26: Site-directed mutagenesis
This method uses PCR to edit specific nucleotides in a DNA sequence. Most often this technique is used to study the importance or function of certain sequences and their resulting proteins.
Also known as
Site specific mutagenesis or oligonucleotide-directed mutagenesis
Samples needed
To perform this method you first need to determine what sequence you want to edit and design forward and reverse primers that correspond to your intended mutations. In addition to primers, this process requires plasmid DNA with the sequence of interest which will be edited via PCR
Method
The first step in this experimental workflow is to perform PCR amplification using your primer pairs of interest combined with template plasmid DNA. The results of this amplification will be many copies of the new mutated DNA sequence as well as the few remaining copies of your original template circular DNA plasmid.
These new mutated products will be linear and must be circularized before transformation into bacterial cells. This process can be accomplished a number of different ways, one of which being treatment with a cocktail of enzymes (KLD mix): kinase, ligase, and dpnI, used to prepare the sample. Within this mixture, kinase phosphorylates the 5’ end of each of your linear strands which allows the ligase to join the two ends together resulting in a circular product.
The last step in this process is degrading the remaining template DNA to increase the number of mutants recovered in future steps. To accomplish this we must have a way to degrade only the template plasmids without damaging the newly amplified PCR products. Luckily, E. coli cells contain specialized enzymes which work to methylate GATC sites as a way to recognize parental vs replicated DNA strands. Knowing this, we can use dpnI, an enzyme which specifically works to cut methylated GATC sites, to clear the unwanted sequences. These products are now ready to be transformed into bacterial cells so the mutation’s function can be assessed.
Controls
It is important to use wild type (WT) primers in your experiment as a control to assess how mutations in the plasmid sequence affect downstream form and function.
Interpretation
Although results of this method are not typically seen until the new plasmids are transformed into bacterial cells (where they will undergo large-scale, in vivo replication), the success of the process could be visualized using Sanger sequencing. This type of DNA sequencing allows researchers to identify changes in small sections of the genome, perfect for identifying mutations such as insertions, deletions or single nucleotide changes.
Image descriptions
Figure 1 image description:
A schematic of the method of site-directed mutagenesis.
Step 1: Amplification. PCR Reaction mix includes template DNA (the sequence you intend to amplify), DNA polymerase, dNTP mix, specific primers (oligonucleotides for either WT or mutation of interest), and reaction buffer.
Step 2: Re-circularizing. This involves treatment with a kinase to phosphorylate the 5' ends of the DNA, a ligase to join the linear PCR products into a circle, and DpnI, to digest the template DNA. ↵
Figure 2 image description:
Images shows headlines from studied utilizing site-directed mutagenesis. These studies can be found in the citations below. The text states: "Site-directed mutagenesis in the news. This method has been popular in optimizing enzyme performance and specificity." ↵
Thumbnail
"Site-Directed-Mutagenesis.gif" ↗ by Stephengregg is licensed under CC BY-SA 3.0(opens in new window) ↗.
Description: An animation showing the basics of site directed mutagenesis.
Author
Emilie Jones, Tufts University
- Chen, Z., J. Chen, W. Zhang, T. Zhang, C. Guang, and W. Mu. 2018. Improving Thermostability and Catalytic Behavior of l-Rhamnose Isomerase from Caldicellulosiruptor obsidiansis OB47 toward d-Allulose by Site-Directed Mutagenesis. Journal of Agricultural and Food Chemistry.
- Meng, T., S. Bezstarosti, U. Singh, M. Yap, L. Scott, N. Petrosyan, F. Quiroz, N. V. Eps, E. K.-W. Hui, D. Suh, Q. Zhu, R. Pei, C. S. M. Kramer, F. H. J. Claas, D. Lowe, and S. Heidt. (n.d.). Site‐directed mutagenesis of HLA molecules reveals the functional epitope of a human HLA‐A1/A36‐specific monoclonal antibody.
- Yamamoto, T., S. Fujiwara, Y. Tachibana, M. Takagi, K. Fukui, and T. Imanaka. 2000. Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis. Journal of Bioscience and Bioengineering 89:206–209.