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Real-Time Multiplex PCR from Genomic DNA Using the iCycler iQ Detection System

ater than 1,000-fold, then the reaction efficiencies should not differ by more than 5% and the individual reaction efficiencies should be greater than 90%. The most powerful tool for adjusting reaction efficiency is to examine the secondary structures of the PCR product, primers, and probe. It is critical to eliminate all secondary structures that might interfere with DNA:DNA interactions, but the secondary structure must be evaluated at the appropriate temperature. Secondary structures that melt below the annealing temperature are not important. It is often necessary to raise the annealing temperature or relocate one or both primers or the probe in order to avoid secondary structure problems.

Cross-reactivity between primers and targets in the separate reactions can occur due to specific gene interaction effects, such as primer recognition, as well as to competition for substrates. It is straightforward to test for cross-reactivity of the individual reaction components by comparing a set of wells containing one reaction alone to a set containing all reaction mixtures. If there is no crossreactivity, the CT of template A when amplified alone will be identical to the CT of template A in the presence of the other components. A labeled probe is not required to test for cross-reactivity.

Addition of extra polymerase, dNTPs, and MgCl2 improves results for 2 or more individual reactions in multiplex. For a typical 50 l reaction, we use 3.253.75 U Taq polymerase, 450 M each dNTP, and 5 mM MgCl2 (compared to 1.25 U polymerase, 200 M each dNTP, and 3 mM MgCl2 for a single reaction). If cross-reactivity is minimal, but efficiencies of multiplex reactions are lower than th
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