“Countless commercially available DNA polymerases make the choice difficult. Here is the guide on how to select a DNA Polymerase for your genetic experiment.”
The function of a DNA polymerase is to synthesize a new DNA strand. We know about it as it’s an integral part of our replication system. It uses a short RNA primer and with the help of various enzymes, it prepares a new DNA strand.
For genetic experiments, we need millions of DNA copies. DNA polymerase again helps us here in our experiment as well. For example, we use Taq DNA polymerase in PCR to amplify the DNA.
The assay requirements differ among various experiments, for instance— some need speed, some precision, and some proofreading while some all of these. Common Genetic experiments in which DNA polymerase is used are PCR, DNA sequencing, cloning, mutagenesis studies and other in vitro studies.
All these experiments need a separate type of DNA polymerase. However, a few known and popular ones are Taq, hot-start, Pfu, T7, Q5, Phusion, Exo(-), Vent and Deep Vent DNA polymerases, and Klenow fragment, etc.
Each DNA polymerase has its own advantages and limitations. Then how to choose one? In this article, I will provide a complete guide using which you can choose a DNA polymerase for your experiment. I will also explain each DNA polymerase in detail so that you can understand its importance.
Stay tuned.
Disclaimer: Information provided here is collected from peer-reviewed resources and re-presented in an understandable language. All the sources are enlisted at the end of the article.
Key Topics:
Commercially Available DNA Polymerase:
In 1956, Arther Kornberg and his co-workers discovered the DNA polymerase from E. coli. DNA Polymerase is a class of polymerase enzymes and has a function to synthesize the DNA. It also has proofreading activities.
Put simply, it synthesizes the DNA using the single-stranded template and if it finds any mismatch, immediately repairs it with the proofreading activity, if needed.
Taq, hot-start, Pfu, T7, Q5, Phusion, Exo(-), Vent and Deep Vent DNA polymerases, and Klenow fragments are 10 common and commercially available DNA polymerases. I am going to explain each DNA polymerase, its source, fidelity, activity, applications and importance.
Taq DNA Polymerase:
Taq DNA polymerase is widely used in the field of molecular biology and is the most popular. Its discovery revolutionized the field of genetics as it enabled us to synthesize DNA in vitro.
Source: In 1966, Thomas D Brook isolated a heat-stable Taq DNA polymerase from Thermus Aquaticus bacteria. The hot water bacteria was derived from the hot spring at Yellowstone National Park.
Activity: Taq DNA polymerase has polymerization activity only. It can work efficiently at higher temperatures between 90 to 95ºC. However, as the temperature increases above the maximum activity zone, the activity decreases.
Fidelity: Taq DNA polymerase usually lacks exonuclease activity (3’ to 5’) and thus has a higher error rate. Notedly, the error rate of Taq DNA polymerase is 1.1 x 10-4 per base pair per amplification.
Applications: As we don’t require proofreading in conventional PCR, Taq DNA polymerase is widely used in conventional and other PCR experiments. It is further used for routine genotyping and DNA sequencing experiments only to amplify the DNA.
Hot start DNA Polymerase:
Hot start DNA polymerase is not a separate type of polymerase, indeed. It’s a modified DNA polymerase (usually Taq DNA polymerase) that can only activate upon heating.
Source: Hot start DNA polymerase is an artificially modified DNA polymerase, for example, if it’s Taq DNA polymerase, it is derived from Thermus Aquaticus.
Activity: It’s linked with a ligand, other enzyme or molecule that allows its activity only during the heating step. Thus an early enzymatic activity can be avoided thereby, the chances of non-specific amplifications can be reduced.
Fidelity: Hot start DNA polymerase is also available in high-fidelity form as well.
Applications: As it increases the precision and sensitivity of the reaction, it is widely used in clinical diagnosis, high-throughput experiments and other research modules in which the specificity and sensitivity matter.
Related article: Polymerase Chain Reaction (PCR)- Definition, Principle, Steps, Procedure, Protocol, Applications and Types.
Q5 DNA Polymerase:
The Q5 DNA polymerase is the registered product of the New England Biolabs. It has polymerase and proofreading activities. The amplification rate of the Q5 DNA polymerase is ~280 times more than the normal Taq DNA polymerase.
Source: Q5 DNA polymerase is usually derived from the natural bacteria, however, is modified to boost its fidelity and polymerization power.
Activity: Q5 DNA polymerase synthesizes the DNA as well as proofreads the newly synthesized DNA strand. It’s an ultra-accurate enzyme. And thus, is the best choice for experiments. It performs polymerization at a high speed.
Fidelity: It has an ultra-high fidelity rate. It carries 3’ to 5’ exonuclease activity. The company suggests having an ultra-low error rate.
Applications: experiments that need minimal error rate use Q5 DNA polymerase. It’s preliminarily used in DNA sequencing, artificial mutagenesis and genetic engineering.
Related article: DNA Sequencing: History, Steps, Methods, Applications and Limitations.
Pfu DNA polymerase:
The present enzyme is a high-fidelity DNA polymerase widely used in various genetic modules. The name ‘pfu’ is derived from where it’s isolated.
Source: The Pfu polymerase is isolated from a species of extremophilic Archaea called Pyrococcus furiosus. It can live at an extremely high temperature.
Activity: It possesses polymerization and proofreading activities. Note that the polymerization rate of Pfu is lower than the Taq DNA polymerase and thus it’s often used in combination with the Taq DNA polymerase.
Fidelity: Pfu DNA polymerase has 3’ to 5’ exonuclease activity. Meaning it can correct the nucleotide errors during the polymerization. Notedly, the present enzyme is known for its ultra-high fidelity and very low error rate.
Applications: The main application of a pfu enzyme is in the sequencing of short DNA fragments accurately. It’s also used for difficult PCR DNA templates oftentimes.
Phusion DNA polymerase:
The Phusion DNA polymerase is supplied by TermoFisher and New England Biolabs. It has 50 times more fidelity than the normal DNA polymerase. Minimal optimization steps are required to use it.
Source: Phusion DNA polymerase is not derived from any natural bacteria. It’s an artificially synthesized enzyme, prepared using genetic engineering. It’s prepared to achieve very high fidelity.
Activity: It does have polymerization and proofreading activity with a very high fidelity rate.
Fidelity: It has a 3’ to 5’ exonuclease (proofreading) activity.
Applications: The present synthetic enzyme has crucial applications in high-fidelity genotyping, library preparation for NGS and long-range PCR amplification. As long-range PCR reactions are prone to error, the Phusion enzyme is commonly used here.
T7 DNA Polymerase:
T7 DNA polymerase is derived from the phage T7. it’s a DNA-dependent DNA polymerase and is widely used in genetic research.
Source: T7 DNA polymerase is derived from the E. coli invader bacteriophage T7. It’s a high-fidelity enzyme derived from a viral source.
Activity: it has both polymerization and proofreading activity.
Fidelity: T7 DNA polymerase is a high-fidelity DNA polymerase.
Applications: The present enzyme has crucial and diverse applications including DNA labeling and sequencing. It is also used in in vitro transcription studies to prepare RNA from the DNA template.
Whenever DNA labeling is required, T7 DNA polymerase is the first choice.
Related article: Site-Directed Mutagenesis: Methods and Applications.
Deep Vent DNA Polymerase:
Deep Vent DNA polymerase is another product from the New England Biolabs. It is a thermophilic DNA polymerase that works efficiently at various higher temperatures.
Source: The present enzyme is derived from the thermophilic archaea— Pyrococcus.
Activity: DNA polymerization and proofreading (exonuclease) activity.
Fidelity: It’s a high-fidelity enzyme with a 3’ to 5’ proofreading activity.
Applications: Deep Vent is used particularly in DNA sequencing, reverse transcription and amplification from environmental samples.
Vent DNA Polymerase:
Vent DNA polymerase is yet another thermophilic enzyme derived from the bacteria Thermococcus litoalis. It’s a high-fidelity DNA polymerase that contains polymerization and proofreading activities.
Applications: The present enzyme is used for RNA quantification (RT-PCR) and isothermal amplification.
Klenow Fragment:
A Klenow fragment is a modified form of the DNA polymerase designed for end-labeling experiments.
Source: A Klenow fragment is isolated from the E coli DNA polymerase I.
Activity: It possesses both polymerization and proofreading activities.
Fidelity: It has a low error rate and high-fidelity DNA polymerase.
Applications: The present enzyme is commonly used for DNA probe labeling, DNA probe end-labeling, primer extension reaction and Sanger sequencing.
Related article: Gene cloning: Definitions, Steps, Procedure, Applications and Limitations.
Exo(-) DNA Polymerase:
The present polymerase is genetically engineered DNA polymerase. It contains an inactive exonuclease domain for some critical genetic experiments.
Source: it is derived from a natural source. However, here one exonuclease domain is inactivated and not removed.
Activity: DNA polymerization only.
Fidelity: lacks a proofreading function.
Applications: The present enzyme is applied for studies like second-strand cDNA synthesis, random-primed labeled reaction and mutagenesis studies.
How to select a DNA polymerase for your experiment?
Now, it’s difficult to discuss every genetic experiment and suggest the possible DNA polymerase. Instead, I am giving you a summary. Check out the applications and select in which application category your experiment falls.
Based on that you can choose your DNA polymerase. Keep in mind that, you should have that Taq, vent and exo(-) lack a proofreading activity so if you need a proofreading activity, avoid these options.
| Enzyme (DNA polymerase) | Source | Activities | Applications |
| Taq | Thermus Aquaticus | Polymerization | Conventional PCR, genotyping and sequencing. |
| Hot start | Genetically engineered | Polymerization and proofreading | High throughput, multiplex and diagnostic PCR experiments. |
| Q5 | Derived from natural bacteria but artificially modified | Polymerization and proofreading | High-fidelity PCR, DNA sequencing and mutagenesis studies. |
| Pfu | Pyrococcus furiosus | Polymerization and proofreading | Cloning, sequencing and difficult DNA templates. |
| Phusion | Artificially modified | Polymerization and proofreading | NGS-library preparation, long-range and high-fidelity PCR. |
| T7 | Bacteriophage T7 | Polymerization and proofreading | Sanger sequencing, and synthesizing RNA from the DNA (in vitro transcription) |
| Deep Vent | Pyrococcus | Polymerization and proofreading | PCR for sequencing, Reverse transcription and PCR for environmental samples. |
| Vent | Thermococcus litoralis | Polymerization | Isothermal amplification and RNA quantification. |
| Klenow | Derived naturally | Polymerization and proofreading | DNA labeling and DNA probe end-labeling, primer extension and Sanger sequencing. |
| Exo(-) | Derived naturally modified to inactivate exonuclease | Polymerization | Random primed labeling, second strand cDNA synthesis and mutagenesis studies. |
Wrapping up:
DNA polymerase is an important enzyme for various molecular genetic experiments. In addition, it’s usually found in almost all prokaryotes and eukaryotes. DNA polymerase has an important role in DNA synthesis. And by doing so our genetic code remains intact.
These DNA polymerases that we have discussed are available commercially and create confusion. I have given a brief guide. I hope it helps you.
Sources:
DNA Polymerase Selection Chart from New England Biolabs.
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