11/06/2018
THE STEPS OF PCR
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The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.
The basic steps are:
Denaturation (96°C):
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Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
Annealing (55 - 65°C):
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Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
Extension (72°C):
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Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
This cycle repeats 25- 35 times in a typical PCR reaction, which generally takes 22 - 44 hours, depending on the length of the DNA region being copied. If the reaction is efficient (works well), the target region can go from just one or a few copies to billions.
That’s because it’s not just the original DNA that’s used as a template each time. Instead, the new DNA that’s made in one round can serve as a template in the next round of DNA synthesis. There are many copies of the primers and many molecules of Taq polymerase floating around in the reaction, so the number of DNA molecules can roughly double in each round of cycling.