PCR technology: Taq DNA polymerase characterization!
aq DNA polymerase is isolated from a strain of Thermusaquaticus yT1. yT is a thermophilic fungus that can grow at 70 to 75°C. The bacterium was isolated from the volcanic hot springs of Yellowstone National Forest Park in the United States in 1969. separated in.
1. Enzyme activity and thermal stability
The enzyme gene has a full length of 2,496 bases, encoding 832 amino acids, and the enzyme protein molecule is 94KDa. Its specific activity is 200,000 units/mg. Each enzyme molecule can extend about 150 nucleotides per second at 75 to 80°C. , the elongation rate is greater than 60 nucleotides/second at 70℃ and 24 nucleotides/second at 55℃. Temperature that is too high (above 90℃) or too low (22℃) can affect the activity of Taq DNA polymerase , although this enzyme has almost no DNA synthesis above 90°C.
However, it does have good thermal stability and can still maintain high activity under the high temperature conditions of PCR cycles. At 92.5°C, 95°C, and 97.5°C, the Taq DNA polymerase in the PCR mixture was tested for 130 minutes, 40 minutes, and 5 minutes respectively. After ~6 minutes, 50% activity can still be maintained. Experiments show that the denaturation temperature during PCR reaction is 95°C ~ 20 seconds, after 50 cycles.
Taq DNA polymerase still has 65% activity. The thermal stability of Taq DNA polymerase is a prerequisite for the enzyme to be used in PCR reactions, and is also the reason why PCR reactions can develop rapidly and be widely used. Taq DNA polymerase also has reverse transcription Activity, it acts like reverse transcriptase. This activity temperature is generally 65~68°C. When Mn2+ is present, its reverse transcription activity is higher.
2. Ion dependence
aqDNA polymerase is a Mg2+-dependent enzyme, and the catalytic activity of the enzyme is very sensitive to Mg2+ concentration. Using salmon sperm DNA with very low activity as a template, when the concentration of dNTP is 0.7~0.8mmol/L, PCR is performed with different concentrations of Mg2+ The reaction lasted for 10 minutes. The measurement result showed that the catalytic activity of the enzyme was highest when the Mgcl2 concentration was 2.0mmol/L. This concentration can activate the activity of TaqDNA polymerase to the maximum extent. If Mg2+ is too high, the enzyme activity will be inhibited.
When the Mgcl2 concentration is 10mmol/L, it can inhibit 40-50% of the enzyme activity. Since Mg2+ can combine with dNTPs and affect the free Mg2+ concentration in the PCR reaction solution, the concentration of Mgcl2 should be appropriately adjusted and optimized in different reaction systems. Concentration. In general reactions, the Mg2+ concentration should be at least 0.5~1.0mmol/L higher than the total dNTP concentration. An appropriate concentration of KCL can increase the catalytic activity of Taq DNA polymerase by 50~60%.
The optimal concentration is 50mmol/L. When it is higher than 75mmol/L, the activity of the enzyme is obviously inhibited.
3. Loyalty
TaqDNA polymerase has 5'→3' polymerase activity and 5'→3' exonuclease activity, but no 3'→5' exonuclease activity. It does not have the 3'→5' proofreading activity of Klenow enzyme. Therefore, If certain base mismatches occur during the PCR reaction, the enzyme has no correction function. The probability of base mismatch of Taq DNA polymerase is 2.1×10-4.
4. Inhibitors
Low concentrations of urea, formamide, dimethylformamide (DMF) and dimethyl sulfoxide (DMSD) have no effect on the catalytic activity of Taq DNA polymerase. Very low concentrations of ionic surfactants such as deoxycholine acid Sodium (less than 0.06%), sodium lauryl sarcosinate (less than 0.02%), and sodium dodecyl sulfate (SDS, less than 0.01%) can almost completely inhibit the activity of the enzyme. Non-ionic surfactants are used in Higher concentrations (Tween20, NP-40. and Tritox-100) such as >5% can inhibit the activity of the enzyme.
