Thermus aquaticus
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Thermus aquaticus is a species of bacterium that is the source of the enzyme Taq DNA Polymerase, one of the most important enzymes in molecular biology. It is one of several thermophilic bacteria that belong to the Deinococcus-Thermus group.
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Biology
In 1969, Thomas Brock and Hudson Freeze of Indiana University reported a new species of bacteria which they named Thermus aquaticus [1] (http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=5781580). This thermophilic organism was isolated from hot springs in Yellowstone National Park
Enzymes from T. aquaticus
T. aquaticus was to eventually become famous as a source of thermostable enzymes, particularly the "Taq" DNA Polymerase, as described below.
- Aldolase
- Studies of this extreme thermophilic bacterium that could be grown in cell culture was initially centered on attempts to understand how protein enzymes (which normally inactivate at high temperature) can function at high temperature in thermophiles. In 1970 Freeze and Brock published an article describing a thermostable aldolase enzyme from Thermus aquaticus.
- RNA polymerase
- The first polymerase enzyme isolated from Thermus aquaticus was a DNA-dependent RNA polymerase (G.M. Air and J.I. Harris, 1974). This was the year that DNA sequencing was made practical by the invention of the labeled-chain termination method. (Sanger F, Donelson JE, Coulson AR, Kossel H, Fischer D. "Determination of a nucleotide sequence in bacteriophage f1 DNA by primed synthesis with DNA polymerase" Journal of Molecular Biology 5;90(2):315-33.) DNA sequencing typically depends on the use of DNA-directed DNA polymerases, enzymes that make a new strand of DNA starting with an existing strand. "Primed synthesis" refers to the fact that DNA polymerases usually need a region of DNA double helix as a start point for attaching to the DNA strand that is to be replicated. In the test tube, the start point can be determined by providing a primer, a short strand of DNA that will attach by base pairing to the target strand.
- DNA polymerase ("Taq pol")
- DNA polymerase was first isolated from Thermus aquaticus in 1976. (Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus (http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=8432) by A Chien, D B Edgar, and J M Trela in Journal of Bacteriology 127 (3): 1550–1557.) The first advantage that was found for this thermostable (temperature optimum 80°C) DNA polymerase was that it could be isolated in a purer form (free of other enzyme contaminants) than could the DNA polymerase from other sources. A molecular model of the structure of this enzyme is shown to the right.
- Taq I restriction enzyme
- Most molecular biologists probably became aware of Thermus aquaticus in the late 1970s or early 1980s because of the isolation of useful restriction endonucleases from this organism. In 1978 S. Sato published an article called "A single cleavage of Simian virus 40 (SV40) DNA by a site specific endonuclease from Thermus aquaticus, Taq I." in J. Biochem. (Tokyo). 1978 Feb;83(2):633-5.
- Note: The term "Taq" to refer to Thermus aquaticus arose from the convention of giving restriction enzymes short names such as Sal and Hin, names that come from the genus and species names of the source organisms.
The 300 Million Dollar Man
In the early 1980s Kary Mullis was working at Cetus on the synthesis of DNA. There was interest there in developing methods for detecting gene mutations that would be useful in disease screening. A major problem was that the available techniques (such as oligomer restriction) relied upon having a lot of DNA copies of the mutated gene. Mullis was familiar with the idea of using DNA oligonucleotides and hybridizing them to target DNA strands.
Idea Number One: PCR
In order to advance a project he was involved with, he began to think about using two oligonucleotides, one to hybridize to each strand of a DNA double helix. At first, his only reason for adding DNA polymerase to his experiments was as a way of making sure that deoxynucleoside triphosphates would be removed from his samples. But he then realized that the enzyme might make useful copies of the oligonucleotide-primed DNA strands. He immediately realized this was a potential way to amplify a region of DNA. It was 1983.
- Note: If you are not familiar with the polymerase chain reaction procedure, you may want to read about it before continuing with this article.
The main problem was, that after a round of strand copying, the DNA would have to be heated to near boiling in order to denature the newly formed double stranded DNA, allowing the strands to separate and open new templates for another round of amplification. This heating step would denature and inactivate the DNA polymerase, requiring that new enzyme be added at each amplification step.
First PCR article:
- Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia (http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/034) by Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. in Science 1985 Dec 20;230(4732):1350-4.
This original PCR technique was slow and labor-intensive. The "inside-the-box" thinkers at Cetus began to automate the process. The first PCR machine, "Mr. Cycle" automatically added more enzyme after every heating and cooling step.
Idea Number Two: Thermostable Taq Polymerase
Those who know Mullis such as Thomas J. White agree that it was Mullis who came up with the idea of using Taq polymerase in order to avoid having to add polymerase to the PCR reaction during the thermocycling process. This was the key idea that made the PCR technique available to an army of molecular biologists.
Roche Molecular Systems eventually bought the PCR patents from Cetus for $300,000,000. Kary Mullis got $10,000 from Cetus and a Nobel Prize from his scientific peers. Research scientists and biotechnology companies spend hundreds of millions of dollars each year for Taq polymerase.
A 1988 article describing the use of Taq polymerase for PCR (Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase (http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009) Science 1988 239 487-91.) led to Science magazine naming Taq polymerase its first "Molecule of the Year" in 1989.
By 1989 the PCR technique was being used in all areas of modern biology research from AIDS to Z. Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection. (http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=2650862) by J C Guatelli, T R Gingeras, and D D Richman in Clin Microbiol Rev. 1989 2 217–226.
See also
Figure: PCR Publications by Year.
The PCR technique became a major tool for molecular biology after the Taq DNA Polymerase and thermocyclers became available.
PCRpublication.jpg
PCR publication graph