In general, a single PCR run will undergo 25-35 cycles. The first step for a single cycle is the denaturation step, in which the double-stranded DNA template molecule is made single-stranded. The temperature for this step is typically in the range of 95-100°C, near boiling. The high heat breaks the hydrogen bonds between the strands (Figure: Denaturation). The second step is a primer annealing step in which the primers bind to complementary sequences in the single-stranded DNA template. The general temperature range for this step is 45-55°C.
In this annealling step the temperature is much lower, allowing the primers to bind to the single-stranded DNA template (Figure: Anneal). It is important to understand that the primers will bind (anneal)only to that segment of the template which contains a sequence complementary to the primer.
For example, if the primer sequence is 3’ AACTGGA 5’, then it will only bind to the template where the sequence 5’ TTGACCT 3’ is found. If that sequence cannot be found, no binding will take place and no DNA will be copied. Therefore, primers can be designed which are gene specific, allowing cloning of only a specific portion of DNA. If only one primer binds and not also the other, there will not be an efficient exponential increase of DNA copies. Therefore, no end product will be detected. Both primers are needed for successful PCR.
Finally, the DNA extension step is when the DNA polymerase enzyme (i.e. Taqpolymerase) adds bases complementary to the template to the bound primers (Figure: Extension). The chemical mixture is heated to around 75°C and the newly synthesizing DNA strand is extended to the end of the template area to be copied. The completion of this step is also the completion of one cycle.
For cycle 2, the denaturation, annealing, and extension steps are repeated again. This time, though there will be twice as many DNA template molecules compared to what there was at the beginning of cycle 1. In other words, copies are being made of the original template and of the copies made in the previous cycle. Therefore, if everything is working correctly, at the end of a cycle there is double the amount of that particular DNA sequence as what was found at the beginning of the cycle.
The instrument which heats and cools the DNA samples is called a thermal cycler (Figure: Thermal cycler). Each small tube contains all chemical components needed for a single PCR reaction. A thermal cycler can be programmed for specific temperatures and the amount of time spent at each temperature. The amount of time it takes to heat to a temperature or cool down to another is called the 'ramp time'. This amount of time can also be controlled on some thermal cycler models.