Taq Polymerase Basics
What is Taq Polymerase?
Taq DNA Polymerase, or Taq polymerase, is a biological catalyst involved in the attachment of nucleotides to synthesize DNA––like any other polymerase.
A homolog of the Pol I DNA polymerase found in Escherichia coli (E.coli), Taq polymerase is an 832-amino acid protein with a molecular weight of 94 kDa (approx).
But what’s peculiar about this polymerase is that it is a thermostable enzyme that can stand high-temperature conditions, unlike most known enzymes that work properly only at 37°C (body temperature).
The optimum temperature for Taq polymerase to be most active is 70-75°C, and it shows thermal stability even at 92°C.
How and where was this enzyme found?
It was in the 1960s when Thomas D. Brook conducted an experiment in which he was sampling bacteria in the lakes and springs of the Yellowstone National Park.
Out of his observations, he discovered threads of bacteria thriving in a hot spring at above 80 degrees. This was the first-ever bacterial species found to withstand such extreme temperature conditions.
He named it Thermus aquaticus––abbreviated as “Taq”. Thermus was used for “stable at a high temperature” and aquaticus because it was found in “hot waters.”
Later in 1976, Chien et al. isolated a protein molecule from this heat-stable bacteria called Taq polymerase, specifically, Taq DNA Polymerase.
This polymerase can withstand extremely high temperatures and presents an advantage to biologists to perform DNA replication under heated conditions.
The most common application of Taq polymerase is its utilization in conducting Polymerase Chain Reaction (PCR), which will be discussed next.
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What is Polymerase Chain Reaction (PCR)?
Polymerase Chain Reaction (PCR) is a well-known molecular biology technique used to amplify specific regions of DNA.
Just how a photocopy machine might duplicate pages in a book, PCR facilitates the production of billions of copies of a single DNA segment.
Kary Mullis invented this DNA photocopy technique and revolutionized research in the field of molecular biology. Replicating DNA in vitro became possible, and researchers could now make new copies from separate DNA templates in just a few reaction cycles.
Many different types of PCR techniques are available based on which nucleic acid (RNA or DNA) has to undergo PCR amplification; the most common PCR technique is Real-Time PCR, also called quantitative PCR (qPCR).
How does it work?
To carry out in vitro amplification of DNA using PCR, a PCR machine (thermocycler) is required, along with several core ingredients. These core ingredients, or PCR reagents, include:
- Template DNA: DNA fragment to be copied.
- PCR Buffer: This is to ensure optimum conditions for the reaction. It is composed of Tris-HCl, potassium chloride (KCl), and magnesium chloride (MgCl2).
- DNA Nucleotides (dNTPs): Needed to construct the new DNA strand.
- PCR Primers: Short stretches of DNA (oligonucleotides) that initiate PCR.
- Thermostable DNA polymerase (Taq polymerase): For the extension of DNA along the template strand.
These components are then put in a sterile PCR tube in a sequential manner, and the PCR reaction is set up.
PCR is a temperature-dependent technology that involves three steps to complete one cycle of the reaction:
- Denaturation (94°C)
The template DNA is heated such that two complementary strands separate.
- Annealing (55-65°C)
The temperature is decreased to enable the attachment of primers to serve as a starting point for DNA synthesis.
- Extension (72°C)
This final step is where Taq polymerase comes into action. Once primers are attached, the Taq polymerase takes its position on the strand to produce the new strands by adding the dNTPs. This leads to the production of new complementary DNA (cDNA) strands.
The newly synthesized strands thus act as templates in the next cycle of PCR. After each cycle, the DNA doubles. The cycle is repeated about 25-35 times, taking approximately 2-4 hours to complete the entire procedure and obtain the PCR product.
The next step is to perform Agarose Gel Electrophoresis and obtain billions of copies of DNA!
Taq Polymerase in PCR: What does it do?
Taq DNA polymerase is the backbone of PCR. No Taq polymerase. No PCR.
PCR amplification works on the principle of temperature variation—heating and cooling reactions—which makes Taq polymerase a highly advantageous enzyme.
The major reason behind this is that Taq polymerase can work at high temperatures with high efficiency and amplification capacity, which other bodily enzymes cannot. It can add 150 nucleotides per second and is known to replicate a 1000 bp strand of DNA in less than 10 seconds at 72°C.
The extension stage of PCR is where the Taq DNA polymerase gets employed. It binds at the primer and DNA template junction, using it as substrate. It reads the DNA sequence and starts to add nucleotides (dNTPs) in the 5’ to 3’ direction.
Taq polymerase is optimally active at 72°C, which is why the extension step is carried out at that exact temperature.
In addition, for the proper functioning of Taq polymerase, Mg2+ is added in the form of MgCl2 to the PCR buffer. Mg2+ is a cofactor that binds to the active site of Taq polymerase and catalyzes the formation of phosphodiester bonds in order to incorporate new dNTPs, making it a mandatory requirement.
A standard PCR experiment is said to be successful when Taq polymerase is added in the presence of Mg2+.
Taq Polymerase Advantages and Disadvantages
Though the discovery of Taq polymerase has been a great benefit to scientific research, it has its disadvantages, too.
Taq polymerase can withstand high temperatures, which enables its application in performing PCR.
A typical DNA polymerase has mainly two roles to play: 5’ to 3’ exonuclease activity and a 3’ to 5’ exonuclease proofreading activity. Taq DNA polymerase lacks the power to proofread. It cannot detect and correct mismatched nucleotides, making it hard to obtain an error-free DNA strand.
At optimum temperature conditions, this polymerase effectively uses nucleotides to synthesize a new DNA strand in a complementary fashion.
As Taq polymerase is a temperature-dependent enzyme, a slight shift in the temperature can affect the polymerase activity. This can result in changes in enzyme configuration and even mismatching of nucleotides, making the polymerase less specific. It is known to add one wrong nucleotide per 9000 nucleotides.
|High Amplification Capacity|
During amplification, Taq polymerase holds the capacity to add 150 nucleotides per second.
Unlike other polymerases, Taq polymerase functions only in the 5’ to 3’ direction and does not perform excision repair in the 3’ to 5’ direction. Hence, its movement is unidirectional.
At 92°C, its half-life is more than 2 hrs, higher than any other polymerase. This makes Taq Polymerase a power tool for in vitro amplification of target DNA.
|Bivalent Cation Requirement|
An additional cofactor is needed for the effective functioning of Taq polymerase (the metal ion Mg2+). Without this bivalent cation, Taq polymerase will not be of much use.
In a nutshell, Taq polymerase shows high thermostability and processivity but low fidelity and specificity.
Fortunately, the advances in recombinant DNA technology have made it possible to overcome the above-mentioned drawbacks. There are Taq polymerases that are now commercially available that show high specificity, high fidelity, high sensitivity, and can be modified according to the PCR type.
Moreover, modifications of conventional PCR, as seen in Hot Start PCR, have changed the course of accuracy and specificity of DNA amplification, thereby reducing non-specific binding.
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PCR is an in vitro DNA amplification technique that requires a thermally stable DNA polymerase called Taq polymerase to resist inactivation during high temperatures. It’s efficient in cloning DNA segments and has become an integral part of molecular research.
With the increasing demands of PCR, a huge supply of Taq polymerase, as well as an effective PCR setup, is needed.
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