How to Design Primers for Site Directed Mutagenesis

Site-directed mutagenesis (SDM) is an in vitro method of creating a mutation in a known sequence. It is often performed by PCR-based methods. Typically, one or two bases are changed in site-directed mutagenesis. Primers can be designed with the desired mutations for introducing small changes to the nucleotide sequence. Primer extension and inverse PCR can be used to introduce large-scale mutations. This approach may change the amino acid composition of a particular protein. Site-directed mutagenesis is used to study the changes of the activity of proteins. It is also used to create fusion proteins as well.

Key Areas Covered

1. What is Site-Directed Mutagenesis (SDM)
      – Definition, Role, Method
2. How to Design Primers for Site-Directed Mutagenesis
     – Substitutions, Deletions, Insertions

Key Terms: Deletions, Insertions, Mutations, Site-Directed Mutagenesis, Substitutions, Traditional PCR

How to Design Primers for Site Directed Mutagenesis

What is Site-Directed Mutagenesis

Site-directed mutagenesis is a molecular biology technique used to introduce specific changes to a nucleotide sequence of a gene. It is used to change the amino acid sequence of a protein, introduce or remove restriction sites, destroy transcription binding sites, and create fusion proteins.


During site-directed mutagenesis, the mutations are introduced to the plasmids using primers, consisting of the desired mutation. The entire template is amplified by PCR, incorporating the mutation into the template. Then, the parent template is removed from the sample with the use of an enzyme, methylation-dependent endonuclease. The PCR product or the nicked plasmid molecule with the desired mutation is transformed into bacteria. The plasmids can be isolated from the bacteria with desired modifications. The process of site-directed mutagenesis is shown in figure 1.

How to Design Primers for Site Directed Mutagenesis_Figure 1

Figure 1: Site-Directed Mutagenesis

Three main types of methods are used to introduce desired mutations in site-directed mutagenesis. They are traditional PCR, primer extension, and inverse PCR. Primer extension and inverse PCR can be used to introduce large-scale nucleotide changes.

Traditional PCR

Traditional PCR can be used to introduce one or two nucleotide changes to the target sequence with modified primers. The changes can be nucleotide substitutions, deletions or additions. The mutation is incorporated into the amplicon during PCR. Hence, the original sequence is replaced by the mutated sequence in the primer.

Primer Extension

In primer extension, the desired mutation is incorporated during a nested PCR. Here, the target sequence is flanked by two primers. The desired mutation is incorporated into the internal primer and, the mutation is introduced in the second PCR round. Generally, the specificity of a PCR reaction decreases with the increased number of mismatched nucleotides in the primers. However, long internal primers with large-scale mutations can be used in primer extension as nested PCR can increase the specificity of the PCR reaction.

Inverse PCR

Inverse PCR is a method of amplifying unknown DNA fragments by designing primers to a known DNA sequence. It can be used to substitute, delete, or insert nucleotides in large-scale.

The applications of site-directed mutagenesis are described below.

  1. To study the structure, function, and catalytic properties of proteins
  2. To improve the properties of proteins (protein engineering)
  3. To introduce or remove restriction endonucleases sites.

How to Design Primers for Site-Directed Mutagenesis

Site-directed mutagenesis is a process of introducing the desired mutation by means of a primer. The mutation can be a substitution, insertion or deletion. As the specificity of PCR decreases with the increasing mismatched nucleotides in the primer, traditional PCR can only introduce one or two base pair changes to the target sequence. Other methods such as primer extension and inverse PCR can be used to introduce large-scale mutations. The primer design in site-directed mutagenesis is shown in figure 2.

How to Design Primers for Site Directed Mutagenesis

Figure 2: Primers for Site-Directed Mutagenesis


For substitutions, one of the two primers should contain the desired mutation in the middle of the primer. Here, the site that contains the mutation does not anneal to the target sequence since it forms a distortion.


For deletions, the sequence to be deleted from the target can be neglected during the primer design. As this sequence is situated apart from the flanked region by primers, it would not be amplified during PCR.


For insertions, the sequence to be added is entangled to the 5’ end of one of the primers during primer design. Therefore, the inserted sequence may remain attached to the amplicon as well.


Site-directed mutagenesis is a technique used to introduce mutations into a DNA sequence. These mutations can be substitutions, insertions or deletions. Small-scale mutations can be introduced in the traditional PCR. Large-scale mutations can be introduced in primer extension or inverse PCR. The introduction of the mutation is done by incorporation of the desired mutation to the primer. 


1. “Methods for site-Directed mutagenesis.” INTEGRATED DNA TECHNOLOGIES, .

Image Courtesy:

1. “Site Directed Mutagenesis” By Knbusby – Own work (Public Domain) via

About the Author: Lakna

Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things

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