992) Methods Enzymol 216:279-303.

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