# Thermostable DNA Polymerase

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

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.
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

Frequently used B-type DNA polymerases are the Pfu polymerase, the Pwo polymerase, the KOD polymerase, the Tli polymerase (also called Vent), which originates from various archaea, the Tag polymerase, the Tce polymerase, the Tgo polymerase, the TNA1 polymerase, the Tpe polymerase, the Tthi polymerase, the Neq polymerase and the Pab polymerase.
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. In archaeal polymerases, the error rate suffers when an analogue Klenow fragment is generated, as the correcting exonuclease activity is removed in the process. Some archaeal DNA polymerases are characterised less by their suitability for standard PCR than by their reduced inhibition in the amplification of A-DNA.

## 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. A fusion protein of the PCNA homologue from Archaeoglobus fulgidus was also generated with archaeal thermostable DNA polymerases. 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). A modified Pfu polymerase was also generated by protein design (Pfu Ultra). 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, e.g. Herculase as a commercial mixture of Taq and Pfu polymerase.
The baseline synthesis rates of various polymerases (processivity, productivity) have been compared. 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). Among the modified thermostable DNA polymerases, various mutations have been described that increase the synthesis rate. 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.
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. 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).
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. This polymerase can also be used for the incorporation of fluorescence-labelled dideoxynucleotides.

## Other DNA polymerases

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

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

* 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.

## Weblinks

* Promega: Properties of Thermostable DNA Polymerases (PDF; 208 kB). Accessed september 27, 2012.
* NEB Polbase. Accessed september 27, 2012.
* Fermentas DNA-Polymerasen. Accessed september 27, 2012.