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TAQ Polymerase

TAQ Polymerase


TAQ Polymerase

A polymerase enzyme is essential in reactions such as the polymerase chain reaction (PCR).  This is because it attaches the nucleotides to the DNA during the PCR process which helps to form the new strands. This is one of the essential parts of the process.
The PCR technique was originally developed in California by the Cetus Corporation. Briefly, this technique allows DNA to be duplicated using a series of cycles which incorporate heating and cooling to allow the DNA molecules to denature and then be reassembled. A thirty cycle PCR can produce upwards of a billion new copies of the DNA in question.

The Original Reaction
The PCR technique was developed slowly over time. It was originally used by Kary Mullis, working for the Cetus Corporation to synthesise oligonucleotide probes for sickle cell anaemia mutations. Much of his work was a laborious, manual intervention, which involved heating the sample, then cooling, and adding polymerase by hand when the mixture was at the correct temperature. Mainly, he was using a modified version of Frederick Sanger’s Nobel Prize-winning work on DNA sequencing.

Because polymerase, itself a large and labile protein, will completely denature at higher temperatures, it had to be added only when the temperature was absolutely correct. It took several years for Mullis to perfect this technique. Initially he used the Klenow fragment, which is a hydrolytic product of polymerase taken chiefly from Escherichia Coli bacteria. This is readily available in large numbers and is cheap and easy to manufacture. The optimum working temperature of this enzyme is 37 degrees C.

Unfortunately, this means that it is completely destroyed during one cycle of PCR and involved, for Mullis and his team, the onerous task of adding a fresh supply each time the cycle was restarted. This results in a poor technique, which is unstable, unreproducible and open to contamination and other errors.

Thermus aquaticus

Thermus Aquaticus is a thermophilic bacterium which is found in hot springs. It was discovered in the hot geysers of Yellowstone National Park in Wyoming, USA completely by accident by Thomas Brock and his student, Hudson Freeze in 1966. 

It has been known since ancient times that there are organisms which can flourish in hot, even boiling, water because they can be clearly seen. Ancient Greek and Roman scholars commented on the different colours of the organisms. Much of the later research has been conducted by food technologists to ensure the absence of all bacteria in food processing and canning procedures.

Brock and Freeze were attempting to culture pink bacterium from Octopus Spring in Yellowstone Park by culturing it in a salt medium at 70 degrees C. Their efforts were unsuccessful, but in doing so, they discovered a new, previously unknown strain of a yellow bacterium which Brock later named Thermus aquaticus. Strains of this have been found to occur all over the world. Brock notes that nearly thirty years later, ‘a literature survey showed that more than a 1000 papers had been published on T. aquaticus’.

It is because of the ability to thrive at temperatures over 75 degrees C that the polymerase isolated from T. aquaticus (Taq) was adopted by the PCR developers. In 1976, Chien described for the first time, a 94kD DNA polymerase isolated from Taq. Using this newly discovered thermostable enzyme, it became possible to incorporate all the constituents at the start of the PCR cycle into a closed tube and let the whole reaction run through all the cycles without having to open the tubes at all. This allowed for a more stable and more reproducible reaction.

Once the enzyme had been isolated and used, the use of PCR also became widespread. The potential uses of this technology are so ubiquitous that it can encompass many of the scientific disciplines. Commercial productions of Taq polymerase make use of genetic engineering – the very process which it facilitates. The Taq gene is incorporated into E. coli and is now cheaply and readily made for use across the world.

Advantages of Taq

Taq polymerase is thermostable up to 92 degrees C. It has a half life of up to 130 minutes in a normal cycle. But it can also withstand high-stringency cycles where the temperature does not go lower than 70 degrees C, albeit with a lower half life.  

Because it is able to work at higher temperatures, there is less mis-priming. That is, there is reduced non-speicific binding to non-complementary base-pairs.

At high temperatures, the enzyme has high processivity. This is the number of nucleotides which can be incorporated in a certain amount of time. At 72 degrees C, Taq polymerase can work up to 60 nucleotides per second, and only about 1.5 nucleotides per second at 37 degrees C. This incredibly high temperature-specificity means that the enzyme works only when it is meant to. This is extremely important for controlling, what is in essence, a highly specialised and sensitive reaction.

Types of Taq polymerase

AmpliTaq is a variation of Taq which has 5’-3’ exonuclease activity. It can be easily expressed in E. coli and it does not contain any contaminants such as other stable nucleases. 
Hot start Taqs require an initial heating step of 95 degrees C for 5 minutes before the actual cycles can begin. This denatures any contamination and thus prevents non-specific, unwanted amplification. The initial heating step activates the polymerase. Many of these hot start Taqs are avaialbe such as Thermostart, Proofstart and RedHot DNA.

For more specialised PCR, an AMPLITaq Stoffel fragment is available. This is a shortened version of the enzyme which does not have any 5’-3’ exonuclease activity. It is especially useful for amplifying GC-rich regions of the genome. The advantages of this shorter fragment is that it can work around thermally stable secondary structures and so is useful for cloning plasmids and ligated DNA. It is also very useful in multiplex PCR because it can withstand a wider range of magnesium concentrations.

In contrast to normal Taq polymerase which can amplify up to 10kb, LongAmpTm Taq has been developed to amplify an extremely large sequence of up to 40kb.