DNA polymerase helps in replicating DNA properly, in PCR, it incorporates dNTPs on the DNA strand. Taq DNA polymerase is the first choice for it.
However, the Taq DNA polymerase is not the only performer in the PCR. Other polymerases such as hot start DNA polymerase, High fidelity DNA polymerase and Phusion are some of the alternatives of Taq DNA polymerase used in the polymerase chain reaction.
Hotstar can only work at a higher temperature while the high fidelity DNA polymerase incorporates correct nucleotides at a correct position.
The high fidelity polymerase accurately replicates or amplify the DNA of our interest by incorporating the correct nucleotide as well as by removing the wrong nucleotides from the DNA.
But where to use which polymerase?
In this article, we are discussing the story of DNA polymerase and its role in “in vitro replication” aka PCR.
What is polymerase?
Now, let us start the story with polymerase itself,
The DNA polymerase is an enzyme which synthesised a long chain of the nucleic acid (RNA/DNA). The RNA polymerase manufactures the RNA while the DNA polymerase manufactures the DNA.
Here, we are talking about the polymerase in PCR, hence we will focus on the DNA polymerase only.
In the year 1956, Arther Kornberg and his coworkers discovered the DNA polymerase from E.coli.
The polymerase moves from the 3′ to 5′ direction and adds nucleotide with the help fo short single-stranded RNA (Replication). The DNA polymerase synthesises the DNA during the process of replication in vivo.
However, this DNA polymerase cannot be used in the process of PCR, why?
Because first, here we are using the DNA primers instead of RNA primers. Also, normal DNA polymerase cannot work at a higher temperature. It can only work efficiently at the lower temperature (at body temperature).
Read our article on DNA replication: General process of DNA replication
Then which polymerase we can use in the PCR?
The polymerase we are using in the PCR is the Taq DNA polymerase which can work efficiently at a higher temperature. Read more on Taq DNA polymerase: Function of taq DNA polymerase in PCR
What is Taq DAN polymerase?
In the year 1966, Thomas D Brook discovered the Taq DNA polymerase from E.coli at the Yellowstone national park, named it as Thermus aquaticus. Because the bacteria can survive at a higher temperature, the Taq DNA polymerase isolated from the bacteria can work at a higher temperature too.
After the discovery of the PCR, the Taq DNA polymerase become the first choice for the PCR reaction.
The Taq DNA polymerase is thermostable. It can work smartly even at a temperature of more than 90°C.
Further, it can incorporate dNTPs very efficiently even after so many incubations and that is the reason the Taq DNA polymerase is useful and a smarter choice for front end PCR experiments.
Taq DNA polymerase is the first choice for any PCR reaction, still, it has several limitations.
The higher activity of the Taq is reported near 70°C and it also works at a temperature of more than 90°C or 100°C. Interestingly, The Taq can be work very preciously at a lower temperature too.
Yes, believe me, even at a temperature between 20°C to 40°C it has notable activity. Now, this is something very serious.
Why it is a problem?
Because while preparing the reaction, the Taq actively involved in the polymerisation, it adds a random nucleotide to any of the available DNA fragment whether it is primer or DNA.
The results are dirty, so many, nonconclusive DNA bands with primer-dimers appear in the agarose gel electrophoresis.
Don’t blame Taq for that.
Even if it is a superior choice in the conventional PCR, it has another major drawback.
The Taq DNA polymerase does not have the proofreading activity, hence the error rate of noncomplementary base pairing is reported nearly 1.1 x 10-4 per base pair per amplification.
For very basic and conventional PCR experiments, the use of the Taq is an OK decision but for some advance and sensitive experiments in which the correct base pairing matters a lot, Taq cannot work.
The experiments such as SNP genotyping, Cloning and NGS based PCR protocols, accuracy in the dNTP addition is crucial.
So it is sad to say that we have to let go of our Taq DNA polymerase.
Now, what are the enzymes used instead of Taq? and How it works?
High fidelity DNA polymerase helps to overcome this problem.
Before praising the high fidelity DNA polymerase one other class of polymerase is as important as the high fidelity DNA polymerase, the “hot-start DNA polymerase.”
Remember the first limitation of Taq? it can work even at a lower temperature, this problem can be solved by using the host start DNA polymerase.
Read further,
- Site-Directed Mutagenesis: Methods and Applications
- A Complete Guide of the Polymerase Chain Reaction
Hot start DNA polymerase:
It actually starts at hot. Once the PCR reaction entered into the heating step, this DNA polymerase is activated.
Different techniques are available for performing hot-start PCR, but among them all, the use of the hot-start DNA polymerase is a good decision.
Here, the DNA polymerase is an antibody linked. The antibody linked with the enzyme makes it inactive during the reaction preparation but once the reaction enters into the heating step, the antibodies are degraded and the polymerase is directly inserted into the reaction.
The DNA polymerase is not available during the reaction preparation, therefore the chance of non-specific banding is reduced.
In another approach, the highly specific oligonucleotides are attached with the Taq DNA polymerase, once the reaction is heated, the oligomers are released and the Taq can be available for the amplification.
Wax beads are also used for the hot-start PCR reaction. In this method, wax beads are used as a barrier for Taq DNA polymerase.
The detailed explanation of each method is described in our previous article: What is hot start PCR?
Conclusively, we can say that the hot-start DNA polymerase can facilitate accurate amplification.
The main advantage of hot-start DNA polymerase is its specificity and the sensitivity which is ideal for the diagnostic as well as research.
It also amplifies the low abundant DNA templates.
Now, the first problem of the Taq DNA polymerase is solved by using the hot-start DNA polymerase.
Nonetheless, the addition of the wrong dNTPs during the amplification still persist by using the hot-start DNA polymerase. Even this problem can be solved by using another polymerase.
Due to the exonuclease activity present in the high fidelity DNA, the chance of incorporation of the wrong dNTPs can be eliminated.
Note: The hot-start DNA polymerase cannot be applicable for the longer PCR or long-range PCR and for DNA with higher GC content and repetitive DNA sequences.
High fidelity DNA polymerase:
The base pairing between G:C and A:T or C:G and T:A is called Watson and Crick base pairing. The main objective of any replication process (whether it is in vivo or in vitro) is to insert correct nucleotide to the growing DNA strand.
The fidelity of DNA polymerase is the accurate replication or amplification of template DNA. This types of DNA polymerase has polymerase as well as proofreading activity.
The high fidelity DNA polymerase completes the amplification process in the following steps:
- Reading the template DNA.
- Select the appropriate nucleoside triphosphate to place it on the DNA.
- Incorporates the correct nucleotide triphosphate to the 3′ end of the primer.
- Proofread the growing DNA strand from 5′ to 3′ direction.
- Removes the wrong nucleotide monophosphate from the DNA by sliding back.
The rate of the incorrect nucleotide addition is very low in high fidelity DNA polymerase and if an incorrect nucleotide is inserted, the polymerase moves back, identify the wrong dNTP and remove it by correct nucleotide.
This types of activity of DNA polymerase is referred to as exonuclease proofreading activity. See the figure below,
However, the cost of the high fidelity DNA polymerase is more as compared with normal Taq DNA polymerase.
So the question arises in mind that in which experiments we can use the high fidelity DNA polymerase?
In the FAQs section of this article, we will answer this question along with three other questions,
When to use Taq DNA polymerase?
When to use hot-start DNA polymerase?
When to use high fidelity DNA polymerase?
Now, continue the discussion on the high fidelity DNA polymerase.
Pfu polymerase, Phusion polymerase and Q5 polymerase are some of the examples of commercially available high fidelity DNA polymerase.
Pfu polymerase:
The Pfu polymerase is isolated from a species of Archaea called pyrococcus furiosus. This type of Archaea can live at extremely high temperature hence called as extremophilic.
The Pfu contains both the polymerase as well as proofreading activity. Therefore it is the best choice for cloning and DNA sequencing.
On the downside, the speed of the polymerisation is very low by using the Pfu so instead of Pfu only, it is used in combination of Taq DNA polymerase.
The combination increases the speed and accuracy of DNA synthesis. The Taq synthesise DNA faster, and the Pfu removes the misincorporated dNTPs if so.
Pfu is one of the best polymerases for the amplification of shorter DNA for DNA sequencing.
Phusion DNA polymerase:
The Phusion DNA polymerase is supplied by TermoFisher and New England Biolabs. It has 50 times more fidelity than the normal Taq.
Interestingly, if we follow the standard protocol of the company, minimum optimisation is required to achieve amplification.
By using the Phusion, the yield of the PCR reaction is increased using less enzyme.
With the Proofreading perspective, the exonuclease activity rate of Phusion is higher than the Pfu with 6 fold lower error rate.
It is applicable in PCR for DNA sequencing, cloning, amplification of GC-rich templates, long-range PCR, in vitro mutagenesis and DNA microarray.
The Phusion DNA polymerase is also available in the hot start version, in which it is supplied along with one other DNA polymerase.
Q5 DNA polymerase:
The Q5 DNA polymerase is the registered product of the New England Biolabs. This polymerase is the best option available in recent days.
The amplification rate of the Q5 DNA polymerase is ~280 times more than the normal Taq DNA polymerase.
The fidelity of Q5 is very high and the error rate is ultra-low.
The Q5 is ultra-fast, reliable, accurate and has a broader range of amplification. It can even amplify the high GC rich regions efficiently.
The robust amplification rate is supported by the processivity-enhancing Sso7d domain. The Q5 has 3′ to 5′ exonuclease activity.
They said “fidelity at its finest” about the Q5 DNA polymerase.
The Q5 DNA polymerase is also available in the hot start version, in which it is supplied along with one other DNA polymerase which increases the specificity of the reaction by reducing the non-specific amplification and primer-dimer formation.
How to select DNA polymerase?
Several points in addition to this explanation are important while selecting the DNA polymerase such as length of the amplicon, type of PCR reaction, required specificity, Fidelity and required sensitivity.
Amplicon length: If our PCR product is too long, the standard Taq DNA polymerase might not work efficiently. For that, we need a high fidelity DNA polymerase.
Types of PCR reaction: PCR reaction is one of the important factors, depending upon the types of PCR the DNA polymerase is selected.
If the reaction is simple PCR we can use Taq otherwise, for longer PCR, high GC template DNA and multiplex PCR use only high fidelity DNA polymerase or hot-start DNA polymerase.
Required specificity: Some PCR reactions are performed just for confirming the amplification in which fidelity is not required. Successful amplification can be achieved in this type of reaction by using only Taq DNA polymerase or hot-start DNA polymerase.
Fidelity: Higher fidelity and precision is required for cloning, sequencing and microarray. So for this assays use only high fidelity DNA polymerase (no other option).
Required sensitivity: Sensitivity is one of the crucial factors for diagnostic PCR, hence use only hot-start DNA polymerase during PCR for diagnostic purposes.
If you are OK with so many DNA bands, you can even use Taq DNA polymerase to reduce the cost of the experiment.
Quick FAQs:
What is polymerase?
The polymerase is an enzyme which synthesizes the nucleic acid (DNA/RNA).
What is DNA polymerase?
The polymerase which synthesizes particularly the DNA is called a DNA polymerase.
What is Taq DNA polymerase and when to use it?
The Taq DNA polymerase is a thermostable DNA polymerase which incorporates dNTPs even at a higher temperature during the PCR amplification.
The Taq DNA polymerase is used in the simple or conventional PCR with shorter DNA templates in which precision is not required.
What is a hot-start DNA polymerase and when to use it?
The hot-start DNA polymerase is used to increase the specificity of the PCR reaction by remains inactive at a lower temperature.
The hot-start DNA polymerase is used to increase the specificity of the reaction. It is used in the amplification of DNA for sequencing.
What is high fidelity DNA polymerase and when to use it?
The high fidelity DNA polymerase is a type of DNA polymerase having polymerase as well as exonuclease activity.
It removes wrong nucleotides from the growing DNA strand. It is used in DNA sequencing, cloning, DNA microarray and site-directed mutagenesis.
Additional resource related to this topic:
Conclusion:
Specificity and the sensitivity of the PCR reaction matters a lot for downstream applications. Always prefer to use hot-start DNA polymerase, for sequencing using one of the high fidelity DNA polymerase enlisted above.
For standard protocols and simple PCR reaction, Taq DNA polymerase minimises the cost with high amplification rate.
Choosing right DNA polymerase can minimise the difficulty in the reaction, However, which types of DNA polymerase to be used in PCR reaction is depends on us and our experiment.
