“Quantitative PCR is a technique for nucleic acid quantification. TaqMan Probe and SYBR Green Dye are two common techniques used for qPCR.”
In our previous comprehensive article on real-time PCR, we discussed the concept, principle, process, steps, applications and limitations. We did mention the two types of chemistry behind quantitative analysis but didn’t explain much.
The two common types of chemistry behind quantitative PCR analysis are probe-based hybridization and dye-based qPCR analysis. TaqMan is a common probe while SYBR green is a dye used for probe-based hybridization and dye-based intercalation, respectively.
While both achieve the same ultimate goal which is to quantify the target nucleic acid levels– their principle, working, advantages, limitations and applications differ significantly.
So in this article, I compare these two chemistry, again extensively; and guide you to choose the best technique for your quantitative PCR analysis. Explore the concept, principle, advantages, disadvantages and applications of each technique in the present blog.
TaqMan Probe-based hybridization:
The TaqMan probe relies on the principle of hybridization and subsequent hydrolysis. Again, I planned to write a dedicated article on the TaqMan probe, so here I am only giving you a rough idea.
So the probe having a fluorescent dye- the reporter dye, binds upstream to the primer. Usually under normal conditions, the reporter dye remains in close association with the quencher dye and can’t emit fluorescence.
The special type of Taq DNA polymerase having the exonuclease activity, dissociates the probe from the target sequence which eventually releases fluorescence and is recorded as a positive amplification.
A TaqMan probe is a short, single-stranded, labeled and sequence-specific probe used for accurate quantification in PCR. It’s highly specific and accurate.
Dye-based intercalation technique:
In the dye-based qPCR approach, instead of a probe, a fluorescent dye is used for the quantification. SYBR Green is a fluorescent dye commonly used in this technique. Now, here the dye only binds to the dsDNA and releases fluorescence.
Meaning, it gives us a positive signal only when amplification occurs and dsDNA forms. The dye-based method is cost-effective and handy but at the same time is not so accurate. It provides signals in the case of non-specific amplification as well.
Moving ahead, let’s discuss some of the differences between the two.
The TaqMan probe-based method uses a short, single-stranded and labeled DNA probe while the SYBR Green dye-based method only uses a dye for quantification. No such probes are used in the dye-based approach.
The probe-based quantification relies on the principle of hybridization in which the probe binds only with its highly complementary location on the target sequence. Conversely, in the dye-based quantification, a dye can directly bind to the double-stranded DNA, and hybridization doesn’t occur here.
In the probe-based quantification the fluorescence signal increases when the reporter dye is unquenched and detaches from the sequence, while in the dye-based quantification, the fluorescence signal increases when the dye binds to the dsDNA.
The TaqMan probe-based quantification is highly specific as it only hybridizes with the complementary location, on the other hand, the dye-based quantification is not so specific as it can bind to any double-stranded DNA, whether it is specific or non-specific, in the reaction.
Henceforth, in order to achieve high-sequence complementation, prior probe-designing is required in the probe-based method but is not required in the dye-based method.
A reporter dye is incorporated on the 5’ end while a quencher dye is incorporated on the 3’ end of the TaqMan probe, however, no such labeling is required in the dye-based quantification. A DNA-binding dye can directly be added to the reaction.
Studies suggest that the probe-based technique doesn’t inhibit the reaction while the dye oftentimes can inhibit the PCR reaction and so lacks precision.
At the assay level, as different probes can be designed for different template DNA regions, multiplexing can be performed in probe-based chemistry. Contrary, the dye-based approach doesn’t support multiplexing.
>> Learn more about multiplexing: Multiplex PCR- Principle, Process, Protocol, Advantages, Limitations and Application.
Nonetheless, probe-based quantification is a complex, tedious and complicated process that needs prerequisites and high-end expertise while dye-based quantification is a simple, straightforward and easy method. It doesn’t need any prerequisites or high-end expertise.
So the TaqMan probe-based approach is costly and time-consuming while the SYBR Green dye-based approach is relatively cheaper and faster.
Due to its high sequence specificity and accurate quantification, the TaqMan probe-based technique has been utilized in gene expression, quantification, viral load estimation, SNP genotyping, Multiplex analysis, pathway analysis, copy number variation studies, mutation detection and sequencing-based studies.
While the dye-based approach has been used in gene expression studies and pathogen detection only.
Using the TaqMan probe-based approach, as low as 1 to 10 copies of template DNA can be detected. However, the copy detection efficiency of the dye-based approach is variable and lacks precision in low-abundant DNA detection.
Because of these numerous advantages, a large number of pre-designed assays are available for TaqMan probe-based quantification. Contrary, fewer standard pre-designed assays are available for dye-based quantification.
|TaqMan probe-based qPCR||SYBR Green dye-based qPCR|
|A short, single-stranded and labeled probe is used.||Any sequence-specific probe is not used here.|
|No dsDNA binding dye is used.||dsDNA binding dye is used.|
|Use the principle of hybridization and probe displacement by Taq DNA polymerase.||Doesn’t use the principle of hybridization or probe displacement.|
|Highly specific.||Comparatively less specific.|
|Less chances of non-specific signals.||High chances of non-specific signals.|
|Require extensive probe design and validation.||Doesn’t require probe designing and validation.|
|A probe can bind to its exact complementary sequence only.||Dye can bind to any dsDNA present or produced during the amplification.|
|It can detect as low as 1 to 10 copies of the template present in the sample.||The detection sensitivity is variable and can’t detect low-copy number DNA effectively.|
|Allows multiplexing.||Doesn’t allow multiplexing.|
|Complex and tedious process.||Simple and easy process.|
|Applied for gene expression studies, disease diagnosis, pathogen detection and quantification, CNV and low abundant DNA detection.||Applied for gene expression studies and pathogen detection and characterization.|
How to choose between TaqMan probe-based vs SYBR Green dye-based qPCR assay?
So far we have understood the concept of each type of qPCR assay and are trying to differentiate between the same. Now, let’s discuss the main point of this article. How can you choose the technique for your experiment?
However, let me tell you if you read the article thoroughly you will get the answer! Anyway, I will simplify things here.
TaqMan Probe-based assay is highly accurate, sequence-specific and allows multiplexing. So the first point is clear, if you want accurate results, high-sequence specificity and want to quantify many templates in a single reaction, you have to choose TaqMan probe-based PCR quantification.
On the other hand, if you want a cost-effective, simple, quick and flexible option, definitely you can go with the dye-based assay. However, less customized and ready-to-use options are available for dye-based quantification.
In another scenario, if you are an experienced peer, doing extensive research, or clinical analysis, disease diagnosis or low abundant DNA analysis (which requires high precision and accuracy) you should go with the TaqMan probe-based assay.
However, for your Master’s/ PhD research or some non-critical research work, you can try the dye-based approach. But in my opinion, if costume and ready-to-use options are available for any level of molecular genetic experiment, you must go with it.
The reason is! The qPCR experiment is not as simple as the conventional one, so it’s better to go with the flow and use some standard kits.
Now, let’s make the picture more clear.
Use TaqMan probe-based qPCR assay for,
- High-sensitive reactions.
- Multiplex reaction.
- High accuracy and sequence specificity.
- Gene expression analysis.
- Clinical studies.
- Disease diagnosis.
- SNP genotyping.
- GMO studies.
- Low abundant DNA analysis.
Use SYBR Green Based qPCR assay for,
- If you are a budding scientist.
- Low-cost option.
- Single reaction.
- Simple and easy reaction.
- Primary screening in genetic experiments.
- High-throughput data validation.
- eDNA and microbial DNA studies.
>> Related article: What is Environmental DNA?
Quantitative PCR is a crucial technique for quantitative analysis in molecular genetic labs. These two types are commonly employed depending on the requirement of the scientist. However, each type of assay has its own advantages and limitations.
I strongly recommend working with both techniques, if you get an opportunity to explore so that you can get an idea about the platform. Notwithstanding, the TaqMan probe-based approach is widely accepted in scientific experiments and the clinical diagnosis industry.
I hope this article will add more value to your PCR learnings. Do share this article and bookmark the page.
P.S. Strengthen your PCR knowledge by enrolling in our PCR mastery course.