Thermostable DNA polymerases areDNA polymerases that originate fromthermophiles, usually bacterial or archaeal species, and are thereforethermostable. They are used for thepolymerase chain reaction and related methods for the amplification and modification ofDNA.

Several DNA polymerases have been described with distinct properties that define their specific utilisation in a PCR, inreal-time PCR or in anisothermal amplification. Being DNA polymerases, the thermostable DNA polymerases all have a 5'→3' polymerase activity, and either a 5'→3' or a 3'→5' exonuclease activity.

DNA polymerases are roughly shaped like a hand with a thumb, palm and fingers.^[12][13] The thumb is involved in binding and movingdouble-stranded DNA.^[12] The palm carries the polymeraseactive site, whereas the fingers bind substrates (template DNA andnucleoside triphosphates).^[12][14] Theexonuclease activity is in a separateprotein domain.^[12]Mg²⁺ is acofactor.

The polymerase active site in the palm catalyses the prolongation of DNA, starting from aprimer bound to a template DNA single strand:

deoxynucleoside triphosphate + DNA_n ⇌pyrophosphate + DNA_n+1.

Thermostable DNA polymerases of natural origin are found in thermophilicbacteria,archaea and their pathogens. Among the bacterial thermostable DNA polymerases,Taq polymerase, Tfl polymerase, Tma polymerase, Tne polymerase, Tth and Bst polymerase are used.^{[4][15][16][2]}

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 anadenosine overhang (sticky ends) at the 3' end of the newly generated strand. TheKlenow fragment of Bst (BF) has a strand displacement activity which allows for use in isothermal amplification without the necessity ofdenaturation of the DNA in athermocycler, and its 5'→3' exonuclease activity is deleted for higher yield.^[2]

Frequently used B-type DNA polymerases are thePfu polymerase,^[4] the Pwo polymerase,^[17] the KOD polymerase,^[3] the Tli polymerase (also called Vent), which originates from various archaea,^[18] the Tag polymerase,^[19] the Tce polymerase,^[20] the Tgo polymerase,^[8] the TNA1 polymerase,^[21] the Tpe polymerase,^[22] the Tthi polymerase,^[23] the Neq polymerase^[24] and the Pab polymerase.^[25]

The archaeal variants (belonging to the B-type) produceblunt 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.^[26][27] In archaeal polymerases, the error rate suffers when a Klenow fragment analogue is generated, as the correcting exonuclease activity is removed in the process.^[4] Some archaeal DNA polymerases are characterised less by their suitability for standard PCR than by their reduced inhibition in the amplification ofA-DNA^[28] or DNA with modified bases.^[29][30]

Variousfusion 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 theDNA clamp of the thermostable DNA-binding proteinSSo7d byprotein design.^[31] A fusion protein of thePCNA homologue fromArchaeoglobus fulgidus was also generated with archaeal thermostable DNA polymerases.^[32] Similarly, fusion proteins of thermostable DNA polymerases with the thermostable DNA-binding protein domain of atopoisomerase (type V, withhelix-hairpin-helix motif, HhH) fromMethanopyrus kandleri were generated (TopoTaq andPfuC2).^[33][34] A modified Pfu polymerase was also generated by protein design (Pfu Ultra).^[35] 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,^[22][36] e.g.Herculase^[8] andTaqPlus^[10] as a commercial mixture of Taq and Pfu polymerase,Expand as a commercial mixture of Taq and Pwo,^[37]Expand High Fidelity as a commercial mixture of Taq and Tgo,^[10]Platinum Taq High Fidelity as a commercial mixture of Taq and Tli (Vent),^[10] andAdvantage HF 2 as a commercial mixture of Titanium Taq and an unnamed proof-reading polymerase.^[10] These mixtures can be used for long-range PCR to synthesize products of up to 35kb length.^[36][38] Other additives are used to help against difficultGC-rich sequences, avoid or neutralise the negative effects ofPCR inhibitors (like blood components or detergents^[39] ordUTP^[40]), or alter thereaction kinetics.^[41]

The baseline synthesis rates (speed, productivity) of various polymerases 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).^[11] Among the modified thermostable DNA polymerases, various mutations have been described that increase the synthesis rate.^[42][43][44] KOD polymerase and some modified thermostable DNA polymerases (iProof/Phusion,Pfu Ultra,Velocity orZ-Taq) are used as a PCR variant with shorter amplification cycles (fast PCR, high-speed PCR) due to their high synthesis rate. Processivity describes the average number of base pairs before a polymerase falls off the DNA template. The processivity of the polymerase limits the maximum distance between the primer and the probe in some forms of real-time quantitative PCR (qPCR).

The error rates of various polymerases (fidelity) have been described. The error rate of Taq polymerase is 8 × 10⁻⁶ errors per base, that ofAdvantage HF 6.1 × 10⁻⁶ errors per base, that ofPlatinum Taq High Fidelity 5.8 × 10⁻⁶ errors per base and doubling, that ofTaqPlus 4 × 10⁻⁶ errors per base and doubling, that of KOD polymerase 3.5 × 10⁻⁶ errors per base and doubling, that of Tli polymerase andHerculase 2.8 × 10⁻⁶ errors per base and doubling, that ofDeep Vent 2.8 × 10⁻⁶ errors per base and doubling, that of Pfu,Phusion DNA Polymerase (identical withiProof DNA Polymerase) andHerculase II Fusion 1.3 × 10⁻⁶ errors per base and doubling and that ofPfu Ultra andPfu Ultra II 4.3 × 10⁻⁷ errors per base and doubling.^[4][8][10] A newer analysis found slightly different error rates:Deep Vent (exo-) polymerase (5.0 × 10⁻⁴ errors per base and doubling), Taq polymerase (1.5 × 10⁻⁴ errors per base and doubling),Kapa HiFi HotStart ReadyMix (1.6 × 10⁻⁵ errors per base and doubling), KOD (1.2 × 10⁻⁵ errors per base and doubling),PrimeSTAR GXL (8.4 × 10⁻⁶ errors per base and doubling), Pfu (5.1 × 10⁻⁶ errors per base and doubling),Deep Vent DNA polymerase (4.0 × 10⁻⁶) errors per base and doubling,Phusion (3.9 × 10⁻⁶ errors per base and doubling), andQ5 DNA polymerase (5.3 × 10⁻⁷ errors per base and doubling).^[5] Yet another found error rates of 3–5.6 × 10⁻⁶ for Taq, 7.6 × 10⁻⁶ for KOD, 2.8 × 10⁻⁶ for Pfu, 2.6 × 10⁻⁶ forPhusion, and 2.4 × 10⁻⁶ for Pwo.^[6] To reduce the number of mutations in the PCR product (e.g. formolecular cloning), more template DNA and less cycles can be used in the PCR.^[10]

Bacterial thermostable DNA polymerases generally produce higher product concentrations than archaeal, but with more copy errors. In the bacterial thermostable DNA polymerases, a Klenow fragment (Klen-Taq) or aStoffel fragment can be generated by deleting the exonuclease domain in the course of protein design, analogous to the DNA polymerase fromE. coli, which results in a higher product concentration.^[45][15] Twoamino acids required for the exonuclease function of Taq polymerase were identified bymutagenesis asarginines at positions 25 and 74 (R25 and R74).^[46] Ahistidine toglutamic acid mutation at position 147 (short: H147E) in KOD polymerase lowers the relatively high exonuclease activity of KOD.^[27]

The favouring of individualnucleotides by a thermostable DNA polymerase is referred to as nucleotide specificity (bias). In PCR-basedDNA sequencing with chain termination substrates (dideoxy method), their uniform incorporation and thus unbiased generation of all chain termination products is often desired in order to enable higher sensitivity and easier analysis. For this purpose, aKlenTaq polymerase was generated by deletion and aphenylalanine at position 667 was exchanged fortyrosine bysite-directed mutagenesis (short: F667Y) and namedThermo Sequenase.^[47][48] This polymerase can also be used for the incorporation of fluorescence-labelled dideoxynucleotides.^[49]

The template specificity of the polymerases is increased by using hot-start polymerases, to avoid binding of primers to unwanted DNA templates or to each other at low temperatures before the beginning of the PCR.^[50] Examples are theantibody-inhibited Pfu polymerasePfu Turbo, thePlatinum Pfx as a commercial KOD polymerase with an inhibiting antibody and thePlatinum Taq as an antibody-inhibited Taq polymerase.^[8] Hot-start polymerases are either inhibited by inactivation withformaldehyde^[51][52] (ormaleic anhydride, exo-cis-3,6-endoxo-Δ4-tetrahydropthalic anhydride, citraconic anhydride, 3,4,5,6-tetrahydrophthalic anhydride, cis-aconitic anhydride, or 2,3-dimethylmaleic anhydride),^[53] by complexing the magnesium with phosphates^[54] or by binding an antibody to their active site.^[55][56] Upon heating to 95 °C, the formaldehyde dissociates from proteins,^[57][58][59] or the magnesium ions are released,^[54] or the antibody is denatured and released in the process.^[60][61] Furthermore, polymerases can be inhibited withaptamers that denature upon heating.^[62][63] A fifth variant is a polymerase adsorbed onlatex beads viahydrophobic effects, which dissolves with increasing temperature. In the sixth and oldest variant, the reaction mixture without polymerase is coated withwax and the polymerase is added on top of the cooled wax. When heated, the wax layer melts and the polymerase mixes with the reaction mixture.^[64]

Some DNA polymerases used in isothermal DNA amplification, e.g. inloop-mediated isothermal amplification,multidisplacement amplification,recombinase polymerase amplification orisothermal assembly, for the amplification of entiregenomes (e.g. theφ29 DNA polymerase from thebacteriophage phi29, B35DNAP from the phageBam35) are not thermostable, while others like the Bst Klenow fragment are thermostable.^[65] The T4, T6 andT7 DNA polymerases are also not thermostable.

The standardreverse transcriptases (RNA-dependent DNA polymerases) ofretroviral origin used forRT-PCR, like theAMV- and theMoMuLV-Reverse-Transcriptase, are not thermostable at 95 °C. At the lower temperatures of areverse transcription unspecifichybridisation ofprimers to wrong sequences can occur, as well as unwantedsecondary structures in the DNA template, which can lead to unwanted PCR products and less desired PCR products. The AMV reverse transcriptase may be used up to 70 °C.^[66] Also, some thermostable DNA-dependent DNA polymerases can be used as RNA-dependent DNA polymerases by exchanging Mg²⁺ ascofactors withMn²⁺, so that they may be used for an RT-PCR.^[67] But since the synthesis rate of Taq with Mn²⁺ is relatively low, Tth was increasingly used for this approach.^[68] The use of Mn²⁺ also increases the error rate and the necessary amount of template, so that this method is rarely used. These problems can be avoided with the thermostable3173-Polymerase from a thermophilicbacteriophage, which can withstand the high temperatures of a PCR and prefers RNA as a template.^[69]

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 forRT-PCR variants, qPCR in different variants, site-specific mutagenesis and DNA sequencing. They are also used to producehybridisation probes forSouthern blot andNorthern blot by random priming. The 5'→3' exonuclease activity is used fornick translation andTaqMan, among other things, without DNA replication (amplification).

Alice Chien and colleagues were the first to characterise the thermostable Taq polymerase in 1976.^[70] The first use of a thermostable DNA polymerase was byRandall K. Saiki and colleagues in 1988, introducing Taq polymerase for PCR.^[71][72] The thermostability of Taq polymerase obliviated the need to add a non-thermostable DNA polymerase to the reaction after everymelting phase of the PCR, because the Taq polymerase is not denatured by heating to 95 °C during the melting phase of each cyle. In 1989, the Taq polymerasegene was cloned and the Taq polymerase was produced inEscherichia coli as arecombinant protein.^[73][72] DNA of up to 35,000 basepairs was synthesized byWayne M. Barnes by using different mixtures of A and B type polymerases,^[36][72] thereby creating the long-range PCR. The high synthesis rate of KOD polymerase was published in 1997 byMasahiro Takagi and colleagues,^[3][72][14] thereby creating the fundamentals of high speed PCR. Other optimisations to the PCR were developed in the following years, e.g. circumventing PCR inhibitors and amplifying difficult GC-rich DNA sequences,^[41] as well as modifying thermostable DNA polymerases by protein design. In 1998 the loop-mediated isothermal amplification was developed by Tsugunori Notomi and colleagues atEiken Chemical Company, using Bst polymerase at 65 °C.^[74][75]

• J. Sambrook, T. Maniatis, D. W. Russel:Molecular cloning: a laboratory manual.Cold Spring Harbor Laboratory Press; 3rd edition (2001), ISBN 0-87969-577-3.
• F. Akram, F. I. Shah, R. Ibrar, T. Fatima, I. U. Haq, W. Naseem, M. A. Gul, L. Tehreem, G. Haider:Bacterial thermophilic DNA polymerases: A focus on prominent biotechnological applications. In:Analytical biochemistry. Volume 671, June 2023, p. 115150,doi:10.1016/j.ab.2023.115150,PMID 37054862.
• K. Terpe:Overview of thermostable DNA polymerases for classical PCR applications: from molecular and biochemical fundamentals to commercial systems. In:Applied Microbiology and Biotechnology. Volume 97, issue 24, December 2013, p. 10243–10254,doi:10.1007/s00253-013-5290-2,PMID 24177730.

• NEB Polbase. Accessed September 27, 2012.

• DNA replication
• Biochemistry
• Molecular biology
• Polymerase chain reaction

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