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July 01, 2010 (Vol. 30, No. 13)

Design Tools for Use in Assay Optimization

Sensitive and Specific Primers and Probes Are Goals of New Modeling Platform

  • Luminex xTAG Technology

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    Figure 2. Luminex xTAG data from monoplex (purple) and multiplex (magenta) PCR assays using primers designed in Visual OMP. Each of the RNA sequences was spiked into the reverse transcription reaction at a different concentration, resulting in the projected cDNA copy numbers shown. xTAG data shown are normalized average values of the median fluorescence intensity (MFI) obtained from PCR assays repeated in triplicate, after 28 cycles of PCR.

    Multiplex real-time PCR is unable to detect all PCR targets in highly multiplexed assays, due to the limited number of channels that can be detected in a real-time assay. The Luminex xTAG® detection technology can circumvent this limitation and is appropriate for situations that require a high degree of multiplexing.

    Multiplex PCR reactions are difficult to design, however, because amplicons are often synthesized with unequal efficiencies due to unequal hybridization thermodynamics, product inhibition of DNA polymerase, and primer-dimer interactions. Visual OMP is well suited for multiplex PCR because it designs primers so that all amplicons are synthesized with equal efficiency, while simultaneously avoiding undesired thermodynamic effects.

    To validate the Visual OMP and Luminex xTAG technologies for designing and quantifying multiplex PCR experiments, an eight-plex PCR assay was designed in Visual OMP for detection by Luminex xTAG technology. cDNA targets were obtained following reverse transcription (using polyT primers) of eight polyadenylated RNAs spiked into human liver total RNA at different concentrations.

    PCR primers were designed in Visual OMP and scanned with ThermoBLAST against the human transcriptome database. Simulating all primers together with their targets at actual reaction conditions in Visual OMP confirmed that the primers were specific (less than 0.01% primer binding to each possible off-target sequence) and sensitive for their targets, binding at greater than 99.9% efficiency, suggesting that all targets should amplify equally well.

    Figure 2 shows data obtained by Luminex xTAG technology from Visual OMP-designed monoplex and multiplex PCR assays. The singleplex and multiplex reactions resulted in similar trends in intensity with respect to copy number, indicating that assay design in Visual OMP resulted in a reliable multiplex PCR assay.

    PCR assay design and optimization is typically a complex and time-intensive process, particularly for multiplexed assays where difficulties scale with the number of targets in the reaction. Enabled by accurate simulation capabilities, PCR Pathfinder, Visual OMP, and ThermoBLAST allow users to design optimal PCR assays without the need for trial-and-error optimization.

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