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.

Diagram showing the three steps in PCR. The first step is denaturation. In the denaturation step, a section of two complementary DNA strands called the target section is shown. The strand fragments are both read in the 5 prime to 3 prime direction. Arrows show that the complementary strands move apart. The next step in PCR is annealing. In this step, small primers attach to the 3 prime end of both strand segments. The third step is extension. Here, new DNA that is complementary to each existing strand is copied in the 3 prime to 5 prime direction. The result is two sets of the target section of DNA. The steps in PCR are then repeated, to continue to replicate more copies of the targeted DNA section. The complementary strands are separated during denaturation, primers are attached during annealing, and new DNA is copied during extension.

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.