In the evolution of the Polymerase Chain Reaction (PCR), two developments have greatly simplified the procedure: automation of temperature cycling and the use of a thermostable DNA polymerase. The original method, using the Klenow fragment of E. colf DNA polymerase I, was very tedious because of the thermal lability of the enzyme. The initial PCR process required moving the samples to be amplified between two heat sources: one at high temperature (94°–95°C), required to denature the double-stranded DNA, and one at relatively low temperature (37°C), needed for both the annealing of the primers and their extension. Because the Klenow fragment is irreversibly denatured every time the sample temperature is raised to 94–95°C, the enzyme has to be replenished every cycle, after the sample temperature is restored to 37°C, in order to extend the annealed primers. Thus, the requirements of automating the PCR method at the time of its invention were actually two-fold: 1) cycling the temperature between 94°C and 37°C, and 2) adding fresh enzyme every cycle.
KeywordsBrittle Milling Polystyrene Dial Editing
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