“The function of Taq DNA polymerase in PCR is to amplify or synthesise DNA or gene of interest for various downstream application. It’s a type of thermostable DNA polymerase, can work at higher temperature as well.”

The present article is focused on the function and importance of Taq DNA polymerase and how it amplifies the DNA. In addition to this, the advantages and disadvantages of it are also included.

Let’s start with the basics,

What is DNA polymerase?

DNA polymerases are the class of enzymes function to synthesise DNA during DNA replication. Replication is a process of synthesising or copying DNA. It forms two different daughter DNA using DNA polymerase.

The DNA polymerase is an important biological molecule of the central dogma specifically, in 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).

We have covered an amazing article on DNA polymerase. Read it here: Multifunctional DNA polymerase.

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 mismatched nucleotide and reinsert the exact nucleotide match. The mechanism is called base excision repair.

All enzymes work properly at body temperature which is 37°C. so DNA polymerase’s highest activity is at 37°C, however, as temperature increase, the activity decreases. Here, a question immediately strikes in mind that how 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.

The polymerase can’t be used in PCR because it can not work at a higher temperature, however, a specialised DNA polymerase known as Taq DNA polymerase helps to overcome the present problem.

In 1966, during one summer, at Yellowstone national park in the USA, Thomas D Brook discovered a bacteria. 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 was found in hot water- name aquaticus had given to it. It is called as Thermous aquatiqus, “A hot water bacteria.”

Later on, in the year 1976, Chien et al., isolated polymerase from Thermus aquaticus and named it as Taq DNA polymerase. Now, this is an enzyme which can solve the problem for PCR. The reason behind its use in PCR is that stability of the enzyme at a higher temperature.

Several advantages and disadvantages of it are enlisted below,

Advantages of Taq DNA polymerase:

Obviously, Thermostable : the enzyme is thermostable polymerase hence it can even work at a higher temperature

Efficiency : It is highly efficient. As it is reached at its optimum temperature, the thermostable polymerase becomes fully functional and adds nucleotides to the growing DNA strand.

Hight amplification capacity : It can insert 150 nucleotides per seconds during amplification.

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 an Mg 2+ 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.

Read our article on it: Choosing the right DNA polymerase for your PCR experiment.

Comparison of DNA polymerase and Taq DNA polymerase:

Properties DNA polymerase Taq DNA polymerase Activity 5’ to 3’ polymerase activity and 3’ to 5, proofreading activity 5’ to 3’ polymerase activity Optimum temperature 37°C 72°C Thermostability Stable at 37°C Stable at 92°C Fidelity Higher Low Additional cofactor Not require Require Applicability In vivo In vitro

My ultimate guide for using Taq DNA polymerase

The commercially available Taq DNA polymerase is stable at room temperature as well as higher temperature. The issue with using the Taq is its specificity. It incorporates mismatch during amplification and has no power to repair it. This is the reason, the chances of non-specific bindings are very high using the present game-changer.

Interestingly, the Taq DNA polymerase remains inactive at a lower temperature. Thus to avoid unnecessary amplification, prepare your PCR reaction on ice.

Always, prefer to add Taq DNA polymerase at the end of the reaction, after the addition of all ingredients. As it reaches in reaction at the end, the chance of early amplification decreases.

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.

The Taq is highly demanding!

It requires some of the cofactors such as MgCl 2 for performing well during the reaction. Although the ready to use PCR buffer already has MgCl 2 in it, still, adding some amount of it can increase amplification.

Read our article on how to use MgCl 2 in PCR: Role of MgCl2 in PCR reaction

Remember! too much Taq decreases reaction specificity, you will get more bands in agarose gel. Thus always use Taq as per manufactures instruction.

Hot start PCR is the best option for the 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.

Conclusion:

This is all about the Taq DNA polymerase and function of it in PCR reaction. But I have to ask something to you. The Taq has limited activity, it can add nucleotides up to 1500bps So what are the option to perform the long range PCR? Is there any other alternative of Taq commercially available?

Please do comment and let me know.

Articel written by: Dr Tushar Chauhan

Article covered by: Tushar Kachhadiya and Ravi Parmar