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Dye-based qPCR

Dye-based qPCR Kits

Fast, highly-sensitive and reproducible qPCR results

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.

SensiFAST™ SYBR® Kits

SensiFAST™ SYBR® Kits from our partner Meridian (Bioline) have been developed for fast, highly-sensitive and reproducible qPCR and have been validated on commonly-used real-time PCR instruments. The use of an antibody-mediated hot-start DNA polymerase minimizes amplification from primer-dimers, thereby improving assay specificity and sensitivity.

To find the optimal SensiFAST kit for your real-time PCR instrument and application, you can also use this Real-Time PCR Selection Chart.