2 Note Taq DNA polymerase was isolated from thermophilic fungus yT 1. YT is a thermophilic fungus that can grow at 70 ~ 75℃. It was separated from the volcanic hot springs in Yellowstone National Forest Park in the United States in 1969. The total length of the enzyme gene is 2496 bases, encoding 832 amino acids, and the enzyme protein molecule is 94KDa. Its specific activity is 200,000 units/mg. At 75 ~ 80℃, each enzyme molecule can extend about 150 nucleotides per second, and at 70℃, it can extend more than 60 nucleotides per second. At 55℃, it is 24 nucleotides/second. Too high temperature (above 90℃) or too low temperature (22℃) will affect the activity of Taq DNA polymerase. Although there is almost no DNA synthesis above 90℃, the enzyme does have good thermal stability and can still maintain high activity under the high temperature condition of PCR cycle. At 92.5℃, 95℃ and 97.5℃, Taq DNA polymerase in PCR mixture can still maintain 50% activity after 65438±0.30min, 40min and 5 ~ 6 min respectively. The experiment shows that the denaturation temperature of PCR reaction is 95℃ ~ 20 seconds, and after 50 cycles, Taq DNA polymerase still has 65% activity. The thermal stability of Taq DNA polymerase is a prerequisite for its application in PCR reaction. It is also the reason for the rapid development and wide application of PCR. Taq DNA polymerase also has reverse transcription activity, similar to reverse transcriptase. The temperature of this activity is generally 65 ~ 68℃, and its reverse transcription activity is high in the presence of Mn2.
TaqDNA polymerase is a Mg2-dependent enzyme, and its catalytic activity is very sensitive to the concentration of Mg2. When the concentration of dNTP is 0.7 ~ 0.8 mmol/L, different concentrations of Mg2 are used for PCR reaction 10min. The results show that when the concentration of Mgcl2 is 2.0 mmol/L, the catalytic activity of the enzyme is the highest, which can activate the activity of Taq DNA polymerase to the maximum extent, and when the concentration of Mg2 is too high, it will inhibit the activity of TaqDNA polymerase. When the concentration of Mgcl2 is 10mmol/L, Mg2 can bind to dNTP, which will affect the concentration of free Mg2 in PCR reaction solution, so the concentration of Mgcl2 should be adjusted appropriately in different reaction systems. Optimize the concentration. Generally speaking, the concentration of Mg2 should be at least 0.5 ~ 1.0 mmol/L higher than the total concentration of dNTP. The proper concentration of KCL can improve the catalytic activity of Taq DNA polymerase by 50 ~ 60%, and the optimum concentration is 50 mmol/L. When the concentration of KCL is higher than 75mmol/L, the activity of Taq DNA polymerase is obviously inhibited.