One disadvantage of intercalating dye-based methods is that they detect any dsDNA produced in the reaction. This includes off target amplification products and primer-dimers, which results in inaccurate quantification. Therefore, it is imperative to perform a denaturation (melt) curve after each qPCR experiment to verify reaction specificity and ensure that only the desired target was amplified. Additionally, unlike with probe-based qPCR that permits multiplexing, only one target can be interrogated at a time in a dye-based qPCR experiment.
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