Taq Polymerase – Definition, Role, and Limitations of Taq Polymerase
Taq polymerase enzyme is an important DNA polymerase enzyme that is isolated from the bacterium Thermus aquaticus. In molecular biology laboratories, it is mandatory to use the most abundant and cheapest polymerase that can be used to perform Polymerase Chain Reaction (PCR). The polymerase is an enzyme that is capable to generate a complementary strand from a template strand by adding dNTPs (deoxyribonucleotide triphosphate), nitrogenous bases.
What is Taq Polymerase?
- Taq polymerase is a thermostable DNA polymerase enzyme that is used in PCR for the synthesis of DNA.
- In 1968 Dr. Thomas D. Brock isolated the Taq polymerase enzyme from bacteria and in the early 1980s, Kary Mullis used this enzyme in a polymerase chain reaction.
- It is isolated from the bacterium Thermus aquaticus.
- The natural habitat of the Thermus aquaticus is hot thermal water temperatures ranging up to 70-75°C.
- The weight of the Taq polymerase enzyme is near about 94 kD.
- Taq DNA polymerase is a DNA-dependent DNA polymerase, which is homologous to DNA polymerase 1 in E. coli.
- Therefore we can say Taq polymerase is composed of the 3′-OH nucleotide addition site, the dNTP / DNA binding site, and the 5 ‘to 3’ exonuclease site.
- However, it lacks a 3 ‘to 5’ exonuclease domain for screening incoming nucleotides.
- Taq Polymerase enzyme is capable to generate a complementary strand from a template strand by adding dNTPs (deoxyribonucleotide triphosphate), nitrogenous bases.
History of Taq polymerase:
- In 1968 Dr. Thomas D. Brock isolated this enzyme from bacteria and in early 1980 by Kary Mullis working with DNA oligonucleotides began using Escherichia coli DNA polymerases to replicate or sequence DNA.
- The problem is that the strands once copied had to be separated by putting them at more than 90ºC to allow them to be copied again.
- These temperatures denatured enzymes and stopped working.
- The incorporation of the Thermus aquaticus polymerase was essential to automate the reaction since this polymerase could perfectly withstand temperatures greater than 90ºC.
- In 1993 Kary Mullis received the Nobel Prize in chemistry “for his invention of the polymerase chain reaction method. “
What is Thermus aquaticus?
- Thermus aquaticus is a thermophilic gram-negative bacteria.
- The nature of the Thermus aquaticus is aerobic and heterotrophic.
- This bacterium is generally found in hot springs.
- This bacterium lives at temperatures between 50 and 80ºC.
- It was described by Thomas D. Brock in 1969 from waters collected from a spring in Yellowstone.
- In non-thermophilic living beings, proteins tend to become denatured, and destructured, at such high temperatures.
- The structure of proteins is given by their folding, which is maintained by electromagnetic forces between their amino acids.
- As the temperature increases, these forces weaken and the protein unfolds. However, the proteins of thermophilic beings, such as Thermus aquaticus, have a conformation especially adapted to these temperatures, so they do not unfold and allow them to live in these conditions that are impossible for others.
What is the role of Taq polymerase in PCR?
- The main role of Taq Polymerase is an enzyme used in the polymerase chain reaction (PCR) to synthesize DNA in vitro and the Taq polymerase enzyme is analogous to E.coli DNA polymerase 1.
- Before the discovery of Taq polymerase the Klenow fragment of DNA-dependent DNA polymerase 1 is used for the first time in PCR reaction. which is not a thermostable enzyme due to the enzyme is denatured during the PCR process.
- The Klenow fragment lacked the 5 ‘to 3’ exonuclease activity. The optimal temperature of the Klenow fragment is 37°C. Therefore, a new enzyme had to be added to the PCR reaction in every cycle.
- The remaining Klenow fragment was denatured during the PCR denaturation step that occurs at 95°C. Furthermore, the Klenow fragment could only synthesize fragments of less than 400 bp.
- In 1968 Dr.Thomas D. Brock isolated the Taq DNA polymerase enzyme from bacteria and in the early 1980s, Kary Mullis was used in a polymerase chain reaction.
- This Taq DNA polymerase Enzyme performs an enzymatic activity is 75-80 °C. Once it reaches its optimum temperature, it becomes fully functional and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C which is much more than the Klenow fragment.
- The error rate of Taq polymerase is also lower than the Klenow fragment approximately one in every million base pairs.
- Pfu is another type of thermostable DNA polymerase used in place of Taq polymerase in PCR. It can correct tests. Therefore, it is more accurate than Taq.
- Taq polymerase’s essential characteristic is its 5 ‘-> 3’ polymerase activity, adding nitrogenous bases in that sense to the DNA strand.
- Although the Taq polymerase was isolated from Thermus aquaticus and produced in laboratories from E.coli bacteria by inserting the Taq polymerase gene. These bacteria grow in conditions much easier to obtain, for what is saved in its production.
- The advantages of the Taq DNA polymerase enzyme are that it does not denature at high temperatures, is inexpensive, and synthesizes DNA very quickly.
What are the limitations of Taq polymerase?
Despite the enormous application of the Taq polymerase enzyme in PCR reactions, this enzyme still has some limitations.
● Low Accuracy: The most important limitation of Taq polymerase is that it’s not the most accurate polymerase enzyme. Taq has an error rate of around one error per 100,000 base pairs as compared to the Pfu polymerase enzyme which has lower accuracy.
● Lack of Proofreading Activity: The disadvantage is that Taq lacks a proofreading activity, so it has a very high mutation rate. The Pfu polymerase enzyme which has proofreading activity is about 10 times more accurate than the Taq polymerase enzyme. If you’re using PCR upstream of cloning or DNA sequencing, that difference in error rate can be very significant.
● Low specificity: Taq DNA polymerase has a lower specificity than the normal ones. Taq polymerase can amplify DNA sequences longer than about 1 kB. Relative to other types of polymerases, Mismatches nucleotides could be added to the sequence by Taq polymerase.
● Low fidelity: Taq polymerase does not have 3’ to 5’ exonuclease proofreading activity, therefore mismatched nucleotides could not be corrected.
● Monovalent / Bivalent cation requirement: Taq polymerase requires cofactors, such as Mg2+ ion, to work properly. The activity of the enzyme is dependent on divalent cations, such as Mg2+. The optimum concentration of Mg2+ is 2 mM. Monovalent cations also have an effect on the activity of Taq Polymerase. The monovalent cation is K+ in the form of KCl when it is used with the optimum concentration 50 mM, when KCl concentration is more than 75 mM it can inhibit the activity of Taq polymerase. Another monovalent cation, such as NaCl, NH4Cl, and NH4 acetate, cannot substitute KCl without a decrease in a specific activity.
Frequently Asked Questions about Taq Polymerase
1. Why is Taq polymerase thermostable?
Answer: The bacteria Thermus aquaticus which produces the Taq polymerase enzyme occurs naturally in thermophilic hot springs. It lives in environments which has extreme temperatures. Due to that Taq polymerase is highly thermostable. Bacterium Thermus aquaticus is first isolated from the effervescent hot springs of Yellowstone National Park in the United States.
2. Why is Taq polymerase preferred in PCR?
Answer: The advantages of the Taq DNA polymerase enzyme is that it does not denature at high temperatures, is inexpensive, and synthesizes DNA very quickly, Taq polymerase’s essential characteristic is its 5 ‘-> 3’ polymerase activity that’s why Taq polymerase is preferred in PCR.
3. Why is Taq polymerase used in PCR rather than other DNA polymerases?
Answer: Same as FAQ No. 2
4. What does Taq stand for?
Answer: Thermostable DNA polymerase (Taq polymerase): For the extension of DNA along the template strand.
5. What is Taq in Taq polymerase?
Answer: Taq in Taq polymerase is Thermus aquaticus which is a thermophilic gram-negative bacteria, aerobic and heterotrophic in nature, and found in hot springs.