How Does PCR Work?
A polymerase chain reaction requires two types of reagents, DNA polymerase and primers, and the process of thermal cycling. These ingredients make possible the chain reaction that exponentially copies the starting DNA sample.
DNA polymerase is an enzyme that synthesizes molecules from deoxyribonucleotides, the building blocks of DNA, in order to create two identical DNA segments from a single original.
Taq polymerase is the specific enzyme typically used in PCR because it can withstand the extreme temperature changes of thermal cycling, eliminating the need to add a new, non-temperature-stable DNA polymerase after every PCR cycle. Its source is the bacterium Thermus aquaticus, which was first isolated from geothermal springs in Yellowstone National Park around the time PCR was discovered.
Primers are short pieces of single-stranded DNA, pairs of which must be specifically designed to prompt the DNA polymerase to initiate amplification of the targeted DNA segment.
Thermal cycling refers to the repeated heating and cooling cycles that facilitate the temperature-dependent reactions of DNA amplification. A thermal cycler is a laboratory instrument that performs temperature-dependent chemical reactions, including PCR.
One cycle of PCR doubles the amount of DNA with which it began. Each cycle involves three steps: denaturation, annealing, and extension.
The first step in a PCR cycle heats the starting sample to almost boiling. The high temperature causes DNA melting, or denaturation—the process of physically breaking down the hydrogen bonds between the two complementary strands of the DNA double helix. The result is two single strands of DNA that will later be synthesized into two complete DNA double helixes.
After denaturation, the temperature of the sample is reduced. This allows the primers to bind to their complementary sequences on each of the separated DNA strands. The primers set the stage for Taq polymerase to begin DNA formation in the final step.
In the final step, the sample is reheated to the temperature at which Taq polymerase is most active—about 162 °F (72 °C)—so that the enzyme can latch onto the base created by the primers during the annealing stage. Taq polymerase assembles two complete DNA segments from the original one.
A new cycle of PCR is then ready to begin with double the amount of DNA of the previous cycle. Cycles continue until the requisite quantity of DNA is achieved.