Polymerase Chain Reaction
Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 8 July 2010, last major update July 2010
Copyright: (c) 2008-2010, PathologyOutlines.com, Inc.
● Initially the Klenow fragment from the E. coli DNA polymerase I was used in PCR reactions
● While the Klenow fragment worked in PCR, it rapidly denatured at the 90°C or higher temperatures required for denaturation, requiring the addition of new Klenow polymerase after every denaturation step
● The need to open the reaction chamber with every PCR thermocycle made PCR cumbersome and greatly increased the chances for contamination with unwanted DNA
● The solution to this problem came from the discovery and cloning of a thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus
● The polymerase, known as the Taq polymerase, has many attributes that are well suited for PCR:
● (a) the enzyme works best at 75-80°C, allowing the elongation step to occur at temperatures which make non-Watson-Crick base paring a rare event
● (b) its half-life at 95°C is over 40 minutes
● (c) at 72°C it can add 1,000 nucleoside triphosphates to a growing DNA strand
● Most PCR reactions done with Taq can be completely closed through all the thermocycles
● Taq lacks 3' to 5' exonuclease proofreading activity, allowing a roughly 1 per 9,000 error rate in nucleoside triphosphate incorporation
● Additionally, Taq often adds an adenine to the end of its polymerase reaction (sometimes used to facilitate TA cloning)
● Taq was named “The Molecule of the Year” by Science in 1989
End of Molecular Pathology > Polymerase Chain Reaction > Taq polymerase
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