Taq polymerase

Last updated
DNA polymerase I, thermostable
PDB 1ktq EBI.jpg
Large (Klenow) fragment of Taq polA, containing the polA and vestigial domains
Identifiers
Organism Thermus aquaticus
SymbolpolA
UniProt P19821
Search for
Structures Swiss-model
Domains InterPro

Taq polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism Thermus aquaticus, from which it was originally isolated by Chinese scientist Alice Chien et al. in 1976. [1] Its name is often abbreviated to Taq or Taq pol. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.

Contents

T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified [1] as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. [2] Therefore, it replaced the DNA polymerase from E. coli originally used in PCR. [3]

Enzymatic properties

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. [4] 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. [5] 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, while optimal Mg2+ concentration is determined by the concentration of nucleoside triphosphates (dNTPs). High concentrations of KCl and Mg2+ inhibit Taq's activity. [6] The common metal ion chelator EDTA directly binds to Taq in the absence of these metal ions. [7]

One of Taq's drawbacks is its lack of 3' to 5' exonuclease proofreading activity [4] resulting in relatively low replication fidelity. Originally its error rate was measured at about 1 in 9,000 nucleotides. [8] 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. [9] Fidelity can vary widely between Taqs, which has profound effects in downstream sequencing applications. [10]

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 PCR

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, [11] greatly amplifying discrete segments of DNA between the primers. [3]

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 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. [2]

Patent issues

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. [12] 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 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. [13]

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. [14]

Domain structure

Taq polymerase, exonuclease
Taq.png
Full Taq DNA polymerase bound to a DNA octamer
Identifiers
SymbolTaq-exonuc
Pfam PF09281
InterPro IPR015361
SCOP2 1qtm / SCOPe / SUPFAM
Available protein structures:
Pfam   structures / ECOD  
PDB RCSB PDB; PDBe; PDBj
PDBsum structure summary

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. [15] 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. [16]

Exonuclease domain

Taq polymerase exonuclease is a domain found in the amino-terminal of Taq DNA polymerase I (thermostable). It assumes a ribonuclease H-like motif. The domain confers 5'-3' exonuclease activity to the polymerase. [17]

Unlike the same domain in E. coli, which would degrade primers and must be removed by digestion for PCR use, [9] this domain is not said to degrade the primer. [18] 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. [19]

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. [20] 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. [21]

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. [9] 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. [22]

Taq Polymerase Taq polimerase.png
Taq Polymerase

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." [23] 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. [24]

The reliance upon Taq polymerase as a catalyst for the PCR replication process has been highlighted during the COVID-19 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. [25]

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. [26]

See also

Related Research Articles

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<i>Thermus aquaticus</i> Species of bacterium

Thermus aquaticus is a species of bacteria that can tolerate high temperatures, one of several thermophilic bacteria that belong to the Deinococcota phylum. It is the source of the heat-resistant enzyme Taq DNA polymerase, one of the most important enzymes in molecular biology because of its use in the polymerase chain reaction (PCR) DNA amplification technique.

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<span class="mw-page-title-main">Exonuclease</span> Class of enzymes; type of nuclease

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References

  1. 1 2 Chien A, Edgar DB, Trela JM (September 1976). "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus". Journal of Bacteriology. 127 (3): 1550–7. doi:10.1128/jb.127.3.1550-1557.1976. PMC   232952 . PMID   8432.
  2. 1 2 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. (January 1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239 (4839): 487–91. Bibcode:1988Sci...239..487S. doi:10.1126/science.2448875. PMID   2448875.
  3. 1 2 Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (December 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science. 230 (4732): 1350–4. Bibcode:1985Sci...230.1350S. doi:10.1126/science.2999980. PMID   2999980. Archived from the original on 2008-12-19.
  4. 1 2 Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, Gelfand DH (May 1993). "High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity". PCR Methods and Applications. 2 (4): 275–87. doi: 10.1101/gr.2.4.275 . PMID   8324500.
  5. ↑ PCR protocols : a guide to methods and applications. Innis, Michael A. San Diego: Academic Press. 1990. ISBN   978-0123721808. OCLC   19723112.{{cite book}}: CS1 maint: others (link)
  6. ↑ PCR technology : principles and applications for DNA amplification . Erlich, Henry A., 1943-. New York: Stockton Press. 1989. ISBN   978-0333489482. OCLC   19323242.{{cite book}}: CS1 maint: others (link)
  7. ↑ Lopata A, Jójárt B, Surányi ÉV, Takács E, Bezúr L, Leveles I, et al. (October 2019). "Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity". Biomolecules. 9 (10): 621. doi: 10.3390/biom9100621 . PMC   6843921 . PMID   31627475.
  8. ↑ Tindall KR, Kunkel TA (August 1988). "Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase". Biochemistry. 27 (16): 6008–13. doi:10.1021/bi00416a027. PMID   2847780.
  9. 1 2 3 van Pelt-Verkuil E, van Belkum A, Hays JP (2008). "Taq and Other Thermostable DNA Polymerases". Principles and Technical Aspects of PCR Amplification. pp. 103–18. doi:10.1007/978-1-4020-6241-4_7. ISBN   978-1-4020-6240-7.
  10. ↑ Brandariz-Fontes C, Camacho-Sanchez M, Vilà C, Vega-Pla JL, Rico C, Leonard JA (January 2015). "Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results". Scientific Reports. 5: 8056. Bibcode:2015NatSR...5E8056B. doi:10.1038/srep08056. PMC   4306961 . PMID   25623996.
  11. ↑ Mullis KB (April 1990). "The unusual origin of the polymerase chain reaction". Scientific American. 262 (4): 56–61, 64–5. Bibcode:1990SciAm.262d..56M. doi:10.1038/scientificamerican0490-56. PMID   2315679.
  12. ↑ Fore J, Wiechers IR, Cook-Deegan R (July 2006). "The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study". Journal of Biomedical Discovery and Collaboration. 1: 7. doi: 10.1186/1747-5333-1-7 . PMC   1523369 . PMID   16817955.
    Detailed history of Cetus Corporation and the commercial aspects of PCR.
  13. ↑ Guatelli JC, Gingeras TR, Richman DD (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". Clinical Microbiology Reviews. 2 (2): 217–26. doi:10.1128/CMR.2.2.217. PMC   358112 . PMID   2650862.
  14. ↑ Curran, Chris, Bio-Medicine, Dec. 7, 1999
  15. ↑ Eom SH, Wang J, Steitz TA (July 1996). "Structure of Taq polymerase with DNA at the polymerase active site". Nature. 382 (6588): 278–81. Bibcode:1996Natur.382..278E. doi:10.1038/382278a0. PMID   8717047. S2CID   4372845.
  16. ↑ 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". Proceedings of the National Academy of Sciences of the United States of America. 102 (49): 17646–51. Bibcode:2005PNAS..10217646B. doi: 10.1073/pnas.0503388102 . PMC   1345721 . PMID   16306270.
  17. ↑ Li Y, Mitaxov V, Waksman G (August 1999). "Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation". Proceedings of the National Academy of Sciences of the United States of America. 96 (17): 9491–6. Bibcode:1999PNAS...96.9491L. doi: 10.1073/pnas.96.17.9491 . PMC   22236 . PMID   10449720.
  18. ↑ "Will the 5'→3' flap endonuclease activity of Taq DNA Polymerase degrade primers?". NEB. Retrieved 28 March 2019.
  19. ↑ TaqMan Gene Expression - NCBI Projects
  20. ↑ Park Y, Choi H, Lee DS, Kim Y (June 1997). "Improvement of the 3'-5' exonuclease activity of Taq DNA polymerase by protein engineering in the active site". Molecules and Cells. 7 (3): 419–24. PMID   9264032.
  21. ↑ Villbrandt B, Sobek H, Frey B, Schomburg D (September 2000). "Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase". Protein Engineering. 13 (9): 645–54. doi: 10.1093/protein/13.9.645 . PMID   11054459.
  22. ↑ Ishino S, Ishino Y (2014). "DNA polymerases as useful reagents for biotechnology - the history of developmental research in the field". Frontiers in Microbiology. 5: 465. doi: 10.3389/fmicb.2014.00465 . PMC   4148896 . PMID   25221550.
  23. ↑ Menon PK, Kapila K, Ohri VC (July 1999). "Polymerase Chain Reaction and Advances in Infectious Disease Diagnosis". Medical Journal, Armed Forces India. 55 (3): 229–231. doi:10.1016/S0377-1237(17)30450-1. PMC   5531883 . PMID   28775636.
  24. ↑ "Polymerase chain reaction (PCR)". stanfordhealthcare.org. Retrieved 2020-04-23.
  25. ↑ "FDA chief warns of supply 'pressure' on reagents for coronavirus tests". MedTech Dive. Retrieved 2020-04-23.
  26. ↑ Overbaugh J, Jackson SM, Papenhausen MD, Rudensey LM (November 1996). "Lentiviral genomes with G-to-A hypermutation may result from Taq polymerase errors during polymerase chain reaction". AIDS Research and Human Retroviruses. 12 (17): 1605–13. doi:10.1089/aid.1996.12.1605. PMID   8947295.
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