Thermostable DNA polymerases commonly used in laboratories
Thermostable DNA polymerases are mostly used in PCR technology. Various thermostable DNA polymerases have 5'-3' polymerase activity, but not necessarily 3'-5' and 5'-3' exonuclease activities. 3'-5' exonuclease activity can eliminate mismatches and blunt ends; 5'-3' exonuclease activity can eliminate synthesis barriers.
Due to these differences, thermostable DNA polymerases can be divided into three categories:
1. Common heat-resistant DNA polymerase
Taq DNA polymerase
It is isolated and extracted from a strain of Thermus aquaticus yT1, which is the most active one of the thermostable DNA polymerases found, reaching 200,000 units/mg. It has 5'-3' exonuclease activity, but not 3'-5' exonuclease activity, so it has no correction function for certain single nucleotide mismatches in synthesis.
TaqDNA polymerase also has template-independent activity, which can add a single nucleotide tail to the 3' of each chain of the PCR double-stranded product, so that the PCR product can have a 3' protruding single A nucleotide tail; another On the one hand, when only dTTP is present, it can add a single T nucleotide tail to the 3' end of a blunt-ended plasmid, resulting in a single T nucleotide tail overhanging at the 3' end. Using this feature, T-A cloning of PCR products can be achieved.
Tth DNA polymerase
Extracted from Thermus thermophilus HB8, the enzyme can effectively reverse-transcribe RNA under the conditions of high temperature and MnCl2; when Mg2+ is added, the polymerization activity of the enzyme is greatly increased, so that cDNA synthesis and amplification can be catalyzed by an enzyme .
2. High-fidelity DNA polymerase
pfu DNA polymerase
It is a high-fidelity heat-resistant DNA polymerase refined from Pyrococcus furiosis. It does not have 5'-3' exonuclease activity, but has 3'-5' exonuclease activity, which can be used to correct the production of PCR amplification. error, so that the base mismatch rate of the product is extremely low. PCR products are blunt-ended, single A nucleotides without 3'-end overhangs.
Vent DNA polymerase
The enzyme is isolated from Thermococcus Litoralis, does not have 5'-3' exonuclease activity, but has 3'-5' exonuclease activity, can remove mismatched bases, and has proofreading function.
3. Thermostable DNA polymerase used in DNA sequence determination
Sequencing-grade TaqDNA polymerase
It is modified on the basis of TaqDNA polymerase to remove the 5'-3' exonuclease activity, to ensure the high accuracy of sequencing records, and to produce sequencing bands with uniform intensity and clear background.
Bca Best DNA Polymerase
A DNA polymerase purified from Bacillus Caldotenax YT-G strain and depleted of its 5'-3' exonuclease activity. Its elongation performance is superior; it can inhibit the formation of DNA secondary structure and can obtain uniform DNA sequencing bands.
Sac DNA polymerase
It is isolated from acid-heat bath fluidized schizophrenia, has no 3'-5' exonuclease activity, and can be used for DNA sequencing, but the ratio of ddNTP/dNTP in the sequencing reaction is high.
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