The Effect of Amplicon Charcteristics on the success of Fast QPCR

Employing fast QPCR cycling protocols is a simple and effective way of maximising throughput by reducing run durations. Fast cycling protocols can easily be achieved by reducing the temperature step dwell times.

It has been reported that the use of fast QPCR protocols can result in a loss of sensitivity and greater inter-replicate variance with some assays. It is therefore important to realise that speeding up cycling protocols can be detrimental to the quality of results, but not in all cases. The factors which determine whether an assay can be successfully employed using fast cycling conditions are poorly understood.

The effect of three target amplicon characteristics on the success of fast QPCR was investigated using a panel of characterised assays. The amplicon characteristics focused on in particular were base-pair length, GC content and minimum ΔG at 60°C, the latter being a measure of the severity of amplicon secondary structure at the annealing temperature.