The Cloning of DNA Taq Polymerase from Thermus Aquaticus
Nan Sowers
MRGS Mentor: Mrs. Klus
JMU Mentor: Dr. McKown
15 December 2013
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Introduction
Thermus aquaticus is a bacterium found in extremely high temperatures, such as the place
of its discovery: Yellowstone National Park’s geysers. This bacterium is significant because of
its success in polymerase chain reactions (PCR). PCRs denature DNA through a series of rounds
and are used in DNA cloning and amplification. Each round doubles the amount of DNA, but
PCRs cannot clone and amplify DNA without an important enzyme that stays active at high
temperatures. Most polymerases are destroyed in PCR’s successive heating cycles. However,
Taq polymerase is an exception because it survives the incredible heat of PCRs. Taq has created
a 300 million dollar industry because of its, “wide use in medical diagnosis (AIDS, for instance)
and forensics (DNA fingerprinting)” (Brock, 1994). Recent studies show that Taq polymerase
is most effectively cloned from thermus aquaticus to be used in polymerase chain reactions.
Evidence/Body
In supporting this claim, one can look at the experiment of Muskingum College.
According to Belyaevskaya (2008), Muskingum College came up with a project in order to clone
the Taq polymerase gene from Thermus aquaticus using the pGEM-3Zf plasmid and
subsequently transform Escherichia coli with this genetic material. Although Taq polymerase is
very effective, it is also very expensive to buy. This is why the college decided to clone the Taq
polymerase gene from Thermus aquaticus using the pGEM-3Zf plasmid. They used the
restriction enzyme digestion to determine which Escherichia coli bacteria contained the pGEM3zf plasmid with the cloned Taq gene inserted in an open reading frame within the plasmid. They
then tested this using SDS Polyacrylamide Gel Electrophoresis and polymerase chain reaction.
They then used the purified Taq Polymerase for course work and research projects at
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Muskingum College (Belyaevskaya, 2008). The article summarizing this experiment is great
because it provides accurate reasoning that is easy to understand why it is worth going through
the trouble of cloning Taq polymerase. Once obtained, “Taq” can be used in all sorts of other
experiments.
Muskingum College was not the only institution that desired Taq polymerase. According
to Sadeghi (2008), Isfahan Medical School wanted to purify recombinant Taq Polymerase from
Escherichia Coli BL21. They chose this enzyme because of its wide use in PCR reactions as
well. They then used three different protocols to purify this enzyme and compared the results.
The most effective of the three was a protocol called Desai. The bands displayed on gels were in
the area that was expected when this protocol was used: around 94 kDa. The other two protocols
left weak bands. One of the other protocols, nickel-nitrilotriacetic acid (Ni-NTA) resin,
eliminated unwanted bands but also reduced the amount of Taq polymerase (Sadeghi, 2012).
Isfahan Medical School’s experiment did not turn out nearly as successful as Muskingum
College’s because they did not use thermus aquaticus. The Escherichia Coli BL21 was much less
effective, only further supporting the claim that “Taq” is most effectively cloned from thermus
aquaticus.
The University of Sains Malaysia started their experiment much differently than the
previous institutions. They began with only a section of thermus aquaticus already separated
from the rest: pTaq. According to Ang (2008), the University of Sains Malaysia set out on a
mission to determine the over-expression of pTaq by a simple purification step. They then made
the goal of determining the activity of their enzyme in comparison to other commercial sources
of recombinants Taq enzymes. Their method involved transforming pTaq into E.coli strain DH5a
in the ampicillin selective media. They grew the culture overnight, harvested and lysed the cells
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in Tris buffer, heat treated the lysate to denature heat labile proteins, and centrifuged the sample
to remove unwanted debris and denatured proteins. They used PCR to determine the activity of
their recombinant Taq enzyme. In conclusion, they found that their Taq polymerase had
significant enzyme activity when compared to commercial recombinant Taq polymerases. Their
Taq polymerase was highly efficient and cost effective as compared to others (Ang, 2008).
Conclusion
Muskingum College and The University of Sains Malaysia’s experiments are proof that
cloning Taq polymerase from thermus aquaticus is effective for use in polymerase chain
reactions. Moreover, Isfahan Medical School’s failed experiment without the use of thermus
aquaticus only further supports this claim as well. Taq polymerase has proved itself to be worthy
of cloning considering its benefits to the medical and criminal justice worlds. Biotechnology has
been completely transformed by the invention of PCR, and to keep it going, the industry needs a
cheaper way to obtain Taq polymerase. Scientists will continue researching more cost effective
and easier ways to clone Taq polymerase for a long time to come and will hopefully develop this
research into a rich future of DNA amplification.
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References
Ang, K. C., Hoe, C. H., Asma, S., Basir, M., Yahaya, O., Tang, T.H. (2008). Over-expresssion of
an in-house recombinant taq DNA polymerase enzyme. Malaysian Journal Of Medical
Sciences, 19.
Belyaevskaya, A. V., Parrish, J. N., & Santas, A. J. (2008). Cloning and Expression of the Taq
DNA Polymerase Gene from Thermus Aquaticus. Ohio Journal Of Science, 108(1), A21.
Brock, T. D. (1994). Yellowstone association for natural science, history & education, inc.
Sadeghi, H., Rabbani, M., Assarzadeh, S., & Moazen, F. (2012). Optimization of the Production
and Purification of Taq Polymerase Enzyme Containing N666E Mutation in its O-Helix
Region. Journal Of Isfahan Medical School, 30(193), 1-10.