Polymerase chain reaction (PCR) is a laboratory technique used to make multiple copies of a segment of DNA. PCR is very precise and can be used to amplify or copy, a specific DNA target from a mixture of DNA molecules. A PCR reaction consists of three steps: denaturation, annealing, and extension. This is how a basic PCR reaction works:
- First, two short DNA sequences called primers are designed to bind to the start (3’) and end (5’) of the DNA target. The primer nucleic acid sequences are chosen to flank the target region, so that on amplification the target is increased.
- To perform PCR, the DNA template that contains the target is added to a tube that contains primers, free nucleotides (adenine, guanine, cytosine, thymine), [these are also referred to as deoxyribonucleotides (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 (eg, 95°C) to denature, or separate, the double-stranded DNA template into single strands. The mixture is then cooled (eg, 60-65°C) so that 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 aforementioned steps. The newly synthesized DNA segments serve as templates in later cycles, which allow the DNA target to be exponentially amplified millions of times.
