Efficiency calculation from the fluorescence increases in the exponential phase of fluorescence history plot (in log. scale) (Figure 3.4). Fitting can be done by eye, or more reliably by software applications like LinRegPCR (Ramakers et al., 2003) or DART-PCR (Peirson et al., 2003). The investigator has to decide which fluorescence data to include in the analysis and which to omit. A linear regression plot is drawn from at least four data points, where the slope of the regression line represents the PCR efficiency. Therefore this method is more or less arbitrary and dependent on the chosen data points. Resulting efficiencies range between E = 1.45, and E = 1.90, and seem more realistic than the results mentioned above. This efficiency calculation method might be good estimator for the 'real efficiency,' because data evaluation is made exclusively in exponential phase.
The advantage of both direct methods is the independency of the background fluorescence. We know from several applications that a rising
Efficiency calculation in the exponential phase.
trend in the background fluorescence will interfere with the indirect curve fit, like sigmoidal, logistic and exponential models. Probe based detection in particular exhibits high and noisy background levels, whereas SYBR® Green I applications show low and constant background fluorescence (Figure 3.5).
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