Important Considerations for Typical, Quantitative and Real-Time PCR Protocols

Abstract

The most important phase of the PCR protocol is the amplification phase, comprising a pre-determined number of amplification cycles (or thermocycles). In the vast majority of cases, a typical thermocycle comprises three distinct but sequential steps. In sequential order, these steps include: (1) a template nucleic acid/amplified PCR DNA melting step, required to dissociate double stranded DNA ready for primer hybridisation (usually 90–95°C for 30–60 seconds), (2) a primer annealing step performed at a temperature that has been calculated to be optimal for the PCR primer pair being used (usually 40–70°C for 30–60 seconds), and (3) a final DNA chain extension step at an optimum temperature for the enzyme of choice to promote the full extension and amplification of the DNA sequence (usually around 70–75°C for 60–120 seconds). It should be noted that both annealing and elongation steps of each thermocycle are performed at “stringent” temperatures, facilitating a reduction in the likelihood of non-specific hybridization between primers and regions of DNA which contain similar but not identical DNA sequences. Exceptions to this rule do exist for certain PCR protocols, e.g. PCR using “degenerate” primers. Finally, some PCR thermocycle protocols utilise only two (instead of 3) steps, by combining the annealing and DNA chain extension steps into a single step using a single annealing and chain extension temperature.