DNA efficiency

Amplification Efficiency Exceeds 100%? How Is That Possible?

Amplification efficiency higher than 100% would mean that in each qPCR cycle, more than two copies of the target sequence have been generated.

Is this possible?

One way of calculating the amplification efficiency is by making serial dilutions of your target, getting their Cq values, plotting them on the logarithmic scale of target concentrations, drawing the linear regression curve (trend line) through the data points and calculating its slope. Finally, efficiency is calculated using equation: E=(10^(-1/slope))-1. However, if you don’t understand what can influence the slope of the serial dilutions curve, this calculation can be misleading.

Ideally, during each replication cycle, the number of molecules of the target sequence doubles, meaning that the efficiency of amplification is 100%. If there is poor reaction efficiency (below 100%) the number of molecules doesn’t double. Main reason for inefficient amplification is bad primer design (strong secondary structures, not appropriate Tm) on inappropriate template region (secondary structures); or non-optimal reagent concentration. Consequently, bigger differences (then they should) occur between Cq values of serially diluted samples. That happens because for each dilution more cycles are needed to reach the threshold of detection (see the Image below and click on it to enlarge it).

Amplification Efficiency

Efficiency exceeds 100%? How can that be?
The main reason for this artefact is the presence of inhibitors in the concentrated DNA/RNA samples. Inhibitors of the polymerase enzyme can be: excess amount of DNA, ethanol, saccharides, phenols, other secondary metabolites etc.
If the inhibitors are present in the concentrated samples, more cycles are needed to reach the threshold of detection then they would be needed in the absence of inhibitors. In diluted samples, inhibitors are diluted together with DNA/RNA to the stage in which they don’t have an inhibitory effect on the polymerase enzyme anymore. Therefore the amplification is efficient and signal comes out as it should (for a certain dilution).
Consequently, the difference between Cq values of concentrated and diluted sample is smaller as predicted and the amplification seems more efficient.

Amplification Efficiency_2

This artefact usually diminishes when testing more diluted samples. Therefore, if inhibition is present, early Cq values of most concentrated samples should be omitted when calculating efficiency. Similarly, the latest Cq values of the most diluted samples should also be omitted in case they are very variable as a consequence of stochastic effect. Such sample dilutions are then also not appropriate to be included in the quantification study.
Other reasons for higher efficiency can be: the presence of polymerase enzyme activators in the reaction, inappropriate dilution series, pipetting errors, presence of unspecific products and primer dimers when using intercalating dyes (this should be controlled for each reaction separately), contamination etc.

 

By Urska Cepin, PhD, Research and Development Assistant, BioSistemika LLC





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