There are three basic steps to polymerase chain reaction studies:
In the first step, denaturing, the double stranded DNA molecules are converted into single strands. In the second step, primers are annealed to the complementary segments of the single stranded molecules. Annealing is when two types of primers, forward and reverse primers pair by hydrogen bonds to separated DNA template strands. They are then extended by the use of DNA polymerase. These three steps have to be carried out at precise temperatures. The primers must be designed specifically for a given PCR study in order for it to yield usable results.
In addition to the primers and the DNA polymerase, PCR reagents include deoxynucleoside triphosphates, or dNTPs, which are building blocks from which DNA polymerase makes a new DNA strand. Buffer solutions create an appropriate chemical environment for optimized activity and DNA polymerase stability. Magnesium or manganese ions are used in part of the PCR study, as are potassium monovalent cations.
When the PCR process is over, it is important to check whether the process generated the correct DNA fragment. For this purpose, a process called agarose gel electrophoresis is carried out to separate out the PCR products by size. The sizes of the PCR products are determined by comparing them with a DNA ladder, which is a molecular weight marker. This contains DNA fragments of known size and are run on the gel beside the PCR plates and products for comparison purposes.
PCR studies are optimized by lab protocols that are specifically designed to minimize the chances of contamination. Disposable plasticware, and meticulous attention to cleaning of the work surfaces between setups are two techniques used to minimize contamination.