Thermostable DNA polymerase

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Taq DNA polymerase with exonuclease (top left) and polymerase domain with DNA (bottom right)

Thermostable DNA polymerases are DNA polymerases that originate from thermophiles , mostly bacteria or archaea species , and are therefore thermally stable . 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 occur among other things 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.

Belonging to the A-type DNA polymerases bacterial thermostable DNA polymerases have, in addition to the 5 '→ 3' - polymerase - type a 5 '→ 3' exonuclease activity , and produce 3'-end of the new strand produced a Adenosine overhang (English English sticky ends ). The processivity (engl. Processivity ) describes the average number of base pairs before a polymerase from the DNA template (engl. Template ) drops. The processivity of the polymerase used limits the maximum distance between the primer and the probe in real-time quantitative PCR . The processivity of a Taq polymerase is around 200 base pairs.

Archaic polymerases

Pfu polymerase with two magnesium ions (gray spheres)

Frequently DNA polymerases used B-type are derived from various archaea Pfu polymerase , the Pwo polymerase , the KOD polymerase, the Tli polymerase (also Vent called), the Tag polymerase, the Tce polymerase, the Tgo polymerase, TNA1 polymerase, Tpe polymerase, Tthi polymerase, Neq polymerase and Pab polymerase.

The archaea variants belonging to the B-type do not produce an overhang (English. Blunt ends , the Tli polymerase makes an overhang in about 30% of the products) and instead of the 5 '→ 3' exonuclease activity have an activity to correct Synthesis errors ( proof-reading ), the 3 '→ 5' exonuclease activity . In the case of the archaic polymerases, the error rate suffers when generating an analogous Klenow fragment, since the corrective exonuclease activity is thereby removed. Some DNA polymerases from archaea are less suitable for standard PCR than they are for their reduced inhibition of the amplification of aDNA .

Modified polymerases

By protein design was from different thermostable polymerases and DNA clamp of the thermostable DNA-binding protein Sso7d various fusion proteins having the low error rate Achaean and the high rate of synthesis of bacterial thermostable DNA polymerases generated ( Q5 polymerase). Also, a fusion protein of was PCNA - homologue from Archaeoglobus fulgidus with archaic thermostable DNA polymerases generated. Fusion proteins of thermostable DNA polymerases with the thermostable DNA-binding protein domain of a topoisomerase (of type V, with helix-hairpin-helix motif , HhH ) from Methanopyrus kandleri were generated ( TopoTaq and PfuC2 ). A modified Pfu polymerase was also generated through 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 the polymerases of type A and B of 30 to 1, e.g. B. Herculase as a commercial mixture of Taq and Pfu polymerase.

The basic synthesis rates of different polymerases ( 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 the KOD polymerase is above 100 base pairs per second (approx. 120 bp / s). Various mutations that increase the rate of synthesis have been described among the modified thermostable DNA polymerases. The KOD polymerase and some modified thermostable DNA polymerases ( iProof , Pfu Ultra , Phusion , Velocity or Z-Taq ) are used in 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 case of bacterial thermostable DNA polymerases, analogous to the DNA polymerase from E. coli, a Klenow fragment ( Klen-Taq ) or a Stoffel fragment can be generated by deleting the exonuclease domain in the course of a protein design , which is higher Product concentration results. Two amino acids necessary for the exonuclease function of Taq polymerase were identified by mutagenesis as arginines at positions 25 and 74 (R25 and R74).

The preference for individual nucleotides by a thermostable DNA polymerase is known as nucleotide specificity (English bias 'preference', 'bias'). In the case of PCR-based DNA sequencing with chain termination substrates ( dideoxy method ), their uniform incorporation and thus the uniform generation of all chain termination products is often desired in order to enable higher sensitivity and easier evaluation. For this purpose, a KlenTaq polymerase was generated by deletion and, by site-specific mutagenesis, a phenylalanine at position 667 was exchanged for tyrosine (F667Y for short) and designated as Thermo Sequenase . This polymerase can also be used to incorporate fluorescence- labeled dideoxynucleotides .

Other DNA polymerases

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

Applications

In addition to the choice of the thermostable DNA polymerase used, further parameters of a PCR are specifically changed in the course of a PCR optimization.

In addition to PCR, the thermostable DNA polymerases are also used for the variants of RT-PCR , various variants of qPCR , site-specific mutagenesis and DNA sequencing . In addition, hybridization probes for Southern Blot and Northern Blot are produced by random priming . The 5 '→ 3' exonuclease activity is used , among other things, for nick translation and for TaqMan without DNA replication ( amplification ) taking place .

literature

Web links

Individual evidence

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