Dye-based qPCR & RT-qPCR

Dye-based quantitative PCR (qPCR) uses real-time fluorescence of a double-stranded DNA (dsDNA) binding dye (e.g., SYBR® Green) to measure DNA amplification as it occurs during PCR. The dye displays weak background fluorescence that increases dramatically upon binding to dsDNA. Thus, amplification of the target sequence results in an increase of fluorescence that is directly proportional to the amount of dsDNA present at each PCR cycle.  This type of qPCR assay requires only two sequence-specific primers, making it a rapid and cost-effective way to interrogate a large number of samples/targets.

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.

TaqMan® is a registered trademark of Roche Molecular Systems, Inc.
SYBR® is a registered trademark of Molecular Probes, Inc.