Biotechnology Index Glossary
Thermostable DNA Polymerases
It is interesting how some seemingly esoteric or obscure discoveries can, years later, be catapulted to something of immense practical importance. Such is the 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. Currently, the world market for Taq polymerase is in the hundreds of millions of dollars each year.
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 and magnesium ion is necessary. In general, they have maximal catalytic activity at 75 to 80C, and substantially reduced activites at lower temperatures. At 37C, Taq polymerase has only about 10% of its maximal activity.
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:
Source and Properties Taq No From Thermus aquaticus. Halflife at 95C is 1.6 hours. Pfu Yes From Pyrococcus furiosus. Appears to have the lowest error rate of known thermophilic DNA polymerases. Vent Yes From Thermococcus litoralis; also known as Tli polymerase. Halflife at 95 C is approximately 7 hours.
In addition to the native polymerases listed in the table above, a number of mutants have been generated and are available, for example, a form of Vent polymerase that lacks the 3'->5' exonuclease and is thereby more similar to Taq.
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. As would be expected from first principles, polymerases lacking 3'->5' exonuclease activity generally have higher error rates than the polymerases with exonuclease activity. The total error rate of Taq polymerase has been variously reported between 1 x 10-4 to 2 x 10-5 errors per base pair. Pfu polymerase appears to have the lowest error rate at roughly 1.5 x 10-6 error per base pair, and Vent is probably intermediate between Taq and Pfu.
Error rate is not the only consideration in chosing a polymerase for PCR, otherwise Taq polymerase would not continue to be the most widely used enzyme by far for the myriad of PCR applications. Other considerations, including reliability and what might be called "fussiness" enter into the choice, as discussed further in the section on Polymerase Chain Reaction Technology.
Back to the index of Restriction Endonucleases and DNA Modifying Enzymes
Last updated on December 27, 1999
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