Thermostable DNA polymerase

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.

Properties

Several DNA polymerases have been described with distinct properties that define their specific utilisation in a PCR, in real-time PCR or in an isothermal 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.

Properties of thermostable DNA polymerases

Polymerase	Taq	Tfl	Tth	Bst Klenow fragment (BF), strand displacing	Tli (Vent)	P GB-D (Deep Vent)	Pfx (KOD)	Pfu
Organism	Thermus aquaticus	Thermus flavus	Thermus thermophilus	Geobacillus stearothermophilus	Thermococcus litoralis	Pyrococcus sp. strain GB-D	Pyrococcus kodakarensis	Pyrococcus furiosus
Origin	bacterial	bacterial	bacterial	bacterial	archaeal	archaeal	archaeal	archaeal
Molecular weight	80kDa [1]	94kDa [1]	94kDa [1]	67kDa [2]	90kDa [1]	90kDa [3]	90kDa [3]	92kDa [1]
Extension Temperature	74 °C [1]	74 °C [1]	74 °C [1]	65 °C [2]	74 °C [1]	75 °C [3]	75 °C [3]	75 °C [1]
5′→3′ Exonuclease Activity	Yes [1]	Yes [1]	Yes [1]	No [2]	No [1]			No [1]
3′→5′ Exonuclease Activity	No [1]	No [1]	No [1]	No [2]	Yes [1]	Yes [3]	Yes [3]	Yes [1]
Reverse Transcriptase Activity	Weak [1]	Yes [1]	Yes [1]	Weak [2]	No [1]			N/A [1]
PCR Ends	3′-A [1]	3′-A [1]	3′-A [1]	3′-A [2]	70% Blunt; 30% Single-base [1]	Blunt [3]	Blunt [3]	Blunt [1]
Fidelity (errors per base and doubling)	8 × 10−6 [4] 1.5 × 10−4 [5] 3-5.6 × 10−5 [6]			1.5 × 10−5 [7]	2.8 × 10−6 [4]	2.7 × 10−6 [4] 4.0 × 10−6 [5]	3.5 × 10−6 [8] 1.2 × 10−5 [5] 7.6 × 10−6 [6]	1.3 × 10−6 [9] [4] 5.1 × 10−6 [5] 2.8 × 10−6 [6]
Synthesis rate (bases/sec.)	21–47 [2] 61 [10]			191 [2]		23 [10]	120 [11] 106–138 [10]	9.3–25 [10]
Processivity (bases)	10–42 [10]					<20 [10]	>300 [10]	6.4–20 [10]

Structure

DNA polymerases are roughly shaped like a hand with a thumb, palm and fingers. ^{[12] [13]} The thumb is involved in binding and moving double-stranded DNA. ^[12] The palm carries the polymerase active site, whereas the fingers bind substrates (template DNA and nucleoside triphosphates). ^{[12] [14]} The exonuclease activity is in a separate protein domain. ^[12] Mg²⁺ is a cofactor.

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

deoxynucleoside triphosphate + DNA_n⇌ pyrophosphate + DNA_n+1.

Bacterial polymerases

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

Archaeal polymerases

Pfu polymerase with two magnesium ions (grey spheres)

Frequently used B-type DNA polymerases are the Pfu 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) 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. ^{[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 of A-DNA ^[28] or DNA with modified bases. ^{[29] [30]}

Modified polymerases

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. ^[31] A fusion protein of the PCNA homologue from Archaeoglobus 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 a topoisomerase (type V, with helix-hairpin-helix motif, HhH) from Methanopyrus kandleri were generated (TopoTaq and PfuC2). ^{[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] and TaqPlus ^[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] and Advantage 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 difficult G C-rich sequences, avoid or neutralise the negative effects of PCR inhibitors (like blood components or detergents ^[39] or dUTP ^[40] ), or alter the reaction kinetics. ^[41]

Speed & Processivity

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 or Z-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).

Fidelity

The error rates of various polymerases (fidelity) have been described. The error rate of Taq polymerase is 8 × 10⁻⁶ errors per base, that of Advantage HF 6.1 × 10⁻⁶ errors per base, that of Platinum Taq High Fidelity 5.8 × 10⁻⁶ errors per base and doubling, that of TaqPlus 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 and Herculase 2.8 × 10⁻⁶ errors per base and doubling, that of Deep Vent 2.8 × 10⁻⁶ errors per base and doubling, that of Pfu, Phusion DNA Polymerase (identical with iProof DNA Polymerase) and Herculase II Fusion 1.3 × 10⁻⁶ errors per base and doubling and that of Pfu Ultra and Pfu 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), and Q5 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 for Phusion, and 2.4 × 10⁻⁶ for Pwo. ^[6] To reduce the number of mutations in the PCR product (e.g. for molecular cloning), more template DNA and less cycles can be used in the PCR. ^[10]

Yield

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 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. ^{[45] [15]} 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). ^[46] A histidine to glutamic acid mutation at position 147 (short: H147E) in KOD polymerase lowers the relatively high exonuclease activity of KOD. ^[27]

Nucleotide specificity

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 unbiased 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. ^{[47] [48]} This polymerase can also be used for the incorporation of fluorescence-labelled dideoxynucleotides. ^[49]

Hot-start thermostable DNA polymerases

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 the antibody-inhibited Pfu polymerase Pfu Turbo, the Platinum Pfx as a commercial KOD polymerase with an inhibiting antibody and the Platinum Taq as an antibody-inhibited Taq polymerase. ^[8] Hot-start polymerases are either inhibited by inactivation with formaldehyde ^{[51] [52]} (or maleic 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 with aptamers that denature upon heating. ^{[62] [63]} A fifth variant is a polymerase adsorbed on latex beads via hydrophobic effects, which dissolves with increasing temperature. In the sixth and oldest variant, the reaction mixture without polymerase is coated with wax 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]

Other DNA polymerases

Some DNA polymerases used in isothermal DNA amplification, e.g. in loop-mediated isothermal amplification, multidisplacement amplification, recombinase polymerase amplification or isothermal assembly, for the amplification of entire genomes (e.g. the φ29 DNA polymerase from the bacteriophage phi29, B35DNAP from the phage Bam35) are not thermostable, while others like the Bst Klenow fragment are thermostable. ^[65] The T4, T6 and T7 DNA polymerases are also not thermostable.

RNA-dependent DNA polymerases

The standard reverse transcriptases (RNA-dependent DNA polymerases) of retroviral origin used for RT-PCR, like the AMV- and the MoMuLV-Reverse-Transcriptase, are not thermostable at 95 °C. At the lower temperatures of a reverse transcription unspecific hybridisation of primers to wrong sequences can occur, as well as unwanted secondary 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⁺ as cofactors with Mn²⁺, 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 thermostable 3173-Polymerase from a thermophilic bacteriophage, which can withstand the high temperatures of a PCR and prefers RNA as a template. ^[69]

Applications

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).

History

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 by Randall 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 every melting 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 polymerase gene was cloned and the Taq polymerase was produced in Escherichia coli as a recombinant protein. ^{[73] [72]} DNA of up to 35,000 basepairs was synthesized by Wayne 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 by Masahiro 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 at Eiken Chemical Company, using Bst polymerase at 65 °C. ^{[74] [75]}

Further reading

• 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.
• Akram F, Shah FI, Ibrar R, Fatima T, Haq IU, Naseem W, Gul MA, Tehreem L, Haider G (2023). "Bacterial thermophilic DNA polymerases: A focus on prominent biotechnological applications". Anal. Biochem. 671 115150. doi:10.1016/j.ab.2023.115150. PMID   37054862..
• Terpe K (2013). "Overview of thermostable DNA polymerases for classical PCR applications: from molecular and biochemical fundamentals to commercial systems". Appl. Microbiol. Biotechnol. 97 (24): 10243–54. doi:10.1007/s00253-013-5290-2. PMID   24177730..

External links

• NEB Polbase. Accessed September 27, 2012.

References

1. Promega: Properties of Thermostable DNA Polymerases (PDF; 208 kB). Accessed september 27, 2012.
2. Oscorbin I., Filipenko M. (2023). "2023 Bst polymerase - a humble relative of Taq polymerase". Computational and Structural Biotechnology Journal. 21: 4519–4535. doi:10.1016/j.csbj.2023.09.008. PMC   10520511 . PMID   37767105.
3. Takagi M., Nishioka M., Kakihara H., Kitabayashi M., Inoue H., Kawakami B., Oka M., Imanaka T. (1997). "Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR". Appl. Environ. Microbiol. 63 (11): 4504–4510. Bibcode:1997ApEnM..63.4504T. doi:10.1128/aem.63.11.4504-4510.1997. PMC   168769 . PMID   9361436.
4. Cline J, Braman JC, Hogrefe HH (September 1996). "PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases". Nucleic Acids Res. 24 (18): 3546–51. doi:10.1093/nar/24.18.3546. PMC   146123 . PMID   8836181.
5. Potapov V, Ong JL (2017). "Examining Sources of Error in PCR by Single-Molecule Sequencing". PLOS ONE. 12 (1) e0169774. Bibcode:2017PLoSO..1269774P. doi: 10.1371/journal.pone.0169774 . PMC   5218489 . PMID   28060945.
6. McInerney P., Adams P., Hadi M. Z. (2014). "Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase". Molecular Biology International. 2014 287430. doi: 10.1155/2014/287430 . PMC   4150459 . PMID   25197572.
7. Hong Guo Fan, Zhai Feng, Huang Wei-Hua: EP0810288 (A2) ― 1997-12-03: A new DNA polymerase with proof-reading 3'-5' exonuclease activity
8. 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
9. H.H. Hogrefe, M. Borns, High fidelity PCR enzymes, in: C.W. Dieffenbach, G.S. Dveksler (Eds.), PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003.
10. Agilent: High-Fidelity PCR Enzymes: Properties and Error Rate Determinations.
11. 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).
12. Steitz T. A. (1999). "DNA polymerases: structural diversity and common mechanisms". Journal of Biological Chemistry . 274 (25): 17395–17398. doi: 10.1074/jbc.274.25.17395 . PMID   10364165.
13. Rothwell P. J.; Waksman G. (2005). "2005 Structure and mechanism of DNA polymerases". Advances in Protein Chemistry. 71: 401–440. doi:10.1016/S0065-3233(04)71011-6. PMID   16230118.
14. Hashimoto H., Nishioka M., Fujiwara P., Takagi M., Imanaka T., Inoue T., Kai Y. (2001). "Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD". Journal of Molecular Biology. 306 (3): 469–477. doi:10.1006/jmbi.2000.4403. PMID   11178906.
15. Villbrandt B, Sobek H, Frey B, Schomburg D (2000). "Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase". Protein Eng. 13 (9): 645–54. doi:10.1093/protein/13.9.645. PMID   11054459.
16. Abu Al-Soud W, Râdström P (1998). "Capacity of nine thermostable DNA polymerases To mediate DNA amplification in the presence of PCR-inhibiting samples". Appl. Environ. Microbiol. 64 (10): 3748–53. Bibcode:1998ApEnM..64.3748A. doi:10.1128/AEM.64.10.3748-3753.1998. PMC   106538 . PMID   9758794.
17. Ghasemi A, Salmanian AH, Sadeghifard N, Salarian AA, Gholi MK (2011). "Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei". Iran J. Microbiol. 3 (3): 118–22. PMC   3279813 . PMID   22347593.
18. Kong H, Kucera RB, Jack WE (1993). "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". J. Biol. Chem. 268 (3): 1965–75. doi: 10.1016/S0021-9258(18)53949-1 . PMID   8420970.
19. Böhlke K., Pisani F. M., Vorgias C. E., Frey B., Sobek H., Rossi M., Antranikian G. (2000). "PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A motif". Nucleic Acids Res. 28 (20): 3910–3917. doi:10.1093/nar/28.20.3910. PMC   110800 . PMID   11024170.
20. Kim KP, Bae H, Kim IH, Kwon ST (2011). "Cloning, expression, and PCR application of DNA polymerase from the hyperthermophilic archaeon, Thermococcus celer". Biotechnol. Lett. 33 (2): 339–46. doi:10.1007/s10529-010-0434-2. PMID   20953664.
21. Cho Y, Lee HS, Kim YJ, Kang SG, Kim SJ, Lee JH (2007). "Characterization of a dUTPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in polymerase chain reaction amplification". Mar. Biotechnol. 9 (4): 450–8. Bibcode:2007MarBt...9..450C. doi:10.1007/s10126-007-9002-8. PMID   17549447.
22. Lee JI, Kim YJ, Bae H, Cho SS, Lee JH, Kwon ST (2010). "Biochemical properties and PCR performance of a family B DNA polymerase from hyperthermophilic Euryarchaeon Thermococcus peptonophilus". Appl. Biochem. Biotechnol. 160 (6): 1585–99. doi:10.1007/s12010-009-8658-0. PMID   19440663.
23. Marsic D, Flaman JM, Ng JD (2008). "New DNA polymerase from the hyperthermophilic marine archaeon Thermococcus thioreducens". Extremophiles. 12 (6): 775–88. doi:10.1007/s00792-008-0181-7. PMID   18670731.
24. Song JG, Kil EJ, Cho SS, Kim IH, Kwon ST (2010). "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". Protein Eng. Des. Sel. 23 (11): 835–42. doi:10.1093/protein/gzq059. PMID   20851826.
25. Dietrich J, Schmitt P, Zieger M, Preve B, Rolland JL, Chaabihi H, Gueguen Y (2002). "PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi". FEMS Microbiol. Lett. 217 (1): 89–94. doi:10.1111/j.1574-6968.2002.tb11460.x. PMID   12445650.
26. Kennedy EM, Hergott C, Dewhurst S, Kim B (2009). "The mechanistic architecture of thermostable Pyrococcus furiosus family B DNA polymerase motif A and its interaction with the dNTP substrate". Biochemistry. 48 (47): 11161–8. doi:10.1021/bi9010122. PMC   3097049 . PMID   19817489.
27. Kuroita T., Matsumura H., Yokota N., Kitabayashi M., Hashimoto H., Inoue T., Imanaka T., Kai Y. (2005). "Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases". J. Mol. Biol. 351 (2): 291–8. doi:10.1016/j.jmb.2005.06.015. PMID   16019029.
28. McDonald JP, Hall A, Gasparutto D, Cadet J, Ballantyne J, Woodgate R (2006). "Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs". Nucleic Acids Res. 34 (4): 1102–11. doi:10.1093/nar/gkj512. PMC   1373694 . PMID   16488882.
29. Eremeeva E., Herdewijn P. (2018). "PCR Amplification of Base-Modified DNA". Current Protocols in Chemical Biology. 10 (1): 18–48. doi:10.1002/cpch.33. PMID   30040232.
30. Wang X., Zhang J., Li Y., Chen G., Wang X. (2015). "Enzymatic synthesis of modified oligonucleotides by PEAR using Phusion and KOD DNA polymerases". Nucleic Acid Therapeutics. 25 (1): 27–34. doi:10.1089/nat.2014.0513. PMC   4296748 . PMID   25517220.
31. Wang Y, Prosen DE, Mei L, Sullivan JC, Finney M, Vander Horn PB (2004). "A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro". Nucleic Acids Res. 32 (3): 1197–207. doi:10.1093/nar/gkh271. PMC   373405 . PMID   14973201.
32. Motz M, Kober I, Girardot C, Loeser E, Bauer U, Albers M, Moeckel G, Minch E, Voss H, Kilger C, Koegl M (2002). "Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes". J. Biol. Chem. 277 (18): 16179–88. doi: 10.1074/jbc.M107793200 . PMID   11805086.
33. 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
34. Pavlov A. R., Pavlova N. V., Kozyavkin S. A., Slesarev A. I. (May 2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications". Trends in Biotechnology. 22 (5): 253–260. doi:10.1016/j.tibtech.2004.02.011. PMID   15109812.
35. 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.
36. Barnes WM (1994). "PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates". Proc. Natl. Acad. Sci. U. S. A. 91 (6): 2216–20. Bibcode:1994PNAS...91.2216B. doi: 10.1073/pnas.91.6.2216 . PMC   43341 . PMID   8134376.
37. Bruno Frey, Bernhard Suppmann: Demonstration of the Expand TM PCR System's Greater Fidelity and Higher Yields with a lacI-based PCR Fidelity Assay. (2000).
38. Tellier R., Bukh J., Emerson S. U., Purcell R. H. (2003). "Long PCR amplification of large fragments of viral genomes: a technical overview". PCR Protocols. Methods in Molecular Biology. Vol. 226. pp. 167–172. doi:10.1385/1-59259-384-4:167. ISBN   1-59259-384-4. PMID   12958497.
39. Miura M., Tanigawa C., Fujii Y., Kaneko S. (2013). "2013 Comparison of six commercially-available DNA polymerases for direct PCR.". Revista do Instituto de Medicina Tropical de Sao Paulo. 55 (6): 401–406. doi:10.1590/S0036-46652013000600005. PMC   4105087 . PMID   24213192.
40. Hogrefe H. H., Hansen C. J., Scott B. R., Nielson K. B. (2002). "2002 Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation.". Proceedings of the National Academy of Sciences . 99 (2): 596–601. doi: 10.1073/pnas.012372799 . PMC   117351 . PMID   11782527.
41. Karunanathie H., Kee P. S., Ng S. F., Kennedy M. A., Chua E. W. (2022). "2022 PCR enhancers: Types, mechanisms, and applications in long-range PCR.". Biochimie. 197: 130–143. doi:10.1016/j.biochi.2022.02.009. PMID   35231536.
42. US Patent 2013034879A1: DNA Polymerases, 2012-08-02 / 2007-02-14, Fermentas UAB et Al (applicant), Remigijus Skirgaila et al. (inventors).
43. US Patent 2009280539A1: DNA Polymerases and related methods, 2009-04-16 / 2009-11-12, Roche Molecular Systems Inc (applicant), Keith A. Bauer (inventor).
44. Li J., Li Y., Li Y., Ma Y., Xu W., Wang J. (2023). "An enhanced activity and thermostability of chimeric Bst DNA polymerase for isothermal amplification applications". Applied Microbiology and Biotechnology . 107 (21): 6527–6540. doi:10.1007/s00253-023-12751-6. PMID   37672070.
45. Barnes WM (1992). "The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion". Gene. 112 (1): 29–35. doi:10.1016/0378-1119(92)90299-5. PMID   1551596.
46. Merkens, L. S.; Bryan, S. K.; Moses, R. E. (1995). "Inactivation of the 5'-3' exonuclease of Thermus aquaticus DNA polymerase". Biochimica et Biophysica Acta. 1264 (2): 243–248. doi:10.1016/0167-4781(95)00153-8. PMID   7495870.
47. Tabor S, Richardson CC (1995). "A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides". Proc. Natl. Acad. Sci. U. S. A. 92 (14): 6339–43. Bibcode:1995PNAS...92.6339T. doi: 10.1073/pnas.92.14.6339 . PMC   41513 . PMID   7603992.
48. Vander Horn PB, Davis MC, Cunniff JJ, Ruan C, McArdle BF, Samols SB, Szasz J, Hu G, Hujer KM, Domke ST, Brummet SR, Moffett RB, Fuller CW (1997). "Thermo Sequenase DNA polymerase and T. acidophilum pyrophosphatase: new thermostable enzymes for DNA sequencing". BioTechniques. 22 (4): 758–62, 764–5. doi:10.2144/97224pf02. PMID   9105629.
49. Prober JM, Trainor GL, Dam RJ; et al. (October 1987). "A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides". Science. 238 (4825): 336–41. Bibcode:1987Sci...238..336P. doi:10.1126/science.2443975. PMID   2443975.
50. D'Aquila R. T., Bechtel L. J., Videler J. A., Eron J. J., Gorczyca P., Kaplan J. C. (1991). "Maximizing sensitivity and specificity of PCR by pre-amplification heating". Nucleic Acids Research. 19 (13): 3749. doi:10.1093/nar/19.13.3749. PMC   328414 . PMID   1852616.
51. S. Buratowski: Hot Start Taq purification. 2015.
52. Graham T. G., Dugast-Darzacq C., Dailey G. M., Nguyenla X. H., Dis E. Van, Esbin M. N., Abidi A., Stanley S. A., Darzacq X., Tjian R. (2021). "Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection". PLOS ONE . 16 (2) e0246647. Bibcode:2021PLoSO..1646647G. doi: 10.1371/journal.pone.0246647 . PMC   7857565 . PMID   33534838.
53. David Edward Birch, Walter Joseph Laird, Michael Anthony Zoccoli: Nucleic acid amplification using a reversibly inactivated thermostable enzyme. United States Patent 5773258.
54. Barnes W. M., Rowlyk K. R. (2002). "Magnesium precipitate hot start method for PCR". Molecular and Cellular Probes. 16 (3): 167–171. doi:10.1006/mcpr.2002.0407. PMID   12219733.
55. Paul N, Shum J, Le T (2010). "Hot start PCR". RT-PCR Protocols. Methods Mol Biol. Vol. 630. pp. 301–18. doi:10.1007/978-1-60761-629-0_19. ISBN   978-1-60761-628-3. PMID   20301005.
56. Kramer MF, Coen DM (2001). "Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization". Current Protocols in Immunology. 24: 10.20.1–10.20.10. doi:10.1002/0471142735.im1020s24. PMID   18432685.
57. Fraenkel-Conrat H, Brandon BA, Olcott HS (1947). "The reaction of formaldehyde with proteins; participation of indole groups; gramicidin". J. Biol. Chem. 168 (1): 99–118. doi: 10.1016/S0021-9258(17)35095-0 . PMID   20291066.
58. Fraenkel-Conrat H, Olcott HS (1948). "The reaction of formaldehyde with proteins; cross-linking between amino and primary amide or guanidyl groups". J. Am. Chem. Soc. 70 (8): 2673–84. Bibcode:1948JAChS..70.2673F. doi:10.1021/ja01188a018. PMID   18876976.
59. Fraenkel-Conrat H, Olcott HS (1948). "Reaction of formaldehyde with proteins; cross-linking of amino groups with phenol, imidazole, or indole groups". J. Biol. Chem. 174 (3): 827–43. doi: 10.1016/S0021-9258(18)57292-6 . PMID   18871242.
60. Kellogg DE, Rybalkin I, Chen S; et al. (June 1994). "TaqStart Antibody: ""hot start"" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase". BioTechniques. 16 (6): 1134–7. PMID   8074881.
61. Sharkey D. J., Scalice E. R., Christy K. G., Atwood S. M., Daiss J. L. (1994). "1994 Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction". Bio/Technology. 12 (5): 506–509. doi:10.1038/nbt0594-506. PMID   7764710.
62. Yakimovich O. Y., Alekseev Y. I., Maksimenko A. V., Voronina O. L., Lunin V. G. (2003). "Influence of DNA aptamer structure on the specificity of binding to Taq DNA polymerase". Biochemistry. Biokhimiia. 68 (2): 228–235. doi:10.1023/a:1022609714768. PMID   12693970.
63. Noma T., Sode K., Ikebukuro K. (2006). "Characterization and application of aptamers for Taq DNA polymerase selected using an evolution-mimicking algorithm". Biotechnology Letters. 28 (23): 1939–1944. doi:10.1007/s10529-006-9178-4. PMID   16988782.
64. Chou Q, Russell M, Birch DE, Raymond J, Bloch W (April 1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Res. 20 (7): 1717–23. doi:10.1093/nar/20.7.1717. PMC   312262 . PMID   1579465.
65. Ordóñez Carlos D., Redrejo-Rodríguez Modesto (2023). "DNA Polymerases for Whole Genome Amplification: Considerations and Future Directions". International Journal of Molecular Sciences . 24 (11): 9331. doi: 10.3390/ijms24119331 . PMC   10253169 . PMID   37298280.
66. Fuchs B., Zhang K., Rock M. G., Bolander M. E., Sarkar G. (1999). "High temperature cDNA synthesis by AMV reverse transcriptase improves the specificity of PCR". Molecular Biotechnology. 12 (3): 237–240. doi:10.1385/MB:12:3:237. PMID   10631680.
67. Carothers AM, Urlaub G, Mucha J, Grunberger D, Chasin LA (1989). "Point mutation analysis in a mammalian gene: rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method". BioTechniques. 7 (5): 494–6, 498–9. PMID   2483818.
68. Myers T. W., Gelfand D. H. (1991). "Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase". Biochemistry. 30 (31): 7661–6. doi:10.1021/bi00245a001. PMID   1714296.
69. Moser M. J., DiFrancesco R. A., Gowda K., Klingele A. J., Sugar D. R., Stocki S., Mead D. A., Schoenfeld T. W. (2012). "Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme". PLOS ONE. 7 (6) e38371. Bibcode:2012PLoSO...738371M. doi: 10.1371/journal.pone.0038371 . PMC   3366922 . PMID   22675552.
70. Chien A, Edgar DB, Trela JM (September 1976). "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus". Journal of Bacteriology. 127 (3): 1550–1557. doi:10.1128/JB.127.3.1550-1557.1976. PMC   232952 . PMID   8432.
71. Saiki R. K., Gelfand D. H., Stoffel P., Scharf P. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. (January 1988). "1988 Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science . 239 (4839): 487–491. doi:10.1126/science.2448875. PMID   2448875.
72. Ishino P., 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.
73. Lawyer FC, Stoffel S, Saiki RK, Myambo K, Drummond R, Gelfand DH (1989). "Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus". J. Biol. Chem. 264 (11): 6427–37. doi: 10.1016/S0021-9258(18)83367-1 . PMID   2649500.
74. Soroka M., Wasowicz B., Rymaszewska A. (2021). "Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?". Cells. 10 (8): 1931. doi: 10.3390/cells10081931 . PMC   8393631 . PMID   34440699.
75. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000). "Loop-mediated isothermal amplification of DNA". Nucleic Acids Res. 28 (12): 63e–63. doi:10.1093/nar/28.12.e63. PMC   102748 . PMID   10871386.