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Site-directed mutagenesis is a procedure for the introduction of mutations in a target DNA sequence. It means that we can direct the mutation according to our interests. It is one of the most important procedures for the introduction of mutations. Researchers often use it for the study of protein structure and function by altering certain base pairs in the DNA sequence.
Site-directed mutagenesis is PCR-based mutagenesis that combines some extra steps such as methylation and recombination. It involves the use of a forward and a reverse primer. In this method, we need to include the mutation sequence into the primers that will amplify the target DNA. During the PCR amplification, forward and reverse primers bind to the DNA template and amplify it.
After the amplification, DNA will have the desired mutation and is ready for the expression. Basically, PCR-based mutagenesis involves the use of normal PCR using Taq-Polymerase and other necessary components. But we can also commercially available site-directed mutagenesis kits to introduce a mutation. These kits may use other types of DNA pol such as AccuprimeTM Pfx DNA Polymerase. There are two ways of doing site-directed mutagenesis; a) traditional PCR-amplification, b) using commercially available kits. However, both use the primers with a specific mutation sequence.
Using the site-directed mutagenesis, it is possible to introduce three different kinds of mutations into the target DNA. These mutations are an insertional mutation, deletion mutation, and substitution mutation. Here, I am going to explain a simplified protocol for the site-directed mutagenesis that uses GENEART® Site-Directed Mutagenesis System. However, you need to optimize it as per your conditions. GENEART® Site-Directed Mutagenesis System comes with components necessary for mutagenesis reaction, recombination as well as transformation. I have presented a complete list of the components in a table given below.
|Components provided with GENEART Site-Directed Mutagenesis System|
|DNA Methylase (4 U/µL)||20 µL|
|200X SAM (S-adenosyl-methionine)||10 µL|
|10X Enhancer||100 µL|
|0.5 M EDTA||500 µL|
|pUC19WHITE Control Plasmid (20 ng/µL)||100 ng|
|Control Primer Mix (10 µM)||25 µL|
|PCR water||1.8 mL|
|5X Reaction Buffer for recombination||90 µL|
|10X Enzyme Mix||45 µL|
|One Shot® MAX Efficiency® DH5αTM-T1® (contains competent cells and SOC medium)||1 box|
Site-directed mutagenesis protocol
First, you need to prepare the following things and always remember to work on the icebox.
- 20-25 ng solution of target DNA sample per 50 µL reaction mix (Note 1)
- 10µM forward and reverse primers separately
- 25X SAM from the 200X SAM provided with the kit (prepare freshly each time Note 2)
|10X Accuprime Pfx Reaction mix||5 µL||1X|
|10X Enhancer||5 µL||1X|
|Primers (10 µM each)||1.5 µL||0.3 µM each|
|Plasmid DNA (20 ng/µL)||1 µL||20 ng|
|DNA Methylase (4 U/µL)||1 µL||4 units|
|25X SAM||2 µL||1X|
|Accuprime Pfx DNA Pol (2.5 U/µL)||0.4 µL||1 unit|
|PCR water (nuclease free)||total volume to 50 µL|
Mix each of the components carefully as indicated in the table above. Each time, when you add a reagent, mix it by pipetting up and down. But don’t forget to load the target DNA at the last. That is because reaction starts once we add the substrate and template DNA is the substrate of the PCR. It’s good to start with PCR water and then add the components one by one and then load the target DNA at the last. Incubate the reaction mixture for 12 to 20 minutes at 37 ºC and proceeds with the PCR program as indicated in the table below.
|Temperature||Duration||No. of Cycles|
|37 ºC||12-20 minutes||1|
|94 ºC||2 minutes|
|94 ºC||20 seconds||12-18 cycles|
|57 ºC||30 seconds (Note 3)|
|68 ºC||30 seconds/kb of plasmid|
|68 ºC||5 minutes||1|
During the PCR, the DNA template denatures the two separate strands. In the annealing stage, primers bind to their respective strands of the target DNA at the appropriate annealing temperature. In the next step, DNA polymerase amplifies the primer attached to the template DNA strand. This leads to the production of many copies of the target DNA.
Methylation is an important step in the mutagenesis. This is to make the template plasmid distinguishable. Once the template DNA is methylated, it can be cleaved during the recombination reaction. It avoids the expression of an unmutated wild-type plasmid template. DNA methylase catalyzes the DNA methylation, it transfers a methyl group from SAM to the nitrogenous bases of the target plasmid DNA.
DNA methylase catalyzes the DNA methylation, it transfers a methyl group from SAM to the nitrogenous bases of the target plasmid DNA. However, DNA methylation step can be skipped if you are working with plasmid isolated directly from the bacteria. That is because wild-type plasmid isolated from bacteria is already methylated so no need to methylate it again. Therefore, the methylation step can be skipped and during the recombination reaction, the enzyme mix cleaves the unmutated methylated plasmid.
Some important notes
Note 1: SAM or S-Adenosyl Methionine is an excellent methyl group donor. 25X SAM is not stable, and therefore, you need to prepare it freshly each time when you perform the mutagenesis. You should not use 25X SAM if it is older than a few hours.
Note 2: 12-20 minutes of incubation provides enough time for DNA methylase to methylate nitrogenous bases of the target DNA. For 2.8 to 4 kb plasmids, incubate the mixture for 12 minutes and for 4 to 14 kb plasmid incubate the mixture for 20 minutes.
Note 3: 30 seconds of extension per each 1kb of DNA and 12-15 cycles for 2.8 to 4 kb plasmids and 18 cycles for 4-14 kb plasmids. Don’t forget to include positive and negative controls throughout the experiments.
Once you are done with PCR, analyze the amplicons using 1% agarose gel. In some cases, it is possible to have high mutagenesis efficiency even with multiple bands or very faint bands. Once you get the mutation, proceed with the recombination reaction. The recombination reaction cleaves the methylated parental plasmid DNA template and enhances the mutagenesis efficiency by 3 – 10 folds. The cleavage of the parental plasmid DNA template is necessary to avoid the transformation of the unmutated wild-type plasmid.
|5X Reaction Buffer||4 µL||1X|
|PCR water (Nuclease free water)||10 µL||-|
|PCR Sample||4 µL||-|
|10X Enzyme Mix||2 µL||1X|
Prepare the reaction mixture according to the table given above. Add the 10X Enzyme mix at the last. Mix the reaction mixture by pipetting up and down and incubate it for 10 minutes. After incubation, stop the reaction by adding 1 ul 0.5 M EDTA and mix it by pipetting up down and put the reaction tube on ice and immediately proceed to the transformation.
Before starting up the transformation process, make the few things ready
- Heat and equilibrate the water bath at 42 ºC for heat shock.
- Warm up the SOC medium to room temperature.
- Prepare the LB agar plates (20 ml LB agar per plate) with an appropriate antibiotic such as ampicillin (Note 4).
- Spread X-gal onto the LB agar plates (Note 5).
Now, proceed with the transformation process as outlined below;
- Put the 50 µL DH5αTM-T1R competent cells into the ice for thawing. Thaw it for 5-7 minutes long (no more than 20 minutes).
- Transfer 2 µL of the recombination reaction directly into each vial of the cells and mix it by tapping. Be careful; do not mix the transformation reaction and competent cells by pipetting up and down, as it may damage the cells.
- Put the cap back to the vial and cover the vial completely with ice for 12 minutes. You can optimize this step by increasing the time of up to 30 minutes.
- After 12 minutes of incubation, transfer the vials to a tube rack and put it into the water bath of 42 ºC for 30 seconds. You can also optimize it by increasing the heat shock time up to 45 seconds.
- Remove the vials from the water bath and immediately cover the vials with ice for 2 minutes.
- Remove the vials from ice and de-cap the vials. Add 250 µL of SOC medium aseptically into each vial and incubate it for 1 hour at 37 ºC and constant shaking at 225 rpm.
- After one hour of incubation, take the vials out of the incubated and dilute the transformation reaction (Rxn 1) with an appropriate volume of SOC medium as detailed in the table below.
Plasmid size Rxn 2 Plating volume of Rxn 2 3 kb 5 µL Rxn 1 + 95 µL SOC 100 µL 6 kb 10 µL Rxn 1 + 90 µL SOC 100 µL 9 kb 20 µL Rxn 1 + 80 µL SOC 100 µL 14 kb 50 µL Rxn 1 + 50 µL SOC 100 µL
- Transfer 100 µL of diluted transformation reaction (Rxn 2) into each LB plates aseptically. Put it at the center of the plate.
- Spread the Rxn 2 by shaking vigorously in a circular motion. Shake it for 1-2 minutes in a clockwise and anti-clockwise.
- Store the remaining transformation reactions at 4 ºC.
- Wrap each LB plates spread with Rxn 2 using sterilized paper and invert the plate up right down.
- Incubate the plates at 37 ºC for 16 to 20 hours and observe the colony growth.
- Next day, select 3-5 colonies and inoculate separately in LB broth containing an appropriate antibiotic.
- Isolate the plasmid and confirm the sequence by sequencing.
Some important notes for transformation
Note 4: It is possible to select transformants by using an antibiotic-resistant screening method. The gene of interest is normally inserted into the antibiotic-resistant gene. And after transformation, the antibiotic-resistant gene is lost. Therefore, wild-type DH5αTM-T1TM competent cells will not grow but only the cell with recombinant/mutated plasmid will grow on the LB agar plate containing antibiotics.
However, if you are working with a gene already containing a mutation. I mean to say, if you are introducing a mutation into the plasmid that already contains the gene of interest in the antibiotic resistance gene region then you may not be able to distinguish the transformants using antibiotic-resistant screening tests. Usually, it happens when we carry out a double mutation in a single gene.
In the first mutation, the gene of interest will be introduced into the antibiotic resistance gene. That plasmid will be used as a template in the next mutation reaction. Therefore, it is possible to have some colonies with the plasmid carrying the double mutation and some carrying single mutation. They both will give a white colony and the mutation can either be confirmed by colony-PCR or by sequencing.
Note 5: We can encounter the same problem in the blue-white screening as in the case of antibiotic resistance screening. Doing a double mutation in the same gene of interest can be hard to distinguish using X-gal blue-white screening method. Since in both mutations, the gene of interest lies on the LacZ gene and the gene is inactivated that makes it hard to distinguish the colony with one mutation and the one with the double mutation. So, it’s better to exclude the addition of X-gal if you are carrying out the double mutation in the same gene of interest incorporated into the LacZ gene