Function of taq DNA polymerase in PCR
The function of Taq DNA polymerase in PCR reaction is to amplify the DNA for the production of multiple copies of DNA. Taq DNA polymerase is a thermostable DNA polymerase which can also work at a higher temperature.
In this article, we will discuss about the DNA polymerase and its function, Taq DNA polymerase, the function of Taq DNA polymerase in PCR reaction, and advantages and disadvantages of Taq DNA polymerase.
Let’s start with the basics,
What is DNA polymerase
The DNA polymerase is an enzyme work during the process of DNA replication. The replication is a process in which a DNA is copied into two daughter DNA molecules. For more detail of DNA replication read our article: DNA Replication: General process of DNA replication
The DNA polymerase is an important biological molecule of the central dogma specifically, replication. It synthesises new DNA strand from the existing strand by adding dNTPs to the growing DNA. The enzyme is discovered by Arthur Kornberg in 1956 (awarded Nobel prize for that in 1959).
Also read the article: Prokaryotic DNA replication
The DNA polymerase has two important activity during replication: 5’ to 3’ polymerase activity and 3’ to 5’ exonuclease proofreading activity. As the polymerase binds to DNA, it adds nucleotide in a direction of 5’ to 3’. However, the polymerase incorporates some wrong dNTPs during replication results in a mismatch.
The 3’ to 5’ proofreading activity helps in removing this mismatch nucleotides. The polymerase moves back to one nucleotide, removes the mismatch nucleotide and reinsert the exact nucleotide match. The mechanism is called as base excision repair.
The enzyme activity is temperature dependent, it works according to temperature change. Generally, all the enzymes have higher activity at 37°C temperature, a body temperature. And so DNA polymerase has. Hence a question strikes in mind whether it can work in vitro?
PCR, polymerase chain reaction is a temperature dependent, in vitro, DNA amplification process. Denaturation, annealing and extension are three temperature-dependent steps in PCR. Denaturation occurs at 94°C, annealing at 56°C to 63°C and extension at 72°C.
Further read: agarose gel electrophoresis
As PCR is only working at a higher temperature so it proves that the polymerase cannot be used in PCR. And the solution to this problem is Taq DNA polymerase.
During one summer, at Yellowstone national park in the USA, Thomas D Brook discovered a bacteria in 1966. He isolated the bacteria from the hot spring of water and named it as Thermus aquaticus. The bacteria can survive at higher temperature hence the name Thermus was given and as it is found in hot water, name aquaticus given to it. Collectively, Thermous aquatiqus, “ A hot water bacteria.”
Later on, in the year 1976, Chien et al., isolated polymerase from Thermus aquaticus named it as Taq DNA polymerase. Now, this is an enzyme which can solve the problem for PCR because it is stable at a higher temperature.
The thermostable polymerase, Taq has several properties such as thermostability, efficiency and higher amplification capacity are best suited for the PCR. Several advantages and disadvantages of Taq is enlisted here,
Advantages of Taq DNA polymerase:
- Obviously, Thermostable: the enzyme is thermostable polymerase hence it can even work at higher temperature
- Efficiency: It is highly efficient. As it is reached at its optimum temperature, the thermostable polymerase becomes fully function and adds the nucleotide to the growing DNA strand.
- Hight amplification capacity: It adds 150 nucleotides per seconds.
- The half-life of thermostable polymerase is higher than any other commercially available polymerase. Taq has a half-life more than 2 hours at temperate of 92°C.
Disadvantages of Taq DNA polymerase:
- Low Specificity: The specificity of Taq DNA polymerase activity is lower as compared with normal polymerase, it can add even mismatched nucleotides. Because it is a temperature dependent, slight fluctuation in temperature causes an adverse effect in Taq activity.
- Low fidelity: Taq does not have 3’ to 5’ exonuclease proofreading activity so it is unable to correct mismatch nucleotide.
- Unidirectional activity: Again, it has unidirectional activity from 5’ to 3’ therefore it cannot go back for base excision repair.
- Bivalent cation requirement: The enzyme required cofactors for working properly. The Taq DNA polymerase always needs a Mg2+ ion as a cofactor.
However, nowadays, high-fidelity Taq DNA polymerase, specific Taq DNA polymerase and High sensitive DNA polymerases are commercially available depending upon the type of PCR reaction.
Comparison of DNA polymerase and Taq DNA polymerase:
Taq DNA polymerase
5’ to 3’ polymerase activity and 3’ to 5, proofreading activity
5’ to 3’ polymerase activity
Stable at 37°C
Stable at 92°C
My ultimate guide for using Taq DNA polymerase
The specificity of the Taq DNA polymerase is lower, it can incorporate mismatches and even unable to repair it. This will result in non-specific amplification. Furthermore, it can stably work at room temperature as well as higher temperature which can also lead to non-specific amplification.
Always prefer to do reaction on ice. At lower temperature, Taq polymerase remains inactive therefore the chance of nonspecific binding will decrease.
In addition, always prefers to add Taq polymerase at last. This will also increase the specificity of the reaction.
Aliquot the enzyme into different tubes. Taq is an enzyme, a sensitive component of biological reaction so we have to store it in cool. Store one working tube at 4°C and remaining other tubers at -20°C.
Wear gloves to avoid contamination.
It required cofactor so use MgCl2 in the PCR reaction or use ready to use PCR buffer having MgCl2 in it. Again, the concentration of MgCl2 is important as well.
Read the article role of MgCl2 in PCR reaction for more detail on the function and concentration of MgCl2 in PCR reaction.
Use Taq units as per the manufactures instruction or as per your protocol because too much of Taq will decrease the specificity of the reaction. And you will observe more bands during agarose gel electrophoresis.
Hot start PCR is the best option for high sensitive PCR reaction. If you are performing some critical PCR experiments use Hot start PCR in which the Taq specific antibody is used to block the activity of Taq polymerase, initially (during reaction preparation). Once tubes are transferred to PCR and heated at 94°C, the antibody is released and destroyed, and the Taq Polymerase is available for reaction.
This technique is very accurate and specific. Hot start PCR kits are now commercially available, so don’t worry about that.
This is all about the Taq DNA polymerase and function of Taq DNA polymerase in PCR reaction. But I have to ask something to you. The Taq has a limited activity of adding nucleotides and generated fragments of up to 1500 bp. So what are the other options for long-range PCR reaction? And what are other options of Taq available commercially?
Please do comment and let me know.
Articel written by: Tushar Chauhan
Article covered by: Tushar Kachhadiya and Ravi Parmar