Yes the enzyme is active at all temperatures but its accuracy is greatest at the recommended extension temperature 68 or 72 c depending on the enzyme. \when the pri,er anneals at say 55c the enzyme still amplifies at about 100 bases every second but the error rate is greater. In the time taken for the mix to heat from annealing to extension the enzyme is still active. Indeed for short amplimers an extension step may not even be needed. In running DHPLC in the past we always saw more changes close to each primer. The errors are random so usually the correct sequence is the strongest in pcr while the taq induced errors are more scattered

• Agree with Paul about inherent mutations close to primers
• I would add to that by saying that if you are mining SNPs by Big dye sequencing, then make sure the region you are interested in assessing accurately is preferbaly 50bp down stream from your priming site:
• This is due to other confounding factors like lower Sequencing ladder production close to priming site, increasing singnal to noise and unincorporated big dye oligomers obscuring 5@ sequence in that proximal region
• Of course my remarks only apply of you elucidate SNPs by Sanger sequencing and not other methods like Pyro Sequencing etc

Hi,

The probability of error incorporation during amplification, highly depends on primer design and the type of enzyme. Also the rate of amplification (or the rate of nt incorporation during elongation) can influence the mismatch or error rate, which in turn can be impacted by thermocycler efficacy (rate of ramp cooling or heating). The following article might be of help.

Article Examining Sources of Error in PCR by Single-Molecule Sequencing