AAT Bioquest

Should I use probe-based detection or dye-based detection for my qPCR analysis?

Posted May 12, 2023


Understanding the pros and cons of probe-based and dye-based detection can help you determine which is best for your qPCR analysis. 

Dye-based detection

In dye-based detection, an intercalating dye (SYBR Green) is used to bind all double-stranded DNA. 

The advantage of dye-based detection is that it is more cost effective as it only requires the addition of PCR primers. 

On the downside, the intercalating dye detects all dsDNA produced in the reaction, which may result in inaccurate quantification as off-target and non-template amplification (NTC) can be observed for some primer sets. You will need to perform denaturation curves after the PCR to distinguish between correct and nonspecific products. Another downside of dye-based qPCR is the inability to perform multiplex reactions, which means you can only measure a single amplicon. 

Probe-based detection

Probe-based detection uses fluorescent-labeled target-specific probes to measure DNA amplification at each cycle of a PCR.

The biggest advantage of probe-based detection is that it yields increased specificity and sensitivity as only specific DNA molecules are labeled. This method is unlikely to result in inaccurate quantification due to NTC amplification. It also offers the least background fluorescence. Another advantage of probe-based detection is that it allows multiplex reactions by designing different amplicons with unique fluorophores according to the optical capabilities of the qPCR instrument.

The downside of probe-based detection is that it is more expensive as you need to design a sequence-specific fluorescently-labeled probe oligonucleotide in addition to typical PCR primers. 

Overall, probe-based detection is the more sensitive detection method and provides an accurate and reproducible analysis, making it the preferred method for measuring transcript abundance.

Additional resources

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Real-Time PCR (qPCR)

ROX Reference Dye *50X fluorescence reference solution for PCR reactions*