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Properties of Thermostable DNA Polymerase

Properties of Thermostable DNA Polymerase. Yufei TU. Discovery-- history of Taq DNA polymerase. The original report of this enzyme, purified from the hot springs bacterium Thermus aquaticus , was published in 1976.

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Properties of Thermostable DNA Polymerase

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  1. Properties of Thermostable DNA Polymerase Yufei TU

  2. Discovery--history of Taq DNA polymerase • The original report of this enzyme, purified from the hot springs bacterium Thermus aquaticus, was published in 1976. • Roughly 10 years later, the polymerase chain reaction was developed and shortly thereafter "Taq" became a household word in molecular biology circles. *THE DARNDEST PLACES: Scientists isolated the thermostable DNA polymerase Taq, an enzyme that drives PCR, from Thermus aquaticus Yellowstone type-1, a resident of geysers like this one at Yellowstone National Park.

  3. Properties • The thermophilic DNA polymerases, like other DNA polymerases, catalyze template-directed synthesis of DNA from nucleotide triphosphates. • A primer having a free 3‘ hydroxyl is required to initiate synthesis • Magnesium ion is necessary. • In general, they have maximal catalytic activity at 75 to 80℃, and substantially reduced activites at lower temperatures. • At 37℃, Taq polymerase has only about 10% of its maximal activity.

  4. Taq DNA Polymerase • Recombinant Taq DNA Polymerase is the enzyme of choice for most PCR applications. • The half-life of enzyme is >40 minutes at 95°C. • The error rate of Taq DNA Polymerase in PCR is 2.2x10-5 errors per nt per cycle;

  5. Other thermostable Polymerases • In addition to Taq DNA polymerase, several other thermostable DNA polymerases have been isolated and expressed from cloned genes. Three of the most-used polymerases are described in the following table:

  6. The Error Rate • One of the most discussed characteristics of thermostable polymerases is their error rate. • Error rates are measured using several different assays, and as a result, estimates of error rate vary, particularly when the assays are performed by different labs.

  7. Reliability/Fidelity Average error rates(mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10-6) Deep Vent (2.7 x 10-6) Vent (2.8 x 10-6) Taq (8.0 x 10-6) exo- Pfu and UlTma (approximately 50 x 10-6).

  8. Reference • Voet,D, Voet,J. Biochemistry Vol.1 3rd ed. • Alberts, Johnson, Lewis. Molecular Biology of The Cell 4th ed. http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/hotpolys.html http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=146123&rendertype=abstract http://www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm http://www.fermentas.com/techinfo/pcr/pcrprotocolpfu.htm

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