Polymerase chain reaction (PCR) is a laboratory technique used to make multiple copies of a segment of DNA. PCR is precise and can amplify or copy a specific DNA target from a mixture of DNA molecules. A PCR reaction consists of denaturation, annealing, and extension.
Denaturation: First, the reaction mixture containing the DNA template is heated to denature or separate the double-stranded DNA into single strands.
Annealing: The reaction mixture is then cooled to allow the primers to anneal or bind to the DNA template.
Extension: The polymerase synthesizes new DNA strands, starting from the primers (extension).
A primary PCR reaction works in the following manner:
- First, two short DNA sequences called primers are designed to bind to the DNA target's start (3’) and end (5’). The primer nucleic acid sequences are chosen to flank the target region to increase the target on amplification.
- To perform PCR, the DNA template containing the target is added to a tube containing primers, free nucleotides (adenine, guanine, cytosine, thymine), [these are also referred to as deoxyribonucleotide triphosphates (dNTPs)], and an enzyme called DNA polymerase.
This mixture is placed in a PCR machine. The PCR machine increases and decreases the temperature of the sample in automatic, programmed steps.
- Initially, the mixture is heated (e.g., 95°C) to denature or separate the double-stranded DNA template into single strands. The mixture is then cooled (e.g., 60–65°C) so the primers anneal (bind) to the DNA template.
- Finally, the temperature is increased to approximately 72°C to optimize DNA polymerase, catalyzing the addition of nucleotides to synthesize new strands of DNA starting from the primers (extension). At the end of the first cycle, each double-stranded DNA molecule consists of one new and one old DNA strand.
PCR then continues with additional cycles that repeat the steps above. The newly synthesized DNA segments serve as templates in later cycles, which allow the DNA target to be exponentially amplified millions of times.
