Taq polymerase#Mutants

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.{{cite journal | vauthors = Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, Gelfand DH | title = 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 | journal = PCR Methods and Applications | volume = 2 | issue = 4 | pages = 275–87 | date = May 1993 | pmid = 8324500 | doi = 10.1101/gr.2.4.275 | doi-access = free }} 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.{{Cite book|title=PCR protocols : a guide to methods and applications|date=1990|publisher=Academic Press| vauthors = Innis MA |isbn=978-0123721808|location=San Diego|oclc=19723112}} 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.{{Cite book|title=PCR technology : principles and applications for DNA amplification|date=1989|publisher=Stockton Press| vauthors = Erlich HA |isbn=978-0333489482|location=New York|oclc=19323242|url-access=registration|url=https://archive.org/details/pcrtechnologypri0000unse}} The common metal ion chelator EDTA directly binds to Taq in the absence of these metal ions.{{cite journal | vauthors = Lopata A, Jójárt B, Surányi ÉV, Takács E, Bezúr L, Leveles I, Bendes ÁÁ, Viskolcz B, Vértessy BG, Tóth J | title = Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity | journal = Biomolecules | volume = 9 | issue = 10 | pages = 621 | date = October 2019 | pmid = 31627475 | pmc = 6843921 | doi = 10.3390/biom9100621 | doi-access = free }}

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.{{cite journal | vauthors = Tindall KR, Kunkel TA | title = Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase | journal = Biochemistry | volume = 27 | issue = 16 | pages = 6008–13 | date = August 1988 | pmid = 2847780 | doi = 10.1021/bi00416a027 }} 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.{{cite journal | vauthors = Brandariz-Fontes C, Camacho-Sanchez M, Vilà C, Vega-Pla JL, Rico C, Leonard JA | title = Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results | journal = Scientific Reports | volume = 5 | pages = 8056 | date = January 2015 | pmid = 25623996 | pmc = 4306961 | doi = 10.1038/srep08056 | bibcode = 2015NatSR...5E8056B }}

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,{{cite journal | vauthors = Mullis KB | title = The unusual origin of the polymerase chain reaction | journal = Scientific American | volume = 262 | issue = 4 | pages = 56–61, 64-5 | date = April 1990 | pmid = 2315679 | doi = 10.1038/scientificamerican0490-56 | bibcode = 1990SciAm.262d..56M }} 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 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.{{cite journal | vauthors = Fore J, Wiechers IR, Cook-Deegan R | title = The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study | journal = Journal of Biomedical Discovery and Collaboration | volume = 1 | pages = 7 | date = July 2006 | pmid = 16817955 | pmc = 1523369 | doi = 10.1186/1747-5333-1-7 | doi-access = free }}

Detailed history of Cetus Corporation and the commercial aspects of PCR. 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.{{cite journal | vauthors = Guatelli JC, Gingeras TR, Richman DD | title = Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection | journal = Clinical Microbiology Reviews | volume = 2 | issue = 2 | pages = 217–26 | date = April 1989 | pmid = 2650862 | pmc = 358112 | doi = 10.1128/CMR.2.2.217 }}

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 department of biological sciences, as the basis for the ruling.{{cite web | title = Historical information available on Taq polymerase findings at the University of Cincinnati | url = http://news.bio-medicine.org/biology-news-2/Historical-information-available-on-Taq-polymerase-findings-at-the-University-of-Cincinnati-12158-1/ | vauthors = Curran C | work = Bio-Medicine | date = 7 December 1999 | archive-url = https://web.archive.org/web/20200521012242/http://news.bio-medicine.org/biology-news-2/Historical-information-available-on-Taq-polymerase-findings-at-the-University-of-Cincinnati-12158-1/ | archive-date = 21 May 2020 }}

{{Infobox protein family|Symbol=Taq-exonuc|Name=Taq polymerase, exonuclease|Pfam=PF09281|Pfam_clan=|SMART=|PROSITE=|MEROPS=|SCOP=1qtm|TCDB=|OPM family=|OPM protein=|CAZy=|CDD=|InterPro=IPR015361|image=taq.png|caption=Full Taq DNA polymerase bound to a DNA octamer}}

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.{{cite journal | vauthors = Eom SH, Wang J, Steitz TA | title = Structure of Taq polymerase with DNA at the polymerase active site | journal = Nature | volume = 382 | issue = 6588 | pages = 278–81 | date = July 1996 | pmid = 8717047 | doi = 10.1038/382278a0 | bibcode = 1996Natur.382..278E | s2cid = 4372845 }} 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.{{cite journal | vauthors = Bu Z, Biehl R, Monkenbusch M, Richter D, Callaway DJ | title = Coupled protein domain motion in Taq polymerase revealed by neutron spin-echo spectroscopy | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 102 | issue = 49 | pages = 17646–51 | date = December 2005 | pmid = 16306270 | pmc = 1345721 | doi = 10.1073/pnas.0503388102 | bibcode = 2005PNAS..10217646B | doi-access = free }}

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.{{cite journal | vauthors = Li Y, Mitaxov V, Waksman G | title = Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 96 | issue = 17 | pages = 9491–6 | date = August 1999 | pmid = 10449720 | pmc = 22236 | doi = 10.1073/pnas.96.17.9491 | bibcode = 1999PNAS...96.9491L | doi-access = free }}

Unlike the same domain in E. coli, which would degrade primers and must be removed by digestion for PCR use,{{cite book |doi=10.1007/978-1-4020-6241-4_7 |chapter=Taq and Other Thermostable DNA Polymerases |title=Principles and Technical Aspects of PCR Amplification |pages=103–18 |year=2008 |isbn=978-1-4020-6240-7 | vauthors = van Pelt-Verkuil E, van Belkum A, Hays JP }} this domain is not said to degrade the primer.{{cite web |title=Will the 5'→3' flap endonuclease activity of Taq DNA Polymerase degrade primers?|url=https://www.neb.com/faqs/2011/11/05/will-the-5-rarr-3-exonuclease-activity-of-em-taq-em-dna-polymerase-degrade-primers | archive-url = https://web.archive.org/web/20210417220639/https://www.neb.com/faqs/2011/11/05/will-the-5-rarr-3-exonuclease-activity-of-em-taq-em-dna-polymerase-degrade-primers | archive-date = 17 April 2021 | work = New England Biolabs (NEB) |access-date=28 March 2019}} 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.{{cite web | url = https://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/ProjTaqMan.shtml | title = TaqMan Gene Expression | work = National Center for Biotechnology Information (NCBI) Projects }}

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.

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.{{cite journal | vauthors = Park Y, Choi H, Lee DS, Kim Y | title = Improvement of the 3'-5' exonuclease activity of Taq DNA polymerase by protein engineering in the active site | journal = Molecules and Cells | volume = 7 | issue = 3 | pages = 419–24 | date = June 1997 | pmid = 9264032 | url = http://www.molcells.org/journal/download_pdf.php?spage=419&volume=7&number=3 }} 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.{{cite journal | vauthors = Villbrandt B, Sobek H, Frey B, Schomburg D | title = Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase | journal = Protein Engineering | volume = 13 | issue = 9 | pages = 645–54 | date = September 2000 | pmid = 11054459 | doi = 10.1093/protein/13.9.645 | doi-access = free }}

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.{{cite journal | vauthors = Ishino S, Ishino Y | title = DNA polymerases as useful reagents for biotechnology - the history of developmental research in the field | journal = Frontiers in Microbiology | volume = 5 | pages = 465 | date = 2014 | pmid = 25221550 | pmc = 4148896 | doi = 10.3389/fmicb.2014.00465 | doi-access = free }}

File:Taq polimerase.png

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."{{cite journal | vauthors = Menon PK, Kapila K, Ohri VC | title = Polymerase Chain Reaction and Advances in Infectious Disease Diagnosis | journal = Medical Journal, Armed Forces India | volume = 55 | issue = 3 | pages = 229–231 | date = July 1999 | pmid = 28775636 | pmc = 5531883 | doi = 10.1016/S0377-1237(17)30450-1 }} 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.{{cite web |url= https://stanfordhealthcare.org/medical-conditions/sexual-and-reproductive-health/hiv-aids/diagnosis/pcr.html |title=Polymerase chain reaction (PCR) |website=stanfordhealthcare.org |access-date=2020-04-23}}

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.{{Cite web|url=https://www.medtechdive.com/news/fda-chief-warns-of-supply-pressure-on-reagents-for-coronavirus-tests/573999/|title=FDA chief warns of supply 'pressure' on reagents for coronavirus tests|website=MedTech Dive|language=en-US|access-date=2020-04-23}}

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.{{cite journal | vauthors = Overbaugh J, Jackson SM, Papenhausen MD, Rudensey LM | title = Lentiviral genomes with G-to-A hypermutation may result from Taq polymerase errors during polymerase chain reaction | journal = AIDS Research and Human Retroviruses | volume = 12 | issue = 17 | pages = 1605–13 | date = November 1996 | pmid = 8947295 | doi = 10.1089/aid.1996.12.1605 }}

{{Commons category|Taq polymerase}}

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• Biological machines
• DNA polymerase I
• DNA replication
• Enzyme catalysis
• Protein domain dynamics

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{{Kinases}}

{{PCR}}

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Category:DNA replication

Category:Polymerase chain reaction