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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.

Properties[edit]

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.

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
Type 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) 8 × 10-6[4] 1.5 × 10-5[5] 2.8 × 10-6[4] 2.7 × 10-6[4] 3.5 × 10-6[6] 1.3 × 10-6[7][4]
Synthesis rate (bases/sec.) 21–47,[2] 61[8] 191[2] 23[8] 120,[9] 106–138[8] 9.3–25[8]
Processivity (bases) 10–42[8] <20[8] >300[8] 6.4–20[8]

Structure[edit]

DNA polymerases are roughly shaped like a hand with a thumb, palm and fingers.[10][11] The thumb is involved in binding and moving double-stranded DNA.[10] The palm carries the polymerase active site, whereas the fingers bind substrates (template DNA and nucleotide triphosphates.[10][12] The exonuclease activity is in a seperate protein domain.[10]

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, Tth and Bst polymerase are used.[4][13][14][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. 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 real-time quantitative PCR (qPCR). 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[edit]

Pfu polymerase with two magnesium ions (grey spheres)

Frequently used B-type DNA polymerases are the Pfu polymerase,[4] the Pwo polymerase,[15] the KOD polymerase,[3] the Tli polymerase (also called Vent), which originates from various archaea,[16] the Tag polymerase,[17] the Tce polymerase,[18] the Tgo polymerase,[6] the TNA1 polymerase,[19] the Tpe polymerase,[20] the Tthi polymerase,[21] the Neq polymerase[22] and the Pab polymerase.[23]

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.[24][25] In archaeal polymerases, the error rate suffers when an analogue Klenow fragment 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.[26]

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.[27] A fusion protein of the PCNA homologue from Archaeoglobus fulgidus was also generated with archaeal thermostable DNA polymerases.[28] 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).[29][30] A modified Pfu polymerase was also generated by protein design (Pfu Ultra).[31] 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,[20][32] e.g. Herculase[6] and TaqPlus[8] as a commercial mixture of Taq and Pfu polymerase, Expand as a commercial mixture of Taq and Pwo,[33] Expand High Fidelity as a commercial mixture of Taq and Tgo,[8] Platinum Taq High Fidelity as a commercial mixture of Taq and Tli (Vent),[8] and Advantage HF 2 as a commercial mixture of Titanium Taq and an unnamed proof-reading polymerase.[8] These mixtures can be used for long-range PCR to synthesize products of up to 35kb length.[32][34] Other additives are used to help against difficult GC-rich sequences, avoid or neutralise the negative effects of PCR inhibitors, or alter the reaction kinetics.[35]

The baseline synthesis rates of various polymerases (processivity, productivity) have been compared.[6] 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).[9] Among the modified thermostable DNA polymerases, various mutations have been described that increase the synthesis rate.[36][37][38] 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.

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, that of Advantage HF 6.1 × 10-6 errors per base, that of Platinum Taq High Fidelity 5.8 × 10-6 errors per base, that of TaqPlus 4 × 10-6 errors per base, that of KOD polymerase 3.5 × 10-6 errors per base, that of Tli polymerase and Herculase 2.8 × 10-6 errors per base, that of Deep Vent 2.8 × 10-6 errors per base, that of Pfu, Phusion DNA Polymerase (identical with iProof DNA Polymerase) and Herculase II Fusion 1.3 × 10-6 errors per base and that of Pfu Ultra and Pfu Ultra II 4.3 × 10-7 errors per base.[4][6][8] 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.[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.[39][13] 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).[40] A histidine to glutamic acid mutation at position 147 (short: H147E) in KOD polymerase lowers the relatively high exonuclease activity of KOD.[25]

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.[41][42] This polymerase can also be used for the incorporation of fluorescence-labelled dideoxynucleotides.[43]

Other DNA polymerases[edit]

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.[44] The T4, T6 and T7 DNA polymerases are also not thermostable.

RNA-dependent DNA polymerases[edit]

The standard reverse transcriptases 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.[45] 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.[46] But since the synthesis rate of Taq with Mn2+ is relatively low, Tth was increasingly used for this approach.[47] 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.[48]

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).

History[edit]

The first use of a thermostable DNA polymerase was by Randall K. Saiki and colleagues in 1988, introducing Taq polymerase for PCR.[49][50] The thermostability obliviated the need to add a DNA polymerase to the reaction after every melting phase of the PCR. In 1989, the Taq polymerase gene was cloned and the Taq polymerase was produced in Escherichia coli as a recombinant protein.[51][50] DNA of up to 35,000 basepairs was synthesized by Wayne M. Barnes by using different mixtures of a A and B type polymerases,[32][50] thereby creating the long-range PCR. The high synthesis rate of KOD polymerase was published in 1997 by Masahiro Takagi and colleagues,[3][50][12] thereby creating the fundamentals of high speed PCR. Other optimisations to the PCR were developed in the following years, e.g. neutralising PCR inhibitors and amplifying difficult GC-rich DNA sequences.[35] In 1998 the loop-mediated isothermal amplification was developed by researchers including Tsugunori Notomi at Eiken Chemical Company, using Bst polymerase at 65 °C.[52][53]

Literature[edit]

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  • 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.

Weblinks[edit]

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