The product of an amplification reaction is an amplicon. The molecular technique consists of 20 to 30 cycles under three steps:
- Denaturation: Separation of the double-stranded DNA by breaking DNA hydrogen bonds to a single-strand DNA. Occurs at 90–96°C for approximately 30 seconds.
- Annealing: Primers are allowed to bind to their template DNA strand, at 50–68°C for approximately 30 seconds.
- Extension: Step in which Taq Polymerase synthetizes new DNA strands at the 3' end of the primers at 72°C.
A set of controls should be included with each experiment for the interpretation of PCR results:
- Blank Control - Determines if there is any contamination in the reaction. No amplification product.
- Negative Control - Tests the specificity of the reaction.
- Positive Control - Tests the sensitivity of the reaction.
