Like DNA replication in an organism, PCR Kits requires a DNA polymerase enzyme that produces new strands of DNA by using existing strands as a template. The DNA polymerase normally used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus).
T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very thermostable and is most active near 70°C70°C70, °, start text, C, end text (temperature at which human or E. coli DNA polymerase would not work). Taq polymerase is ideal for PCR because of this thermal stability. As we shall see, PCR uses high temperatures repeatedly to denature the DNA template or separate its strands.
Steps of PCR
The key ingredients for a PCR reaction are Taq polymerase, primers, template DNA and nucleotides (the building blocks of DNA). The ingredients are placed in a tube, along with any cofactors needed by the enzyme, and undergo repeated heating and cooling cycles that allow DNA synthesis.
The basic steps are:
- Denaturation (96 °text C96°C96, °, start text, C, end text): the reaction is heated sufficiently to separate, or denature, the DNA strands. This provides the single-stranded templates for the next step.
- Quenching (555555 – 656565° C°C°, start text, C, end text): the reaction is cooled so that the primers can bind to their complementary sequences on the single-stranded DNA template.
- Extension (72 °text C72°C72, °, start text, C, end text): the temperature of the reaction is raised to allow Taq polymerase to extend the primers to synthesise new DNA strands.
This cycle is repeated 252525 – 353535 times in a typical PCR reaction, which generally takes 222 – 444 hours, depending on the length of the DNA region being copied. If the reaction is efficient (works well), it can produce billions of copies from one or a few copies of the target region.
That’s because it’s not just the original DNA that is used as the template for each cycle. In fact, the new DNA produced 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 almost double in each cycle. The image below shows this exponential growth pattern.