Effect of Temperature on DNA Conformation
Polymerase Chain Reaction (PCR)
a method used to amplify a specific DNA sequence in vitro by
repeated cycles of synthesis using specific primers and DNA
polymerase
high temperatures needed to denature double-stranded copies of
DNA
original PCR technique used Escherichia coli DNA polymerase
- due to high temperatures needed to denature the doublestranded copies of DNA, the polymerase was also denatured
and had to be replenished every cycle
isolated DNA polymerase from Thermus aquaticus (Taq) - a bacteria
that lives in hot springs and hydrothermal vents and can tolerate high
temperatures
- stable at 95°C, which is needed for the PCR denaturation step
Taq DNA polymerase can be isolated in a purer form (free of other
enzyme contaminants) than could the DNA polymerase from other
sources
Taq has no proof reading function and makes more mistakes than E.
coli enzyme
DNA polymerase from the hyperthermophilic archaean Pyrococcus
furiosus (Pfu or “vent polymerase”) is more thermally stable than Taq
and has a proof reading function
PCR Process explained in “The PCR Dash”