HOME

TheInfoList



OR:

''Taq'' polymerase is a thermostable DNA polymerase I named after the
thermophilic A thermophile is a type of extremophile that thrives at relatively high temperatures, between . Many thermophiles are archaea, though some of them are bacteria and fungi. Thermophilic eubacteria are suggested to have been among the earliest bact ...
eubacterial microorganism ''
Thermus aquaticus ''Thermus aquaticus'' is a species of bacteria that can tolerate high temperatures, one of several thermophile, thermophilic bacteria that belong to the ''Deinococcota'' phylum. It is the source of the heat-resistant enzyme Taq polymerase, ''Taq' ...
,'' from which it was originally isolated by master's student Alice Chien et al. in 1976. Its name is often abbreviated to ''Taq'' or ''Taq'' pol. It is frequently used in the
polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed st ...
(PCR), a method for greatly amplifying the quantity of short segments of
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
. ''T. aquaticus'' is a
bacterium Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among the ...
that lives in
hot springs A hot spring, hydrothermal spring, or geothermal spring is a Spring (hydrology), spring produced by the emergence of Geothermal activity, geothermally heated groundwater onto the surface of the Earth. The groundwater is heated either by shallow ...
and hydrothermal vents, and ''Taq'' polymerase was identified as an
enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
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.


Enzymatic properties

''Taqs optimum temperature for activity is 75–80 °C, with a
half-life Half-life is a mathematical and scientific description of exponential or gradual decay. Half-life, half life or halflife may also refer to: Film * Half-Life (film), ''Half-Life'' (film), a 2008 independent film by Jennifer Phang * ''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 A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
strand of DNA in less than 10 seconds at 72 °C. At 75–80 °C, ''Taq'' reaches its optimal
polymerization In polymer chemistry, polymerization (American English), or polymerisation (British English), is a process of reacting monomer molecules together in a chemical reaction to form polymer chains or three-dimensional networks. There are many fo ...
rate of about 150
nucleotides Nucleotides are Organic compound, organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both o ...
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 Magnesium is a chemical element; it has Symbol (chemistry), symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 ...
ion (Mg2+) promote ''Taq'''s enzymatic activity. ''Taq'' polymerase is maximally activated at 50mM KCl, while optimal Mg2+ concentration is determined by the concentration of
nucleoside triphosphate A nucleoside triphosphate is a nucleoside containing a nitrogenous base bound to a 5-carbon sugar (either ribose or deoxyribose), with three phosphate groups bound to the sugar. They are the molecular precursors of both DNA and RNA, which are chai ...
s (dNTPs). High concentrations of KCl and Mg2+ inhibit ''Taq'''s activity. The common metal ion chelator
EDTA Ethylenediaminetetraacetic acid (EDTA), also called EDTA acid, is an aminopolycarboxylic acid with the formula . This white, slightly water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-solubl ...
directly binds to ''Taq'' in the absence of these metal ions. One of ''Taqs drawbacks is its lack of 3' to 5' exonuclease
proofreading Proofreading is a phase in the process of publishing where galley proofs are compared against the original manuscripts or graphic artworks, to identify transcription errors in the typesetting process. In the past, proofreaders would place corr ...
activity resulting in relatively low replication fidelity. Originally its error rate was measured at about 1 in 9,000 nucleotides. 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. Fidelity can vary widely between Taqs, which has profound effects in downstream sequencing applications. ''Taq'' makes DNA products that have A (
adenine Adenine (, ) (nucleoside#List of nucleosides and corresponding nucleobases, symbol A or Ade) is a purine nucleotide base that is found in DNA, RNA, and Adenosine triphosphate, ATP. Usually a white crystalline subtance. The shape of adenine is ...
) overhangs at their 3' ends. This may be useful in TA cloning, whereby a cloning vector (such as a
plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...
) that has a T (
thymine Thymine () (symbol T or Thy) is one of the four nucleotide bases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine ...
) 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 PCR

In the early 1980s, Kary Mullis was working at Cetus Corporation on the application of synthetic DNAs to
biotechnology Biotechnology is a multidisciplinary field that involves the integration of natural sciences and Engineering Science, engineering sciences in order to achieve the application of organisms and parts thereof for products and services. Specialists ...
. 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 DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, thymine, cytosine, and guanine. The ...
and cDNA synthesis. In 1983, he began using two primers, one to hybridize to each strand of a target DNA, and adding
DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create t ...
to the reaction. This led to exponential
DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all life, living organisms, acting as the most essential part of heredity, biolog ...
, greatly amplifying discrete segments 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 (sourced from '' E. coli''). ''Taq'' polymerase is well-suited for this application because it is able to withstand the temperature of 95 °C which is required for DNA strand separation without denaturing. 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 A machine is a physical system that uses power to apply forces and control movement to perform an action. The term is commonly applied to artificial devices, such as those employing engines or motors, but also to natural biological macromol ...
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 Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...
problems concerning DNA analysis.


Patent issues

Hoffmann-La Roche eventually bought the PCR and ''Taq''
patent A patent is a type of intellectual property that gives its owner the legal right to exclude others from making, using, or selling an invention for a limited period of time in exchange for publishing an sufficiency of disclosure, enabling discl ...
s 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 Chemistry in 1993, the only one awarded for research performed at a
biotechnology Biotechnology is a multidisciplinary field that involves the integration of natural sciences and Engineering Science, engineering sciences in order to achieve the application of organisms and parts thereof for products and services. Specialists ...
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 Forensic science combines principles of law and science to investigate criminal activity. Through crime scene investigations and laboratory analysis, forensic scientists are able to link suspects to evidence. An example is determining the time and ...
. It also began to find a pressing application in direct detection of the HIV in
AIDS The HIV, human immunodeficiency virus (HIV) is a retrovirus that attacks the immune system. Without treatment, it can lead to a spectrum of conditions including acquired immunodeficiency syndrome (AIDS). It is a Preventive healthcare, pr ...
. 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 John Trela at the
University of Cincinnati The University of Cincinnati (UC or Cincinnati, informally Cincy) is a public university, public research university in Cincinnati, Ohio, United States. It was founded in 1819 and had an enrollment of over 53,000 students in 2024, making it the ...
department of biological sciences, as the basis for the ruling.


Domain structure

''Taq'' Pol A has an overall structure similar to that of '' E. coli'' PolA. The middle 3'–5' exonuclease domain responsible for proofreading has been dramatically changed and is not functional. It has a functional 5'-3' exonuclease domain at the amino terminal, described below. The remaining two domains act in coordination, via coupled domain motion.


Exonuclease domain

''Taq'' polymerase exonuclease is a domain found in the amino-terminal of ''Taq'' DNA
polymerase In biochemistry, a polymerase is an enzyme (Enzyme Commission number, EC 2.7.7.6/7/19/48/49) that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by ...
I (thermostable). It assumes a ribonuclease H-like motif. The domain confers 5' -3' exonuclease activity to the polymerase. Unlike the same domain in ''E. coli'', which would degrade primers and must be removed by digestion for PCR use, this domain is not said to degrade the primer. This activity is used in the TaqMan probe: as the daughter strands are formed, the probes complementary to the template come in contact with the polymerase and are cleaved into fluorescent pieces.


Binding with DNA

''Taq'' polymerase is bound at its polymerase active-site cleft with the blunt end of duplex DNA. As the ''Taq'' polymerase is in contact with the bound DNA, its side chains form hydrogen bonds with the purines and pyrimidines of the DNA. The same region of ''Taq'' polymerase that has bonded to DNA also binds with exonuclease. These structures bound to the ''Taq'' polymerase have different interactions.


Mutants

A site-directed mutagenesis experiment that improves the vestigial 3'-5' exonuclease activity by a factor of 2 has been reported, but it was never reported whether doing so decreases the error rate. Following a similar line of thought, chimera proteins have been made by cherry-picking domains from ''E. coli'', ''Taq'', and '' T. neapolitana'' polymerase I. Swapping out the vestigial domain for a functional one from ''E. coli'' created a protein with proof-reading ability but a lower optimal temperature and low thermostability. Versions of the polymerase without the 5'-3' exonuclease domain has been produced, among which ''Klentaq'' or the ''Stoffel'' fragment are best known. The complete lack of exonuclease activity make these variants suitable for primers that exhibit secondary structure as well as for copying circular molecules. Other variations include using ''Klentaq'' with a high-fidelity polymerase, a ''Thermosequenase'' that recognizes substrates like T7 DNA polymerase does, mutants with higher tolerances to inhibitors, or "domain-tagged" versions that have an extra helix-hairpin-helix motif around the catalytic site to hold the DNA more tightly despite adverse conditions.


Significance in disease detection

Because of the improvements ''Taq'' polymerase provided in PCR DNA replication: higher specificity, fewer nonspecific products, and simpler processes and equipment, it has been instrumental in the efforts made to detect diseases. "The use of Polymerase Chain Reaction (PCR) in infectious disease diagnosis, has resulted in an ability to diagnose early and treat appropriately diseases due to fastidious pathogens, determine the antimicrobial susceptibility of slow growing organisms, and ascertain the quantum of infection." The implementation of ''Taq'' polymerase has saved countless lives. It has served an essential role in the detection of many of the world's worst diseases, including: tuberculosis, streptococcal pharyngitis, atypical pneumonia, AIDS, measles, hepatitis, and ulcerative urogenital infections. PCR, the method used to recreate copies of specific DNA samples, makes disease detection possible by targeting a specific DNA sequence of a targeted pathogen from a patient's sample and amplifying trace amounts of the indicative sequences by copying them up to billions of times. Although this is the most accurate method of disease detection, especially for HIV, it is not performed as often as alternative, inferior tests because of the relatively high cost, labor, and time required. The reliance upon ''Taq'' polymerase as a catalyst for the PCR replication process has been highlighted during the
COVID-19 Coronavirus disease 2019 (COVID-19) is a contagious disease caused by the coronavirus SARS-CoV-2. In January 2020, the disease spread worldwide, resulting in the COVID-19 pandemic. The symptoms of COVID‑19 can vary but often include fever ...
Pandemic of 2020. Shortages of the necessary enzyme have impaired the ability of countries worldwide to produce test kits for the virus. Without ''Taq'' polymerase, the disease detection process is much slower and tedious. Despite the advantages of using ''Taq'' polymerase in PCR disease detection, the enzyme is not without its shortcomings. Retroviral diseases (HIV, HTLV-1, and HTLV-II) often include mutations from guanine to adenine in their genome. Mutations such as these are what allow PCR tests to detect the diseases but ''Taq'' polymerase’s relatively low fidelity rate makes the same G-to-A mutation occur and possibly yield a false positive test result.


See also


References

{{Portal bar, Biology, border=no DNA replication Polymerase chain reaction