|Taq polymerase, exonuclease|
DNA polymerase bound to a DNA octamer
Taq polymerase /
T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. Therefore, it replaced the DNA polymerase from E. coli originally used in PCR. Taq's optimum temperature for activity is 75–80 °C, with a half-life of greater than 2 hours at 92.5 °C, 40 minutes at 95 °C and 9 minutes at 97.5 °C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72 °C.
At 75-80 °C, Taq reaches its optimal polymerization rate of about 150 nucleotides per second per enzyme molecule, and any deviations from the optimal temperature range inhibit the extension rate of the enzyme. A single Taq synthesizes about 60 nucleotides per second at 70 °C, 24 nucleotides/sec at 55 °C, 1.5 nucleotides/sec at 37 °C, and 0.25 nucleotides/sec at 22 °C. At temperatures above 90 °C, Taq demonstrates very little or no activity at all, but the enzyme itself does not denature and remains intact. Presence of certain ions in the reaction vessel also affects specific activity of the enzyme. Small amounts of potassium chloride (KCl) and magnesium ion (Mg2+) promote Taq's enzymatic activity. Taq polymerase is maximally activated at 50mM KCl and just the right concentration of Mg2+ which is determined by the concentration of nucleoside triphosphates (dNTPs). High concentrations of KCl and Mg2+ inhibit Taq's activity.
One of Taq's drawbacks is its lack of 3' to 5' exonuclease proofreading activity resulting in relatively low replication fidelity. Originally its error rate was measured at about 1 in 9,000 nucleotides. The remaining two domains act in coordination, via coupled domain motion. Some thermostable DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelity amplification.
Taq makes DNA products that have A (adenine) overhangs at their 3' ends. This may be useful in TA cloning, whereby a cloning vector (such as a plasmid) that has a T (thymine) 3' overhang is used, which complements with the A overhang of the PCR product, thus enabling ligation of the PCR product into the plasmid vector.
In the early 1980s, Kary Mullis was working at Cetus Corporation on the application of synthetic DNAs to biotechnology. He was familiar with the use of DNA oligonucleotides as probes for binding to target DNA strands, as well as their use as primers for DNA sequencing and cDNA synthesis. In 1983, he began using two primers, one to hybridize to each strand of a target DNA, and adding DNA polymerase to the reaction. This led to exponential DNA replication, greatly amplifying the amounts of DNA between the primers.
However, after each round of replication the mixture needs to be heated above 90 °C to denature the newly formed DNA, allowing the strands to separate and act as templates in the next round of amplification. This heating step also inactivates the DNA polymerase that was in use before the discovery of Taq polymerase, the Klenow fragment of the DNA polymerase I from E. coli.
Use of the thermostable Taq enables running the PCR at high temperature (~60 °C and above), which facilitates high specificity of the primers and reduces the production of nonspecific products, such as primer dimer. Also, use of a thermostable polymerase eliminates the need to add new enzyme to each round of thermocycling. A single closed tube in a relatively simple machine can be used to carry out the entire process. Thus, the use of Taq polymerase was the key idea that made PCR applicable to a large variety of molecular biology problems concerning DNA analysis.
Hoffmann-La Roche eventually bought the PCR and Taq patents from Cetus for $330 million, from which it may have received up to $2 billion in royalties. In 1989, Science Magazine named Taq polymerase its first "Molecule of the Year". Kary Mullis received the Nobel Prize in 1993, the only one awarded for research performed at a biotechnology company. By the early 1990s, the PCR technique with Taq polymerase was being used in many areas, including basic molecular biology research, clinical testing, and forensics. It also began to find a pressing application in direct detection of the HIV in AIDS.
In December 1999, U.S. District Judge Vaughn Walker ruled that the 1990 patent involving Taq polymerase was issued, in part, on misleading information and false claims by scientists with Cetus Corporation. The ruling supported a challenge by Promega Corporation against Hoffman-La Roche, which purchased the Taq patents in 1991. Judge Walker cited previous discoveries by other laboratories, including the laboratory of Professor John Trela in the University of Cincinnati department of biological sciences, as the basis for the ruling.
|Wikimedia Commons has media related to Taq polymerase.|
- Chien A, Edgar DB, Trela JM (1976). "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus". J. Bacteriol. 127 (3): 1550–7. PMC . PMID 8432.
- Saiki, RK; et al. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239 (4839): 487–91. doi:10.1126/science.2448875. PMID 2448875.[permanent dead link]
- Saiki, RK; et al. (1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science. 230 (4732): 1350–4. doi:10.1126/science.2999980. PMID 2999980. Archived from the original on 2008-12-19.
- Lawyer FC, et al. (1993). "High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase ...". PCR Methods Appl. 2 (4): 275–87. doi:10.1101/gr.2.4.275. PMID 8324500.
- PCR protocols : a guide to methods and applications. Innis, Michael A. San Diego: Academic Press. 1990. ISBN 0123721806. OCLC 19723112.
- PCR technology : principles and applications for DNA amplification. Erlich, Henry A., 1943-. New York: Stockton Press. 1989. ISBN 0333489489. OCLC 19323242.
- Tindall KR, Kunkel TA (1988). "Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase". Biochemistry. 27 (16): 6008–13. doi:10.1021/bi00416a027. PMID 2847780.
- Bu Z, Biehl R, Monkenbusch M, Richter D, Callaway DJ (December 2005). "Coupled protein domain motion in Taq polymerase revealed by neutron spin-echo spectroscopy". Proc. Natl. Acad. Sci. U.S.A. 102 (49): 17646–51. doi:10.1073/pnas.0503388102. PMC . PMID 16306270.
- Mullis KB (April 1990). "The unusual origin of the polymerase chain reaction". Sci. Am. 262 (4): 56–61, 64–5. doi:10.1038/scientificamerican0490-56. PMID 2315679.
- Fore J, Wiechers IR, Cook-Deegan R (2006). "The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study". J Biomed Discov Collab. 1: 7. doi:10.1186/1747-5333-1-7. PMC . PMID 16817955.
Detailed history of Cetus Corporation and the commercial aspects of PCR.
- Guatelli JC, Gingeras TR, Richman DD (1 April 1989). "Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection". Clin. Microbiol. Rev. 2 (2): 217–26. doi:10.1128/CMR.2.2.217. PMC . PMID 2650862.
- Curran, Chris, Bio-Medicine, Dec. 7, 1999
- Li Y, Mitaxov V, Waksman G (August 1999). "Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation". Proc. Natl. Acad. Sci. U.S.A. 96 (17): 9491–6. doi:10.1073/pnas.96.17.9491. PMC . PMID 10449720.
<link href="./Category:EC_2.7.7" rel="mw:PageProp/Category" />