Thermostable DNA polymerase

From Wikipedia, the free encyclopedia
Taq-DNA-Polymerase with exonuclease- (top left) and polymerase domain with DNA (bottom right)

Thermostable DNA polymerases are DNA polymerases that originate from thermophiles, usually bacterial or archaeal species, and are therefore thermostable. They are used for the polymerase chain reaction and related methods for the amplification and modification of DNA.

Bacterial polymerases[edit]

Thermostable DNA polymerases of natural origin are found in thermophilic bacteria, archaea and their pathogens. Among the bacterial thermostable DNA polymerases, Taq polymerase, Tfl polymerase, Tma polymerase, Tne polymerase and Tth polymerase are used.[1][2][3]

In addition to 5'→3' polymerase activity, the bacterial thermostable DNA polymerases (belonging to the A-type DNA polymerases) have 5'→3' exonuclease activity and generate an adenosine overhang (sticky ends) at the 3' end of the newly generated strand. Processivity describes the average number of base pairs before a polymerase falls off the DNA template. The processivity of the polymerase used limits the maximum distance between the primer and the probe in real-time quantitative PCR (qPCR). The processivity of a Taq polymerase is around 200 base pairs.

Archaeal polymerases[edit]

Pfu polymerase with two magnesium ions (grey spheres)

Frequently used B-type DNA polymerases are the Pfu polymerase,[1] the Pwo polymerase, the KOD polymerase,[4] the Tli polymerase (also called Vent), which originates from various archaea,[5] the Tag polymerase,[6] the Tce polymerase,[7] the Tgo polymerase,[8] the TNA1 polymerase,[9] the Tpe polymerase,[10] the Tthi polymerase,[11] the Neq polymerase[12] and the Pab polymerase.[13]

The archaeal variants (belonging to the B-type) produce blunt ends (the Tli polymerase produces an overhang in about 30 % of the products) and instead of the 5'→3' exonuclease activity have an activity for correcting synthesis errors (proof-reading), the 3'→5' exonuclease activity.[14][15] In archaeal polymerases, the error rate suffers when an analogue Klenow fragment is generated, as the correcting exonuclease activity is removed in the process.[1] Some archaeal DNA polymerases are characterised less by their suitability for standard PCR than by their reduced inhibition in the amplification of A-DNA.[16]

Modified polymerases[edit]

Various fusion proteins with the low error rate of archaeal and the high synthesis rate of bacterial thermostable DNA polymerases (Q5 polymerase) were generated from various thermostable polymerases and the DNA clamp of the thermostable DNA-binding protein SSo7d by protein design.[17] A fusion protein of the PCNA homologue from Archaeoglobus fulgidus was also generated with archaeal thermostable DNA polymerases.[18] Similarly, fusion proteins of thermostable DNA polymerases with the thermostable DNA-binding protein domain of a topoisomerase (type V, with helix-hairpin-helix motif, HhH) from Methanopyrus kandleri were generated (TopoTaq and PfuC2).[19][20] A modified Pfu polymerase was also generated by protein design (Pfu Ultra).[21] Similar effects are also achieved with mixtures of thermostable DNA polymerases of both types with a mixing ratio of the enzyme activities of type A and B polymerases of 30 to 1,[10][22] e.g. Herculase as a commercial mixture of Taq and Pfu polymerase.[8]

The baseline synthesis rates of various polymerases (processivity, productivity) have been compared.[8] The synthesis rate of Taq polymerase is around 60 base pairs per second. Among the unmodified thermostable DNA polymerases, only the synthesis rate of KOD polymerase is above 100 base pairs per second (approx. 120 bp/s).[23] Among the modified thermostable DNA polymerases, various mutations have been described that increase the synthesis rate.[24][25] KOD polymerase and some modified thermostable DNA polymerases (iProof, Pfu Ultra, Phusion, Velocity or Z-Taq) are used as a PCR variant with shorter amplification cycles (fast PCR, high-speed PCR) due to their high synthesis rate.

The error rates of various polymerases (fidelity) are known and have been described. The error rate of Taq polymerase is 8 - 10-6 errors per base pair, that of KOD polymerase 3.5 - 10-6 errors per base pair, that of Tli polymerase and Herculase 2.8 - 10-6 errors per base pair, that of Pfu polymerase 1.3 - 10-6 errors per base pair and that of Pfu Ultra 4.3 - 10-7 errors per base pair.[1][8]

In the bacterial thermostable DNA polymerases, a Klenow fragment (Klen-Taq) or a Stoffel fragment can be generated by deleting the exonuclease domain in the course of protein design, analogous to the DNA polymerase from E. coli, which results in a higher product concentration.[26][2] Two amino acids required for the exonuclease function of Taq polymerase were identified by mutagenesis as arginines at positions 25 and 74 (R25 and R74).[27]

The favouring of individual nucleotides by a thermostable DNA polymerase is referred to as nucleotide specificity (bias). In PCR-based DNA sequencing with chain termination substrates (dideoxy method), their uniform incorporation and thus uniform generation of all chain termination products is often desired in order to enable higher sensitivity and easier analysis. For this purpose, a KlenTaq polymerase was generated by deletion and a phenylalanine at position 667 was exchanged for tyrosine by site-directed mutagenesis (short: F667Y) and named Thermo Sequenase.[28][29] This polymerase can also be used for the incorporation of fluorescence-labelled dideoxynucleotides.[30]

Other DNA polymerases[edit]

The DNA polymerases used in isothermal DNA amplification, e.g. in multidisplacement amplification, recombinase polymerase amplification or isothermal assembly, for the amplification of entire genomes (e.g. the φ29 DNA polymerase from the bacteriophage phi29) are not thermostable. The T4, T6 and T7 DNA polymerases are also not thermostable.

Applications[edit]

Main article: PCR optimization

In addition to the choice of thermostable DNA polymerase, other parameters of a PCR are specifically changed in the course of PCR optimisation.

In addition to PCR, thermostable DNA polymerases are also used for RT-PCR variants, qPCR in different variants, site-specific mutagenesis and DNA sequencing. They are also used to produce hybridisation probes for Southern blot and Northern blot by random priming. The 5'→3' exonuclease activity is used for nick translation and TaqMan, among other things, without DNA replication (amplification).

Literature[edit]

Weblinks[edit]

References[edit]

  1. ^ a b c d J. Cline, J. C. Braman, H. H. Hogrefe: PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. In: Nucleic Acids Res., Volume 24, Issue 18, 1996, p. 3546–3551. PMID 8836181; PMC 146123.
  2. ^ a b B. Villbrandt, H. Sobek, B. Frey, D. Schomburg: Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase. In: Protein Eng., Vol. 13, Issue 9, 2000, p. 645–654. PMID 11054459.
  3. ^ W. Abu Al-Soud, P. Râdström: Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. In: Appl. Environ. Microbiol., Vol. 64, Issue 10, 1998, p. 3748–3753. PMID 9758794; PMC 106538.
  4. ^ M. Takagi, M. Nishioka, H. Kakihara, M. Kitabayashi, H. Inoue, B. Kawakami, M. Oka, T. Imanaka: Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. In: Appl. Environ. Microbiol., Volume 63, Issue 11, 1997, p. 4504–4510. PMID 9361436; PMC 168769.
  5. ^ H. Kong, R. B. Kucera, W. E. Jack: Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities. In: J Biol Chem., Volume 268, Issue 3, 1993, p. 1965–1975. PMID 8420970.
  6. ^ K. Böhlke, F. M. Pisani, C. E. Vorgias, B. Frey, H. Sobek, M. Rossi, G. Antranikian: PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A motif. In: Nucleic Acids Res., Volume 28, Issue 20, 2000, p. 3910–3917. PMID 11024170; PMC 110800.
  7. ^ K. P. Kim, H. Bae, I. H. Kim, p. T. Kwon: Cloning, expression, and PCR application of DNA polymerase from the hyperthermophilic archaeon, Thermococcus celer. In: Biotechnol Lett. (2011), Volume 33(2), p. 339–46. PMID 20953664.
  8. ^ a b c d Bahram Arezi, Weimei Xing, Joseph A. Sorge, Holly H. Hogrefe (2003-10-15), "Amplification efficiency of thermostable DNA polymerases" (PDF), Analytical Biochemistry, vol. 321, no. 2, pp. 226–235, doi:10.1016/S0003-2697(03)00465-2, PMID 14511688{{citation}}: CS1 maint: multiple names: authors list (link)
  9. ^ Y. Cho, H. p. Lee, Y. J. Kim, p. G. Kang, p. J. Kim, J. H. Lee: Characterization of a dUTPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in polymerase chain reaction amplification. In: Mar Biotechnol (NY), Volume 9, Issue 4, 2007, p. 450–458. PMID 17549447.
  10. ^ a b J. I. Lee, Y. J. Kim, H. Bae, p. p. Cho, J. H. Lee, p. T. Kwon: Biochemical properties and PCR performance of a family B DNA polymerase from hyperthermophilic euryarchaeon Thermococcus peptonophilus. In: Appl Biochem Biotechnol., Volume 160, Issue 6, 2010, p. 1585–1899. PMID 19440663.
  11. ^ D. Marsic, J. M. Flaman, J. D. Ng: New DNA polymerase from the hyperthermophilic marine archaeon Thermococcus thioreducens. In: Extremophiles, Volume 12, Issue 6, 2008, p. 775–788. PMID 18670731.
  12. ^ J. G. Song, E. J. Kil, p. p. Cho, I. H. Kim, p. T. Kwon: An amino acid residue in the middle of the fingers subdomain is involved in Neq DNA polymerase processivity: enhanced processivity of engineered Neq DNA polymerase and its PCR application. In: Protein Eng. Des. Sel., Volume 23, Issue 11, 2010, p. 835–842. PMID 20851826.
  13. ^ J. Dietrich, P. Schmitt, M. Zieger, B. Preve, J. L. Rolland, H. Chaabihi, Y. Gueguen: PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi. In: FEMS Microbiol Lett., Volume 217, Issue 1, 2002, p. 89–94. PMID 12445650.
  14. ^ E. M. Kennedy, C. Hergott, p. Dewhurst, B. Kim: The mechanistic architecture of thermostable Pyrococcus furiosus family B DNA polymerase motif A and its interaction with the dNTP substrate. In: Biochemistry (2009), Volume 48(47), p. 11161–8. PMID 19817489; PMC 3097049.
  15. ^ T. Kuroita, H. Matsumura, N. Yokota, M. Kitabayashi, H. Hashimoto, T. Inoue, T. Imanaka, Y. Kai: Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases. In: J Mol Biol. (2005), Volume 351(2), p. 291–8. PMID 16019029.
  16. ^ J. P. McDonald, A. Hall, D. Gasparutto, J. Cadet, J. Ballantyne, R. Woodgate: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs. In: Nucleic Acids Res. (2006), Volume 34(4), p. 1102–11. PMID 16488882; PMC 1373694.
  17. ^ Y. Wang, D. E. Prosen, L. Mei, J. C. Sullivan, M. Finney, P. B. Vander Horn: A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. In: Nucleic Acids Res. (2004), Volume 32(3), p. 1197–207. PMID 14973201; PMC 373405.
  18. ^ M. Motz, I. Kober, C. Girardot, E. Loeser, U. Bauer, M. Albers, G. Moeckel, E. Minch, H. Voss, C. Kilger, M. Koegl: Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes. In: J Biol Chem. (2002), Volume 277(18), p. 16179–88. PMID 11805086. PDF.
  19. ^ P. Forterre (2006), "DNA topoisomerase V: a new fold of mysterious origin", Trends Biotechnol, vol. 24, no. 6, pp. 245–247, doi:10.1016/j.tibtech.2006.04.006, PMID 16650908
  20. ^ A. R. Pavlov, N. V. Pavlova, p. A. Kozyavkin, A. I. Slesarev (2004), "Recent developments in the optimization of thermostable DNA polymerases for efficient applications", Trends Biotechnol, vol. 22, no. 5, pp. 253–260, doi:10.1016/j.tibtech.2004.02.011, PMID 15109812{{citation}}: CS1 maint: multiple names: authors list (link)
  21. ^ Holly H. Hogrefe, M. Borns: High fidelity PCR enzymes. In: C.W. Dieffenbach, G.p. Dveksler (Eds.): PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003.
  22. ^ W. M. Barnes: PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. In: Proc Natl Acad Sci U S A (1994), Volume 91(6), p. 2216–20. PMID 8134376; PMC 43341.
  23. ^ European Patent 1752534A1: Hochgeschwindigkeits-PCR unter Verwendung von Hochgeschwindigkeits-DNA-Polymerase, 2005-05-12 / 2007-02-14 by Toyo Boseki (applicant) & Masaya Segawa et al. (inventors).
  24. ^ US Patent 2013034879A1: DNA Polymerases, 2012-08-02 / 2007-02-14, Fermentas UAB et Al (applicant), Remigijus Skirgaila et al. (inventors).
  25. ^ US Patent 2009280539A1: DNA Polymerases and related methods, 2009-04-16 / 2009-11-12, Roche Molecular Systems Inc (applicant), Keith A. Bauer (inventor).
  26. ^ W. M. Barnes: The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion. In: Gene (1992), Volume 112(1), p. 29–35. PMID 1551596.
  27. ^ L. p. Merkens, p. K. Bryan, R. E. Moses: Inactivation of the 5'-3' exonuclease of Thermus aquaticus DNA polymerase. In: Biochim Biophys Acta (1995), Volume 1264(2), p. 243–8. PMID 7495870.
  28. ^ p. Tabor, C. C. Richardson: A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. In: Proc Natl Acad Sci U S A., Volume 92, Issue 14, 1995, p. 6339–6343. PMID 7603992; PMC 41513.
  29. ^ P. B. Vander Horn, M. C. Davis, J. J. Cunniff, C. Ruan, B. F. McArdle, p. B. Samols, J. Szasz, G. Hu, K. M. Hujer, p. T. Domke, p. R. Brummet, R. B. Moffett, C. W. Fuller: Thermo sequenase DNA polymerase and T. acidophilum pyrophosphatase: new thermostable enzymes for DNA sequencing. In: Biotechniques, Volume 22, Issue 4, 1997, p. 758–762, 764–765. PMID 9105629.
  30. ^ J. M. Prober, G. L. Trainor, R. J. Dam, F. W. Hobbs, C. W. Robertson, R. J. Zagursky, A. J. Cocuzza, M. A. Jensen, K. Baumeister: A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. In: Science, Volume 238, Issue 4825, 1987, p. 336–341. PMID 2443975.
Retrieved from "https://en.wikipedia.org/w/index.php?title=Thermostable_DNA_polymerase&oldid=1226275779"