Using PCR allows enough DNA to be made so it can be manipulated by the experimenter. In brief the principle of the reaction can be divided into three stages: denaturation (the separation of double stranded DNA to produce single stranded DNA), annealing (the binding of region-specific primers to the DNA) and elongation (the extension of the primers by a DNA polymerase to produce copies of the original material). These stages are generally repeated for 25-30 times resulting in the amplification of a specific region of DNA. To do this PCR requires several basic components which are:
- A DNA template, which contains the region of the DNA fragment to be amplified;
- Two primers, which determine the beginning and end of the region to be amplified;
- A heat stable DNA polymerase such as Taq polymerase, which copies the region to be amplified;
- Deoxynucleotide triphosphates, (dATP, dGTP, dCTP, dTTP) from which the DNA polymerase builds the new DNA;
- Buffer, which provides a suitable chemical environment for the DNA polymerase.
All these components can be made up as a master mix with the exception of the DNA and the primers. The DNA template:
- Is often, but not always, double stranded;
- Contains sequence spanning the area to be amplified;
- Can come from:
- Crude cell lysates;
- Purified cloned DNA;
- A single cell;
- And many other sources.
The PCR is carried out in a thermal cycler. This is a machine that can be programmed to heat and cool the reaction tubes within it to the precise temperature required for each step of the reaction. To prevent evaporation of the reaction mixture (typically volumes between 15-100μl per tube), a heated lid is placed on top of the reaction tubes or a layer of oil is put on the surface of the reaction mixture.