PCR experiment

To prepare billions of DNA copies, many repeated cycles of DNA synthesis are performed in one PCR tube. Each cycle includes three distinct steps defined by the temperature (see figure below):

1. Denaturation step (95ºC): At this high temperature, the hydrogen bonds holding together the two DNA strands are broken, and the DNA strands fall apart. The single-stranded DNA template is now available for copying.
2. Annealing step (5-10ºC below the primer with the lower Tm): At the annealing temperature short DNA pieces called primers bind at complementary sites of the template DNA. The primers define the target sequence, which is the specific region of DNA that will be copied. The annealing temperature is calculated from the primer composition (the number of nucleotides as well as the number of guanines and cytosines). Normally, you would need to calculate the optimal annealing temperature for each primer.
3. Extension step (72ºC): At 72 ºC, an enzyme called DNA polymerase is responsible for copying DNA. It recognizes the 3′ end of a primer bound to a template strand and starts copying the template DNA. In this case it is a thermostable polymerase because it needs to be active in the high temperature utilized.

All cycles are performed without intervention in a PCR machine, which can change the temperature automatically after each step. By the end of one cycle, parts of the initial DNA strands have been doubled in number. By the end of, e.g., 30 cycles, which is the number usually performed when doing PCR, at least 1 billion (2³⁰) copies of the target sequence will be present in the tube.

Figure 1. PCR experiment: PCR consists of three steps: 1. Denaturation, 2. Annealing, and 3. Extension. The steps are repeated many times (often 30), producing billions of DNA copies of specific regions.