3.1 Procedure

The PCR usually consists of a series of twenty to thirty-five cycles. Each cycle consists of three steps: denaturation, annealing and elongation (or extension) (Figure 5).

Denaturation

Annealing

Elongation

The DNA polymerase used in the first experiments was isolated from bacteria growing at temperatures of up to 73C. These polymerases were destroyed by the high temperatures used in the denaturation step and so fresh enzyme had to be added at each cycle making PCR a labour intensive process. The discovery of bacteria living in hot springs led to the isolation of a polymerase that was thermostable. The first enzyme was isolated from Thermus aquaticus and is called Taq polymerase.

Another factor reducing the efficiency of the procedure was the fact that initially water baths set at the required temperatures were used and the reaction tubes had to be moved manually. Now many companies have designed and made PCR machines which can be programmed to carry out the steps as needed. Thus PCR has become automated and is widely used.

The animation shows PCR in action.

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Figure 5: How the Polymerase Chain Reacion Works

The PCR product can be identified by its size using agarose gel electrophoresis. The gel is stained with ethidium bromide (or another dye) which binds to the DNA and then visualised under UV light. In this way the DNA can be seen and the size of the fragments determined by comparison with a DNA marker ladder (Figure 6).

© SCBC 2007