“The site-directed mutagenesis is an artificial (in vitro) technique for introducing mutation or alteration into the target (known) DNA sequence.”


In simple words, we can say that

by using artificial techniques such as PCR a mutation or alteration can be inserted into the target-DNA sequence of our interest.

Site-directed mutagenesis: Site: specific location into the genome, Directedperformed by artificial techniques, mutagenesis–  Introduction of mutation.

In the site-directed mutagenesis, At a specific location on oligonucleotide sequence, the mutation is intentionally created, hence it is also called as oligonucleotide-specific mutagenesis or site-specific mutagenesis. The purpose of introducing a mutation is to study the function of a gene.

Here in the present article, we are going to discussing different methods of introducing mutations or performing site-directed mutagenesis into the DNA, Why to do it and that is the purpose of it.

The content of the article is,

  • Introduction to site-directed mutagenesis
  • Importance of site-directed mutagenesis
  • Different techniques for site-directed mutagenesis
      • Direct/conventional PCR
      • Inverse PCR
      • Nested PCR
  • Role of site-directed mutagenesis in CRISPR-CAS9
  • Application of site-directed mutagenesis
  • Conclusion

Introduction:

The first site-directed mutagenesis (SDM) experiment was performed in the year 1974, in the laboratory of Charles Weissmann. The induced mutation was from GC to AT, however, the mutation was inserted randomly. Scientists from the Charles Weissmann university had incorporated mutations and changed the GC nucleotide with AT.

Before the study of Charles Weissmann, chemical analogs and radiations like mutagens are used to create alteration into the DNA sequence, however, those changes are random and not site-specific.

In 1974, Michael Smith and Clyde Hutchison had performed site-directed mutagenesis using the primer extension method. They had achieved artificial mutagenesis at a specific location the very first time in the history of genetics.

The basic principle of site-directed mutagenesis is simple, DNA primers having the desired mutation are artificially synthesized and used to amplify the gene of interest. This primer set is used to amplify a gene of interest in a polymerase chain reaction.

The DNA polymerase (high fidelity) extends the growing DNA strand having the new mutation. Now the gene of interest is transferred into the plasmid for further downstream research.

Mutations such as SNP (Single nucleotide polymorphism), deletion & insertion of few nucleotides are commonly used for SDM. In the final step, DNA sequencing screens the present or absence of mutation in our DNA.

Related reads: Comparison Between DNA Primer And RNA Primer.

Importance:

Various ways, for instance, nested PCR, inverse PCR, or conventional PCR amplification are used to do mutagenesis. Here, no fancy methods or fluorescent chemistry is needed.

But why do we have to do site-directed mutagenesis? What is the significance of doing it? let see some important uses of present methods.

The site-directed mutagenesis is used to remove the restriction sites. Restriction digestion is a process in which the DNA having the recognition site for a particular restriction endonuclease is cleaved into fragments.

Read more on Restriction digestion: What is Restriction Digestion and how to do it?

Next, the modified sequence is transferred into a plasmid and then again, restriction digestion is performed to validate whether the recognition site is removed or not.

But if the DNA of our interest contains the restriction site, the restriction endonuclease cleaves the target DNA as well the rest of the plasmid DNA which indicates experimental failure.

The funda is simple, if we introduce any mutation at a RE recognition site, the enzyme can’t identify it and can’t cut it. This can only be done using the site-directed mutagenesis.

Next, it is practiced to analyze the structure and function of the protein. The method is powerful enough to study the function of a protein formed by a gene.

For example, suppose a gene encodes one wild type protein and it is required in the metabolic pathway of one organism. By mutating this protein-coding gene, the effect of the absence of that protein in the metabolic pathway of an organism can be studied.

Also, one important function of site-directed mutagenesis is in the study of gene editing or gene manipulation. Through gene manipulation, the activity of a particular protein can be determined as well.

Besides these important applications, the method is often practiced to study protein-protein interaction, enzyme alteration, and enzyme silencing studies.

Different techniques:

PCR is regularly used not only to identify mutations or genotypes but it is also used to insert mutations at a particular location. The same method which is used to screen mutations can be used to insert mutations.

We have enlisted three common approaches to do site-directed mutagenesis:

Conventional PCR:

The conventional PCR method is one of the most popular and easy methods for site-directed mutagenesis. The set up is simple; short, single-stranded oligonucleotide primers are designed in such a way that possess the mutation we wish to study.

The Taq DNA polymerase used in the conventional PCR does not have the exonuclease activity hence it cannot identify mismatch during the amplification. If the mutation is minimal enough to incorporate, the primer binds to the DNA having mutation.

Over a period of some amplification cycle, the original DNA sequence is replaced by the DNA sequence having a mutation. The major recommendation for the conventional PCR based mutagenesis is to insert mutant bases up to several limits at the 5′ end of the primer or in the middle of the primer.

From the 3′ end, the Taq polymerase expands the DNA. So the chance of reaction failure is higher there.  Another limitation of the present method is that it carries mutant as well as nonmutant DNA (mixed types of DNA) due to the presence of template DNA.

Hence the yield of the conventional PCR based site-directed mutagenesis is lower.

For more detail on conventional PCR, read the present article: A Complete Guide of the Polymerase Chain Reaction.

Note: Instead of Taq DNA polymerase, we can use the high fidelity DNA polymerase. But for that, the experimental assay is totally different. Further, the Taq DNA polymerase can be utilized in the conventional PCR approach only and has the power to alter only 2 to 3 nucleotides.

The overview of the present method is shown in the figure below,

Site-Directed Mutagenesis: Methods and Applications

Conventional PCR-based site-directed mutagenesis.

Primer extension or nested PCR:

In the primer extension, 2 sets of primers are used in which a single primer set is nested. Here the mechanism of carrying the mutation is the same, the mutation is introduced into the primer at one of the ends.

Now let’s name the primers for convenience,

Let say the outer forward primer is 1 the outer reverse primer is 4

The inner forward primer is 3 and the inner reverse primer is 2.

The location of all four primers are shown in the figure below,

Site-Directed Mutagenesis: Methods and Applications

Here, the inner primer 2 and 3 contain the mutation/ mismatched bases at the end. The amplification process in the nested PCR is completed in two rounds of amplification.

In the first round of amplification, primer 1-2 and primer 3-4 creates two different PCR products.

primer 2 and 3 are mutant primer sequences therefore in two different PCR products, two different mutations are incorporated.  In the second round of amplification, the primer 1 and 4 amplifies the entire DNA sequence having the desired mutation.

Read more on nested PCR: What is nested PCR?

Inverse PCR:

In the inverse PCR, the primers amplify the fragment other than the target sequence. Which means,  it amplifies in the reverse orientation.

The method is used for inserting mutation into the plasmid having a gene of our interest. The high fidelity DNA polymerase is used to do the amplification as well as to linearise the circular plasmid DNA. Once the mutation is inserted, the 5′ phosphorylated end of the primers allows the ligation of the linear DNA.

By the intermolecular ligation, the plasmid is then recircularized. The present method relies on the flanking region amplification thus it is used so often in deleting sequences from a plasmid. See the figure below,

Site-Directed Mutagenesis: Methods and Applications

We can delete many nucleotides by using the inverse PCR method. See figure below for deletion by inverse PCR,

Site-Directed Mutagenesis: Methods and Applications

Read more our inverse PCR: Inverse PCR: Principle, Procedure, Protocol and Applications

These three methods are most popular for the site-directed mutagenesis. Now take look at some of the components used in the PCR reaction, especially for the site-directed mutagenesis.

Template DNA: 

Here, our template is always a circular plasmid DNA because we want to study the gene of our interest, present in a plasmid. Read more on plasmid: Plasmid DNA- Structure, Function, Isolation and Applications.

The concentration of the plasmid DNA must be between 0.1-1.0 ng/μl and it must be highly purified.

Use a plasmid purification kit to purify the plasmid DNA.

If the GC content of the plasmid is higher, use DMSO in the PCR reaction.

DNA polymerase:

The polymerase used in the site-directed mutagenesis must be special and different from the conventional one. Here, instead of normal Taq DNA polymerase, use high fidelity DNA polymerase.

Also, the high fidelity DNA polymerase used in the present assay must be different from other high fidelity polymerases as well. The polymerase used here should have 3′ to 5′ exonuclease activity (not 5′ to 3′ exonuclease activity).

The high fidelity DNA polymerase with 5′ to 3′ exonuclease activity removes the mismatched nucleotides while the 3′ to 5′ exonuclease activity increases the amplification specificity.

Also, polymerase does have the 5′ to 3′ polymerase activity. DNA polymerases such as Pfu, Vent, and Phusion facilitate a higher amplification rate in site-directed mutagenesis. The Taq DNA polymerase is only used in the conventional PCR based method for introducing mutation.

Primers:

Ideally, the mutation is incorporated at the end of the primer, but if the mutation is in between, make sure that both sides of the primer do have at least 11 base pair complementary sequences.

Also, the primer used in the site-directed mutagenesis does not have repetitive sequences and secondary structures.

3 base mismatch is tolerable in the ideal 22 to 25 nucleotide long primer.

Nuclease: 

Now, this is something very important.

The template DNA is natural DNA, it must contain methyl group which means the template DNA is methylated while the newly formed DNA is unmethylated The methylation-specific endonuclease- a type of nuclease used here which cleaves only the methylated DNA.

Therefore, the template DNA having not the mutation is removed from the reaction (because it is methylated). Only the mutant DNA remains in the reaction. See the image,

Site-Directed Mutagenesis: Methods and Applications

The image shows the Methylation-specific nuclease activity which cleaves the methylated template DNA.

A general outline of the SDM:

The experimental set up is explained here:

  • Design the primers to insert specific mutation (deletion, duplication, or SNP). Check all the primer parameters
  • Synthesis the oligonucleotide primers artificially
  • Prepare PCR reaction based on one of the methods listed above
  • Perform PCR reaction
  • Validate the amplicons using the agarose gel electrophoresis
  • Purify the amplicon using a ready to use DNA purification kit.
  • perform DNA sequencing to establish the mutagenesis results.
  • send the sample for cloning.

Read our articles on,

  1. DNA sequencing
  2. Different methods of nucleic acid purification

Site-Directed Mutagenesis: Methods and Applications

Key to success: Use of endonuclease, selecting a primer set, and designing primers are three important hacks to success in mutagenesis.

Role of site-directed mutagenesis in the CRISPR-CAS9:

In modern times, various methods and ready to use kits for site-directed mutagenesis are available. However, one of the most important merits of the site-directed mutagenesis is in the gene editing, especially in the CRISPR-CAS9.

Any point mutation can be introduced in vivo with the help of the CRISPR-CAS9 system into the genome of a model organism.

Here, in the CRISPR-CAS9, the CAS9 is the nuclease which is used to cleave the DNA. Once it induces a double-stranded break, the mutation is inserted through the homologous-direct repair.

Read more on the homologous-direct repair (HDR): What is gene editing and CRISPR-CAS9?

Applications:

The site-directed mutagenesis helps to improve the quality of the protein by removing harmful elements from it. These genetically modified proteins have high commercial values.

The tool is adopted in the study of gene characteristics. Gene properties, a protein encoded by it, and post-translational modification are studied.

The method is the first choice in gene synthesis and gene-editing technology.

It is used in cloning as well.

By mutating the promoter or the regulatory regions of a gene, one can construct the map of the regulatory elements of a gene.

It is also useful in the screening of SNPs as well.

Conclusion:

Site directed-mutagenesis has its own importance in the field of gene editing and gene manipulation. It facilitates improvement in the wild type genotype to produce a commercially important phenotype. Notably, the use of nuclease is crucial to get success. site-directed mutagenesis is used in the knockout mice construction and gene knockout studies too.