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

In simple words, we can say that,

By using some of the artificial techniques such as PCR, the mutation is introduced or 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 oligonucleotide-specific mutagenesis or site-specific mutagenesis.

Here in the present article, we are going to discussing different methods of introducing site-directed mutagenesis into the DNA, Why to do site-directed mutagenesis 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
  1.               Direct/conventional PCR
  2.               Inverse PCR
  3.               Nested PCR
  • Role of site-directed mutagenesis in CRISPR-CAS9
  • Application of site-directed mutagenesis
  • Conclusion

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Introduction to site-directed mutagenesis

The first site-directed mutagenesis experiment was performed into the year 1974, in the laboratory of Charles Weissmann. The induced mutation was from GC to AT. However, the mutation is not at a specific location.

Before the study of Charles Weissmann, chemical analogues and radiations are used to create alteration into the DNA sequence but the mutations are random and not induced at a specific location.

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

The basic principle of site-directed mutagenesis is simple, the single-stranded, short DNA primer having the desired mutation is synthesised artificially and the DNA primers are complementary to the sequence of our interest.

With the help of the controlled temperature dependent cyclic condition, the primer is used to amplify the target DNA sequence.

The DNA polymerase (high fidelity) extends the growing DNA strand having the new mutation.

Now the gene of interest can be transferred into the plasmid for further downstream applications.

Here, The mutation may be SNP, deletion of several nucleotides, addition or insertion. Once the desired mutant plasmid is generated, it can be sequenced to screen the mutation.

Importance of site-directed mutagenesis

By using the conventional PCR modifications such as nested PCR, inverse PCR or conventional amplification, the desired mutation can be incorporated into the DNA sequence.

Here, No fancy methods or fluorescent chemistry for amplification is required.

But why do we have to do site-directed mutagenesis? What is the significance of doing artificial mutagenesis?

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?

The plasmids are constructed to transfer a gene of our interest. Restriction digestion is performed to confirm the gene transformation experiments.

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 fails the experiment.

If any mutation is introduced at the recognition site of the REase, it can not cut it. This can be done using the site-directed mutagenesis.

It is used to analysing the structure and function of the protein.

At the molecular level, the properties of the particular gene or proteins can be screened or studied with the help of the site-directed mutagenesis.

For example, suppose one wild types protein which is encoded by some gene 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. The activity of a particular protein can be determined as well.

Importantly, it is useful in the study of protein-protein interaction.

It is further applicable in the enzyme silencing and enzyme altering.

Different techniques for site-directed mutagenesis

PCR is one of the important tools for identifying the mutation as well as to introduce the mutation at a particular location. The same method which is used for screening particular mutation can be used for insertion of mutation.

Three approaches are most commonly used for Different techniques for site-directed mutagenesis:

Conventional PCR:

The conventional PCR method is one of the most popular and easy methods for site-directed mutagenesis.

In this method, The Short single-stranded PCR primers are designed in a manner which contains the mutation.

The Taq DNA polymerase used into 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 along with the mutation.

The newly synthesised strand having the mutation of our interest replaces the original sequence over a period of amplification cycles.

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.

At the 3′ end, the Taq polymerase expands the DNA. So the chance of reaction failure is higher at 3′ end.

Another limitation of the present method is that it contains mutant as well as nonmutant DNA (mix 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 utilised in the conventional PCR approach only, which can alter up to 2 to 3 nucleotides.

Site-Directed Mutagenesis: Methods and Applications

Primer extension or nested PCR:

In the primer extension, 2 set of primers are used in which a single set of primer is nested. Here the mechanism of carrying 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 round of amplification.

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

The primer 2 and 3 contains a mutant sequence hence the two different PCR product contains the mutant sequence.

In the second round of amplification, the primer 1 and 4 amplifies the full length of 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, hence it amplify in the reverse orientation.

The method is used for inserting mutation into the plasmid having the 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.

It amplifies the flanking regions therefore, it is the best choice for deleting several nucleotides from the 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 into the PCR reaction, especially for the site-directed mutagenesis.

Template DNA: 

Here our target DNA is always a circular plasmid DNA because we want to study the gene of our interest, inserted into the plasmid.

Read more on plasmid: The Colony PCR article contains information on the plasmid.

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

Use 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 than the normal DNA polymerase.

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.

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 preciously while the 3′ to 5′ exonuclease activity increases the amplification specificity.

Also, the polymerase does have the 5′ to 3′ polymerase activity. DNA polymerases such as Pfu, Vent and Phusion facilitates higher amplification rate in site-directed mutagenesis.

The Taq DNA polymerase does not use in the site-directed mutagenesis because it produces such an end after completion of the amplification that interferes in the recircularization of a plasmid.

However, it can be useful for conventional PCR based site-directed mutagenesis.

Primers:

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

Als,o the primer used in the site-directed mutagenesis does not have repetitive sequences and secondary structure.

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

Read more on How to design primer for PCR,

PCR Primer Design
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 containing the mutation 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. Only the mutant DNA remains into the reaction.

Site-Directed Mutagenesis: Methods and Applications

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

The general outline of the SDM:

The general flow of the experiment is as followed:

  • Design the primers for the introduction of specific mutation (deletion, duplication or SNP). Check all the primer parameters
  • Synthesis of the oligonucleotide primers artificially
  • Prepare PCR reaction based on one of the methods listed above
  • Perform PCR reaction
  • Confirm the amplicons using the agarose gel electrophoresis
  • Purify the amplicon using ready to use DNA purification kit.
  • perform DNA sequencing for confirming 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: The process of selecting and designing primers and the use of the methylation-dependent endonuclease is the key to success in the SDM.

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

In modern time, so many 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 the model organisms.

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

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

Applications of site-directed mutagenesis:

The site-directed mutagenesis helps to improve the quality of the protein by removing harmful elements from it.

Also, the modified, genetically engineered proteins have high commercial value.

The tool is used in the study of a gene characteristics in which by introducing specific mutations, the properties of the DNA, its encoded protein and post-translational modifications of that, can be studied.

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

It is used in the cloning as well.

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

It is also useful in the screening of SNPs.

Additional resource related to this article,

In Vitro MutagenesisYou can read this book for more detail on in vitro or site-directed mutagenesis.

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. However, the nuclease used in the present technique is one of the crucial element of the site-directed mutagenesis.