Multiplex Assay Design

There are a number of inherent challenges involved in designing and running multiplex real-time PCR assays. First and foremost is the problem with unequal amplification efficiencies for different products. This may be a result of differences in primer and probe binding affinities for their targets, different primary and secondary structures of target regions, and interaction or cross-reactivity between oligonucleotides within a reaction. These differences lead to competition for reagents such as nucleotides and the DNA polymerase, and this competition will lower the reaction sensitivities of the less efficient reactions. One simple way to decrease competition is to increase the concentrations of reaction components, such as dNTPs or the polymerase.[9] Choosing primers and probes with very similar length, GC content, and hence Tm values can also minimize variation in reaction efficiencies. Competition can be further abrogated by using limiting amounts of primers, such that the primer supply for a reaction is exhausted before the amplification is able to outcompete less efficient reactions and consume a majority of the reagents. This technique is very successful when using a controlled reaction, such as when an internal positive control amplification is multiplexed in the reaction. However, for other target amplifications, especially with unknown or low concentrations of target, a decrease in primer concentration may decrease the reaction efficiency and sensitivity.

Another form of cross-reactivity that exists in multiplexing, besides interactions between oligonucleotides, is with respect to overlap in the fluorescent spectra of the fluorophores being used. Fluorophores must carefully be chosen such that there are sufficient discrete wavelengths

Table 1 Examples of multiplex real-time PCR methodologies and corresponding applications


Target differentiation method



5' nuclease probe


Pathogen detection/differentiation


5' nuclease probe


Genotyping/allelic discrimination


5' nuclease probe


Normalization of pathogen concentration to


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Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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