PCR technology: Taq DNA polymerase properties

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recombinationDNA technical tool enzyme

taq DNA polymerase is isolated from an aquatic lytator (Thermusaquaticus) yT1 strain. yT is a heat-philing " > that can grow at 70 to 75 degrees C. The bacteria was isolated from the Volcano Hot Springs at Yellowstone National Forest in 1969

(i) Enzyme activity and thermal stability

The enzyme"> gene is 2496 bases in length, encoded 832amino acids, the enzyme protein molecule is 94KDa. Its ratio activity is 20 At 00000 units/mg.75 to 80 degrees C, each enzyme molecule can extend about 150 nucleotides per second, with an elongation of 70 degrees C greater than 60nucleotides/s, and 24 nucleotides/s at 55 degrees C. High temperature (over 90 degrees C) or too low (22 degrees C) can affect the activity of Taq DNA polymerase, although the enzyme is almost no DNA synthesis above 90 degrees C, but it does have good thermal stability, in the PCR cycle of high temperature conditions can still maintain high activity. The Taq DNA polymerase in the PCR mixture remained 50% active after 130min, 40min and 5 to 6min, respectively, at 92.5C, 95C and 97.5C. After 50 cycles, Taq DNA polymerase is still 65% active when the PCR reacts with a denatured temperature of 95 degrees C to 20sec. The thermal stability of Taq DNA polymerase is the prerequisite for the enzyme's use in PCR reaction and the reason why PCR reaction can develop rapidly and widely. Taq DNA polymerase also has reverse transcription activity, which is similar to reverse transcription enzyme. The active temperature is generally 65 to 68 degrees C, and when Mn2 plus is present, its reverse recording activity is higher.

(ii) ion-dependent

aqDNA polymerase is an Mg2-dependent enzyme, the catalytic activity of which is very sensitive to Mg2-plus concentrations. Based on the very low activity of salmon sperm DNA as a template, the concentration of dNTP was 0.7 to 0.8mmol/L, with different concentrations of Mg2 plus PCR reaction 10min, the result was Mgcl2 concentration at 2.2. The enzyme has the highest catalytic activity at 0mmol/L, and this concentration can activate the activity of TaqDNA polymerase to the maximum degree, and Mg2 plus is too high to inhibit enzyme activity, which can inhibit 40 to 50% enzyme activity when Mgcl2 concentration is 10mmol/L. Because Mg2 plus can combine with dNTP and affect the free Mg2 plus concentration in PCR reaction fluid, the concentration of Mgcl2 should be adjusted and optimized in different reaction systems. In general reaction, the concentration of Mg2 plus should be at least 0.5 to 1.0mmol/L higher than the total concentration of dNTP KCL can improve the catalytic activity of Taq DNA polymerase by 50 to 60%, and its suitable concentration is 50mmol/L, higher than 75mmol/L when the activity of the enzyme is significantly inhibited.

(iii) faithfulness

TaqDNA polymerases have 5" and "rarr;3" polymerase activity and 5" and "rarr;3" extase activity, while no 3" and rarr;5" extation activity, it does not have Klenow enzyme 3" and rarr;5" proofing activity. Therefore, in PCR reaction, if certain base mismatch occurs, the enzyme has no correction function. The base mismatch rate of Taq DNA polymerase is 2.1×10-4.

(iv) inhibitors

low concentrations of urea, methylamine, methamphetamine (DMF) and DMSO) had no effect on the catalytic activity of Taq DNA polymerase. Very low concentrations of ions > surfactants e.g. sodium deoxycholinerate (less than 0.06%), sodium tyranyl crealanine (less than 0.02%), sodium decane sulphate (SDS, less than 0.01%) can almost completely inhibit the activity of the enzyme. Non-ion surfactants inhibit the activity of the enzyme at higher concentrations (Tween20, NP-40. and Tritox-100) such as .gt;5%

effects of denaturation agents on the activity of TaqDNA polymerases