The PCR method is used to quantify nucleic acids by amplifying a nucleic acid molecule with the enzyme DNA polymerase. Conventional PCR is based on the theory that amplification is exponential. Therefore, nucleic acids may be quantified by comparing the number of amplification cycles and amount of PCR end-product to those of a reference sample. However, many factors complicate this calculation, creating uncertainties and inaccuracies.
These factors include the following:
Initial amplification cycles may not be exponential
PCR amplification eventually plateaus after an uncertain number of cycles
Low initial concentrations of target nucleic acid molecules may not amplify to detectable levels
Variations in PCR amplification efficiency in various biological samples