( ndary structure interference ...)Real-Time PCR: General Considerations, Rev A

ndary structure interference increased both the sensitivity and efficiency of amplification. Using the formula E = (10^-1/slope)-1 to calculate efficiency (E), a reaction with 100% efficiency will generate a slope of -3.32. The amplification plot of the experiment using primer set A generated a slope of -4.53 or 66% efficiency, with a correlation coefficient of 0.995 (Figure 2, upper panel). Moving the reverse primer inwards to the location of primer B shortened the PCR product and eliminated strong secondary structure. This shift in primer location significantly improved the efficiency of the reaction to 99% (slope = -3.35) and a correlation coefficient of 0.999 (Figure 2, lower panel).

MgCl₂ Evaluation
Typically, real-time PCR requires higher concentrations of MgCl₂ for optimal results. To demonstrate this, an analysis of MgCl₂ concentration effects on amplification efficiency was performed. Replicate reactions at 4 MgCl₂ concentrations (1.5, 2.25, 3, and 4 mM) and final primer concentrations of 300 nM each (for the optimized Primer B pair from above) were prepared. Reaction conditions were 1.25 U Platinum Taq DNA Polymerase and 1x PCR buffer (Life Technologies), 0.2 mM each dNTP (Advantage Ultrapure dNTPs, Clontech), and SYBR Green I (Molecular Probes, 1:75,000 dilution of the 10,000x stock solution).

The magnesium concentration had a significant impact on the amplification efficiency of the PCR reactions as demonstrated in Figure 2 and Table 1. The highest PCR efficiency was achieved with a 3 mM MgCl₂ concentration (Table 1). The importance of maintaining high amplification efficiencies i
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