Polymerase is a key factor in the success or failure of PCR. Choosing the most suitable heat-resistant polymerase is the first problem to be considered in PCR experiments. The main differences of many heat-resistant DNA polymerases lie in several indicators such as specificity, fidelity, heat resistance, amplification rate, and length of amplified fragments.
High temperature resistant DNA polymerases fall into three classes. The first category: high amplification efficiency, but no correction function. It has a strong function of synthesizing DNA in the 5'-3' direction, but has no exonuclease activity in the 3'-5' direction. The synthesis efficiency is high, and errors are easy to make during the synthesis process. The main type is Taq DNA polymerase. The second category: It not only has the function of synthesizing DNA in the 5'-3' direction, but also has the activity and error correction function of the exonuclease in the 3'-5' direction, but the DNA synthesis efficiency is greatly reduced compared with ordinary Taq enzymes. The main type is Pfu DNA polymerase. The third type has the characteristics of both the first and second types. It is enough to mix ordinary Taq enzyme and Pfu DNA polymerase in a certain proportion (such as Taq-Plus, LA-Taq, and Ex-Taq). After mixing, due to the two, the final effect is affected by the competition for the substrate template and the disorder of the enzymatic kinetics.
The Type of PCR Enzymes
There are many kinds of PCR enzymes on the market, including Pfu series, Vent series, KOD series, Taq series, Tth, etc.
| DNA Polymerases |
Description |
Application |
| Taq DNA polymerase |
Taq DNA polymerase is derived from Thermus aquaticus. It has high thermal stability and high amplification efficiency. The amplification speed is 30-60 sec/kb. It lacks 3'-5' exonuclease activity, has no correction function, and is prone to generate mismatched bases. base. The PCR product amplified by this enzyme has an A tail at the 3' end and can be directly used for TA cloning. |
Conventional PCR |
| Tth DNA polymerase |
Tth DNA polymerase has higher heat resistance than Taq polymerase, and it has RTase activity. In the presence of Mn2+, RTase activity will be strengthened. In addition, Tth DNA polymerase has a higher reverse transcription temperature of 60-70°C, and the specificity of reverse transcription is high. |
PCR reactions with high GC content |
| Pfu DNA polymerase |
Pfu DNA polymerase has excellent thermostability, has 5'-3' DNA polymerase and 3'-5' exonuclease activity, and has no 5'-3' exonuclease activity. Pfu enzyme is currently the most widely used high-fidelity thermostable DNA polymerase. |
Gene screening, clone expression, mutation detection, site-directed mutagenesis, etc. |
| Vent DNA Polymerase |
The enzyme has 5'-3' DNA polymerase activity and 3'-5' exonuclease activity, and its fidelity is 5-15 times higher than that of Taq DNA polymerase. |
Highly specific PCR amplification |
| KOD DNA polymerase |
It has super strong 3'~5' exonuclease activity, and its fidelity is higher than that of Pfu DNA Polymerase, which is about 50 times that of Taq enzyme. |
Highly specific PCR amplification |
| Hot-Start DNA Polymerase |
Hot-start DNA polymerase is a class of enzymes that improve the specificity and sensitivity of PCR reactions. This type of enzyme is made by modifying DNA polymerase by antibody method or ligand method, so that the activity of DNA polymerase is activated at the pre-denaturation temperature, which can inhibit the non-specific annealing of primers or the secondary reaction of primers under low temperature conditions. Non-specific amplification caused by aggregation. |
Highly specific PCR amplification |
Key Factors for Choosing a PCR Enzyme
Thermostability: The ability of DNA polymerase to withstand high temperature. The thermostability of different DNA polymerases varies greatly, which is generally reflected by the half-life.
Fidelity: The ability of a DNA polymerase to make precise copies of a DNA template. Some DNA polymerases have 3'-5' exonuclease activity, which can correct wrong bases, and the fidelity can be reflected by the mismatch rate.
Sensitivity: The ability of DNA polymerase to amplify low-copy templates can be reflected by the detection rate.
Specificity: The ability of DNA polymerase to amplify only the target fragment during the PCR process. DNA polymerase is often modified by antibody method or ligand method to improve enzyme specificity.
Amplification speed: The rate at which DNA polymerase incorporates nucleotides into new strands of DNA. High amplification rate DNA polymerases can speed up the overall experiment time.
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