The Use of Thermus Aquaticus in the Polymerase Chain Reaction
Scientists use an understanding of the various biological processes common to all life forms in order to further our ability to research and investigate life. In the Polymerase Chain Reaction (PCR) scientists use DNA polymerase taken from the bacterium Thermus aquaticus to amplify segments of DNA sequences for DNA fingerprinting and other applications. The polymerase chain reaction is a technique that copies a specific segment of DNA quickly (Sadava, Hillis, Heller, Berenbaum 2009). In the scientific field it is necessary to make multiple copies of a DNA sequence in order to study DNA or perform genetic manipulations (Sadava, Hillis, Heller, Berenbaum 2009). DNA amplification is a process in which the polymerase chain reaction automatically replicates DNA multiple times in a test tube. The PCR amplification process involves three steps.
The first step heats the reaction in order to denature the two strands of DNA, in the second step the reaction cools in order for the primers to anneal to the strands of DNA, and in the third step the reaction is warmed in order for the DNA polymerase chain reaction to catalyze the production of the complementary new strands (Sadava, Hillis, Heller, Berenbaum 2009). The three steps cycle and repeat until enough DNA that is needed is produced. The PCR mixture must contain, a double stranded DNA to act as a template, two primers, four dNTPs, a DNA polymerase that can withstand high temperatures, and salts and buffers to maintain a neutral PH (Sadava, Hillis, Heller, Berenbaum 2009). In the first step of the PCR reaction, DNA must be heated to more than 90 degrees Celsius. This is one of the main problems encountered in the PCR reaction because most DNA polymerase also denature at these temperatures, which means that during each cycle DNA polymerase must be added (Sadava, Hillis, Heller, Berenbaum 2009). Thomas Brock investigated this issue and realized that a bacterium called Thermus aquaticus lives in high temperatures, up to 95 degrees Celsius, and the DNA polymerase of Thermus aquaticus is heat resistant, it does not denature at high temperatures (Sadava, Hillis, Heller, Berenbaum 2009). Scientists used Thomas Brock's discovery and used Thermus aquaticus DNA polymerase in PCR. This allowed PCR to not have to be added during each cycle, and the DNA polymerase to withstand the high temperatures (Sadava, Hillis, Heller, Berenbaum 2009). PCR can be used for chemical analysis, identification of a person or organism, and to detect diseases (Sadava, Hillis, Heller, Berenbaum 2009). This essay will review three peer reviewed articles on the PCR reaction, Thermus aquaticus, and research done regarding these two topics, and will conclude with modern applications that use this technique.
"Deoxyribonucleic Acid Polymerase from the Extreme Thermophile Thermus aquaticus" is an article by Alice Chein, David B. Edgar, and John M. Trela. Chein, Edgar, and Trela's article discusses the attributes of thermophiles and attempts to purify the DNA polymerase from the bacterium Thermus aquaticus. The purpose of the article is to discuss the characterization and purification of a thermophilic polymerase compared to DNA polymerases from mesophillic microorganisms(Chein, Edgar, and Trela 1976). A mesophillic microorganism is an organism that is too small to be seen by the naked eye and lives in moderate temperatures(Sadava, Hillis, Heller, Berenbaum 2009). A thermophile is an organism that lives in high, extreme, temperatures(Sadava, Hillis, Heller, Berenbaum 2009). The article explains that thermophiles are ubiquitous in nature and that many prokaryotic species live at temperatures above 45 degrees Celsius(Chein, Edgar, and Trela 1976). The materials that were used were, a strain of Thermus aquaticus, the cells were grown in a defined mineral salt containing glutamic acid, this served as the culture medium(Chein, Edgar, and Trela 1976). The growth conditions consisted of Erlenmeyer flasks in a water bath shaker, which were maintained at a temperature of 75 degrees Celsius, initially. Then transferred to carboys that were placed in hot-air incubators. The cultures were allowed to grow for twenty hours before being collected. Then the enzyme extract was prepared, followed by enzyme asseys, and Polyacrylamide gel electrophoresis.
The results of the article concluded that the attempts to remove the BSA from the enzyme sample resulted in a substantial loss of the catalytic activity of the DNA polymerase(Chein, Edgar, and Trela 1976). The reasoning for this result could of stemmed from the low protein concentration of the DNA polymerase after it was separated from the BSA. The conclusion of the research states that it is unknown whether the DNA polymerase when separated from Thermus aquaticus represents the native form of the enzyme in vivo or if it is a result of proteolytic cleavage during the isolation(Chein, Edgar, and Trela 1976). The article did conclude that the enzyme did function at a temperature of 80 degrees Celsius. This temperature is higher than the DNA polymerase from Bacillus stearothemophilus(Chein, Edgar, and Trela 1976). The article stated that because of the temperature range there is a possibility of using the enzyme in gene synthesis. The article was written April 12 1976. Due to further research on the subject we now know that we can use the enzyme in gene synthesis, such as the PCR reaction, and that this is partially because of the temperature range. I kept this article as a primary research article because even though the information has been further tested and more detailed conclusions have been discovered, this article shows how scientific research is used as a stepping stone for further research. The conclusions about the enzyme functioning at higher temperatures is one of the reasons that Thermus aquaticus DNA polymerase is used in the polymerase chain reaction.
"DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA" is an article by Michael A. Innis, Kenneth B. Myambo, David H. Gelfand, and Mary Ann D. Brow. Innis, Myambo, Gelfand, and Brow were experimenting with modifying the conditions of the PCR reaction for the direct DNA sequencing of PCR products by using Thermus aquaticus DNA polymerase. They expected that because of the preparation of the DNA template and the direct sequencing that it would facilitate automation for larger sequencing projects(Innis, Myambo, Gelfand, and Brow 1988). Innis, Myambo, Gelfand, and Brow realized that Thermus aquaticus DNA polymerase (Taq polymerase) simplified the PCR procedure because it would not denature at higher temperatures, which meant that it was not necessary to resupply the enzyme after each cycle.
The article states that Taq polymerase increases the yield and length of the products that can be amplified, which increases the sensitivity of PCR. The increase of sensitivity of PCR allows for the detection of rare target sequences(Innis, Myambo, Gelfand, and Brow 1988). The materials used in their experiments included a variety of enzymes, such as Taq DNA polymerase, Polynucleotide kinase from T4-infected Escherichia, nucleotides, oligonucleotides, DNA, 3' dideoxynucleotide, 5' triphosphates, and dNTPs (Innis, Myambo, Gelfand, and Brow 1988). Many methods were used within the research, such as, the annealing reaction, labeling reaction, extension-termination reaction, asymmetric PCRs, and sequencing of PCR products. The article concluded that Taq DNA polymerase is very rapid and progressed through the replication process automatically (Innis, Myambo, Gelfand, and Brow 1988). The article also concluded that Taq DNA polymerase is sensitive to free magnetism ion concentration. Innis, Myambo, Gelfand, and Brow found that Taq DNA polymerase incorporated the ddNTPs with varied efficiency. They presented efficient protocols for sequencing using Taq DNA polymerase, and suggest that Taq DNA polymerase does hold advantages in different applications for sequencing. The result's of the article showed that Taq polymerase operated at higher temperatures and lower salt concentrations, this supported the conclusion stating that DNA is highly efficient and superior to previously used products (Innis, Myambo, Gelfand, and Brow 1988).
"Effective amplification of long targets from cloned inserts and human genomic DNA" is an article by Suzanne Cheng, Carita Fockler, Wayne M. Barnes, and Russsell Higuchi. Cheng, Fockler, Barnes, and Higuchi used the PCR reaction to amplify a gene cluster from human genomic DNA and phage A DNA. The purpose of their research was to amplify DNA sequences to make the speed and simplicity of the polymerase chain reaction function to facilitate studies in molecular genetics (Cheng, Fockler, Barnes, and Higuchi 1994). The materials a DNA that contained all four dNTPs, the DNA was from a human placenta, human genomic clones were purchased and grown as directed (Cheng, Fockler, Barnes, and Higuchi 1994). The Themostable DNA polymerases used were AmpliTaq DNA polymerase, rTth DNA polymerase which is from Thermus thermophillus, Pyrococcus furiosus polymerase, rTaq, polymerase buffers, dimethyl sulfoxide, and glycerol (Cheng, Fockler, Barnes, and Higuchi 1994). The methods used were analysis of PCR products, two thermal cycling profiles. During the analysis of PCR products the samples were regularly analyzed on standard gels or by using inversion gel electrophoresis. The research within the article concludes that the PCR was able to amplify longer strands of genomic DNA that was previously thought possible (Cheng, Fockler, Barnes, and Higuchi 1994) . The applications of the articles research can be used in genome maps, the ability to make longer DNA templates without having to do labor intensive cloning, rapid sequencing, and may be able to close gaps that are unclonable. It could lead to automated genome sequencing (Cheng, Fockler, Barnes, and Higuchi 1994).
The three articles I chose all contain experiments and research done with Taq DNA polymerase in the PCR reaction. In the first article, also the earliest article, the authors experiment with separating the DNA polymerase from Thermus aquaticus. The results were inconclusive but did show that Thermus aquaticus thrives at higher temperatures, which could be used in the first step of the PCR reaction, when the DNA strand is denatured by high temperatures. The second article the authors experimented with modifying the conditions of the PCR reaction so that direct sequencing of PCR products could occur. They did this by using Thermus aquaticus DNA polymerase. Innis, Myambo, Gelfand, and Brow thought that because of the preparation of the DNA template and direct sequencing that it would be possible to sequence larger projects. The hypothesis was supported by their research. In the third article , and the latest article, the purpose of the research was to amplify DNA sequences in order to make the speed and simplicity of the PCR reaction function more efficiently in studies in molecular genetics. The research done was supported and also shows the potential processes that can be used due to their findings, such as automated genome sequencing. All three of the articles build on each other, allowing for further progress within the field of study. The PCR reaction has many uses in modern science, it is used not only for scientific studies and experiments but also for crime solving as well as medical diagnosis.
Suzanne Cheng, Cartia Fockler, Wayne M. Barnes, and Russell Higuchi. June 1994. Effective amplification of long targets from cloned inserts human genomic DNA. Proc. Natl. Acad. Sci. Vol. 91, pp. 5695-5699. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44063/?tool=pubmed Accessed Friday February 26 2010.
Alice Chien, David B. Edgar, and John M. Trela. September 1976. Deoxyribonucleic Acid Polymerase from the Extreme Thermophile Thermus aquaticus. Journal of Bactersriology. Vol. 127 No. 3. pp 1550-1557. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC232952/?tool=pubmed Accessed Friday February 26 2010.
Michael A. Innis, Kenneth B. Myambo, David H. Gelfand, and Mary Ann D. Brow. December 1988. DNA sequencing with Thermus aquaticus DNA polymerase direct sequencing of polymerase chain reaction- amplified DNA. Proc. Natl. Acad. Sci. Vol.: 85, pp 9436-9440. http://ukpmc.ac.uk/articlerender.cgi?tool=pubmed&pubmedid=3200828 Accessed Friday February 26 2010.
David Sadava, David M. Hillis, H. Craig Heller, May R Berenbaum. 2009. Life The Science of Biology Ninth Edition. Printed in USA. The Courier Companies Inc. pp. 286.