Thermostable DNA polymerase

[12] The palm carries the polymerase active site, whereas the fingers bind substrates (template DNA and nucleoside triphosphates).

[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.

[31] A fusion protein of the PCNA homologue from Archaeoglobus fulgidus was also generated with archaeal thermostable DNA polymerases.

[36][38] Other additives are used to help against difficult GC-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.

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).

[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 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).

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.

[62][63] A fifth variant is a polymerase adsorbed on latex beads via hydrophobic effects, which dissolves with increasing temperature.

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.

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.

[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.

In 1998 the loop-mediated isothermal amplification was developed by Tsugunori Notomi and colleagues at Eiken Chemical Company, using Bst polymerase at 65 °C.