What Is Taq Polymerase And Primers In PCR

Taq polymerase and primers are the main constituents of polymerase chain reactions.  Without the involvement of both,  the PCR cycles cannot be executed.  Taq polymerase is called the thermostable enzyme that can thrive in elevated temperatures also. While primers are the sequences that are complementary to the strand of the target DNA.  In PCR, the primers are called single strands of DNA sequences. The taq polymerase is derived from the eubacterial microorganism while the primers are the DNA sequences used to amplify DNA sequences.

The Taq Polymerase

It is also known as the thermostable DNA polymerase I  which is named after the thermophilic eubacterial microorganism Thermes aquaticus. This microorganism accounts for the name of the taq polymerase enzyme.  It was originally extracted by Chien et al in 1976.  To some extent, the enzyme is called the taq or taq pol.  The main purpose of the taq polymerase is to amplify the number of short segments of DNA in the cycles. Here, the amplification processes with the use of taq polymerase enzyme.

Thermes aquaticus -  The Enzyme

The enzyme is found in hot springs and hydrothermal vents where the temperature is very high.  Due to the high temperature, the enzyme has kindled a high resistive power to the high temperatures. And because of the high thriving power of Thermes aqauticus,  it is used in the polymerase chain reaction in the step of denaturation of DNA or deoxyribonucleic acid.   At this stage of polymerase chain reaction, high-quality protein denaturation occurs which require high temperature. So,  the enzyme Thermes aquaticus is required for this purpose. E. coli also gives out a polymerase enzyme of the same nature for the purpose. 

The Enzymatic Properties Of Taq Polymerase

Following are the enzymatic properties of taq polymerase :

• The usual optimum temperature for the activity for the taq polymerase is 75-80⁰ C.  Taq polymerase also has a half-life greater than 2 hours at a temperature of 92.5⁰ C and so on. 
• When the reaction mixture reaches 75 to 80 ⁰ C, the taq polymerase starts to polymerize at a high speed. Here, the rate of polymerization is about 150 nucleotides per second per taq polymerase.  The DNA replication also gets doubled in the reaction with the taq polymerase enzyme.
• It is seen that a single taq polymerase enzyme can synthesize about 60 nucleotides per second at the temperature range of 60⁰ C and so on.
• At a temperature higher than 90 ⁰ C, the taq polymerase enzyme becomes inactive marking the inhibition of the enzyme. However, the enzyme does not denature itself. It remains intact in the reaction mixture of PCR
• In the reaction vessel,  the presence of several ions does have some effect on the specific activity of the enzyme.  The ions affect the reactivity of the taq polymerase enzyme. For example, the small amounts of potassium chloride(KCl) and magnesium ions do affect enzyme activity.
• To make the enzyme more active in the PCR cycles,  50mM KCl is appropriate.  But the high amounts of KCl and Mg substrates can inhibit the enzymatic activity of the taq polymerase enzyme. 
• To some extent, the concentration of nucleoside triphosphates (dNTPs)   also decides the amounts of taq polymerase needed in the PCR for extending cycles. 

Taq Polymerase In Polymerase Chain Reaction

In a polymerase chain reaction, each round of DNA replication mixture needs to be heated above a high temperature.  At this temperature, denaturation happens which unfolds the DNA of the target. Here, the newly formed strands of DNA get separated from each other and again act as a template for the next round of gene amplification. At the heating stage of the polymerase chain reaction, the taq polymerase enzyme is used as the enzyme is well suited for thriving at high temperatures.  The enzyme helps in the denaturation of DNA strands without hampering the DNA strands and DNA sequencing.

The use of thermostable enzymes- Thermes aquaticus helps in running the PCR at a high temperature  (60⁰ C or above). With this, the enzyme also facilitates the high specificity of the primers in the process.  In a composite sense, the themes aquaticus enzyme reduces the chances of non-specific products such as primer dimer.

Advantages of Taq Polymerase Enzyme In PCR

1. It helps in running the polymerase chain reaction cycles at god number of times
2. The high temperature of the cycle also helps to give out the required amount of gene amplification in the process.
3. The use of taq polymerase facilitates the high specificity of the primers used in the subsequent steps in the PCR.
4. The thermostable enzyme also discards the addition of any new enzymes in the further process of PCR.
5. Helps in getting a good amount of DNA analysis facts.
6. Taq polymerase also acts as a plasmid vector in the PCR thermal cyclers. 

Primers In PCR

Primers are called small sets of nucleotides made up of DNA.  They are typically 18 to 24 base pairs that flank the target DNA. In PCR,  a primer is used for targeting a locus of genes for the amplification process.  This leads to further DNA analysis in the extension of the DNA molecule.

In a polymerase chain reaction,  custom primers are used to direct DNA elongation. Here, the elongation happens towards opposite poles of sequences of amplified DNA.  The primers are short and must encode for the specific upstream and downstream sequences in the DNA.  The use of primers is done in the polymerase chain reaction to amplify a maximum number of genes at a given time. To get the right amplified DNA or deoxyribonucleic acid sequences, the primer design is done. The pairs of primers used here should have similar melting temperatures. In the annealing stage, the primers are set to action that earns the PCR cycle, the amplified DNA.

The primer melting temperature should not be too high. As in higher temperatures, the primers mishybridze and generate bad copies of the DNA.  It can also leads to misrepresentation of the target DNA copies.  The annealing temperature has to be a little low for the primers to act on the target DNA effectively.

The primer design can also take place in a polymerase chain reaction, as they are necessary for the reaction cycles. With the scientific advancements,  the primer designing can also be done through an electronic medium, through the screen.  While designing primers for the PCR, the additional nucleotides bases are added to the sequences of primers. This results in a customized cap sequence of primers at each end of the amplified DNA end.  

Degenerate Primers

Some of the processes of PCR call for the use of degenerate primers.  These types of primers are a mixture of similar but not that much identical sequences. The primers are convenient when the DNA or deoxyribonucleotide acid is extracted from the same organism. This allows for the detection of similar DNA sequences in the same type of organism.  But, the degenerate primers are not good at hybridizing the target DNA or deoxyribonucleic acid sequences, so, they are not the context of PCR. However, degenerate primers are useful in the study of microbial ecology. They allow the amplification of the DNA extracted from the organism.

Conclusion

The taq polymerase and primers have the most extensive role in the whole process of PCR.  This helps to amplify the target genes from an organism with ease.  The denaturation, annealing, and renaturation with the synthesis process are executed with the help of taq polymerases and primers.