The Properties of Taq Polymerase

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Taq polymerase, an enzyme most commonly used in the polymerase chain reaction (PCR), is isolated from Thermus aquaticus. These thermophillic microorganisms live in hot springs at temperatures of 70 to 73 degrees C. The Taq polymerase used today is isolated from strain YT-1, a culture of T. aquaticus collected from Mushroom Spring, a large spring in the Lower Geyser Basin.

Properties

  • Full-length Taq polymerase has 832 amino acids and a molecular weight of 94,000 daltons. It has a specific activity of 292,000 units, with each unit adding 10 nmoles of deoxyribonucleotide triphosphate (dNTPs) into a product in 30 minutes. Taq was the first polymerase found to retain its activity after exposure to high temperature. Most specifically, Taq polymerase has an activity half-life of 45 to 50 minutes at 95 degrees C and of 9 minutes at 97.5 degrees C.

Activity

  • The optimal polymerization activity of Taq is at temperatures of 80 degrees C, in the presence of 55 mM KCl and 2 mM MgCl2 and a pH of 9.4.

Application

  • The most important factor contributing to the popularity of Taq polymerase in PCR is its ability to function at and withstand a high temperature. This property made it ideal for PCR, which requires a high temperature to denature the double stranded DNA. A PCR is composed of as many as 40 cycles, and the temperature of each cycle ranges from about 50 to 95 degrees C. Escherichia coli (E. coli) DNA polymerase, used for PCR before the isolation of Taq, was destroyed at the high temperature and had to be replenished after each denaturation step. This increased the rate of error and chances of cross contamination in the PCR. The use of Taq polymerase in PCR has not only simplified the technique but also improved its specificity, yield and sensitivity. Because of its widespread use and importance in the field of molecular biology, "Science" magazine named Taq polymerase "Molecule of the Year" in 1989.

Limitation

  • The exonuclease proofreading ability is absent in Taq polymerase. This means that Taq has low replication fidelity, as it cannot correct any errors that may occur during amplification. These errors limit the use of Taq polymerase where high accuracy is required.

References

  • "Genetics";The Value of Basic Research: Discovery of Thermus aquaticusand Other Extreme Thermophiles; Thomas Brock; August 1997
  • "PCR Methods and Applications"; High-level Expression, Purification, and Enzymatic Characterization of Full-length Thermus aquaticus DNA Polymerase and a Truncated Form Deficient in 5' to 3' Exonuclease Activity; Frances Lawyer, et al. May 1993
  • "Science"; Primer-Directed Enzymatic Amplification of DNA with Thermostable DNA Polymerase; Randall Saiki et al.; January 1988
  • Colorado State University: Thermostable DNA Polymerases
  • Photo Credit Jupiterimages/Comstock/Getty Images
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