Biochemical Techniques and Instrumentation

6.1.5. Steps in PCR

PCR consists of a series of 20-40 repeated temperature changes called "Cycles". Most commonly PCR is carried out with cycles that have three temperature steps. Each cycle is often preceded by a single temperature step at a high temperature (>90°C) known as "Hold", and another hold at the end, for final product extension or storage known as "Final hold". The temperature and the duration in each cycle depend the enzyme used for DNA synthesis, the concentration of divalent ions, dNTPs in the reaction and the melting temperature (Tm) of the primers.

1. Initial Hold: This step consists of heating the reaction to a temperature of 94–96 °C for 1 min.
2. Denaturation : This step is the first thermal cycling process. It consists of heating the reaction to 94–98 °C for 20–30 sec. In this step, the two strands in a DNA double helix was physically separated into single strands of DNA and this process is known as "DNA melting". Separation occurs due to disruption of hydrogen bonds between complementary bases in the double helix.
3. Annealing: In this step, the reaction temperature is lowered to 50–65 °C for 20–40 sec. This allows annealing of the primers to the single-stranded DNA template through stable hydrogen bonds. The annealing temperature is generally about 3-5^oC below the melting point (Tm) of the primers. Taq DNA polymerase then binds to the primer-template hybrid and begins the DNA synthesis.
4. Extension/elongation: The temperature at this step depends on the DNA polymerase, particularly the Taq polymerase that has its optimum activity at 72°C. The Taq polymerase synthesizes a new DNA strand complementary to the DNA template strand by the addition of dNTPs that are complementary to the template in 5' to 3' direction by condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the extending DNA strand. The duration of extension depends both on the DNA polymerase and the length of the DNA fragment to be amplified. The DNA polymerase can polymerize a thousand bases per minute at its optimum temperature.
5. Final hold: The temperature at this step is 4–15 °C for an indefinite time. This is employed for short-term storage of the reaction products.