Everything you need to know about dye-based qPCR

There are two options for target detection when doing qPCR - either you use dye or probe-based detection. Today we are going to take a deep dive into the world of dye-based qPCR, which was invented during the early nineties by Russell Higuchi [1] and is still used daily around the world.  

What is dye-based qPCR? 

Dye-based qPCR is a cost-effective qPCR option that measures double-stranded DNA amplification during PCR (Polymerase Chain Reaction) based on the detection of fluorescence of a DNA binding dye [1]. In addition to the qPCR mix, nuclease-free water, and DNA it only requires two sequence-specific primers, making it a cost-effective option when looking at a large number of targets. 

How does it work? 

As mentioned previously, dye-based qPCR uses a DNA binding fluorescent dye. Before binding to the DNA, the dye exhibits low background fluorescence. During amplification the dye binds to any double-stranded DNA product found in the sample. This results in an increase of fluorescence that is proportional to the increase of PCR amplicons present at each cycle. [1] 

Fluorescence is measured after every PCR cycle. The threshold cycle (Ct), also known as quantification cycle (Cq), is reached when enough fluorescent dye has bound to the double-stranded DNA to have higher fluorescence than the background fluorescence. The Ct value is used to quantify the amount of target DNA in the sample (the lower the Ct level the bigger amount of target DNA in the sample). [2][3] 
Dye-based qPCR is considered cost effective, because only two sequence-specific primers are required per target, but since these primers are the only way to determine the specificity of the reaction, it’s not as specific as conventional PCR or probe-based qPCR [4]. However, it is way cheaper than having to buy primers and probes for probe-based qPCR. 
The main problem with primers in dye-based qPCR is that they tend to form primer dimers or non-specific amplicons [5][6]. The dye will unfortunately detect and bind to any double-stranded DNA, which also includes all the off target, i.e., non-specific amplification products. This can result in inaccurate quantification of the target sequence. [5] 
There is however a simple way to identify the problem. It is possible to compare the melting temperatures of amplification products using melt curves. This allows to verify the specificity of amplification and to check for the presence of non-specific amplification products [6].  
Melt curve analysis is based on the fact that different double-stranded DNA strands dissociate from each other at different temperatures depending on various factors (G-C content, SNPs, etc.). The melt curves are produced when the PCR product is heated and the double-strands start to dissociate, during which the fluorescent dye also dissociates from the strands, hence a drop in fluorescence can be detected. [6] 
After the qPCR machine has finished collecting data you will get an amplification plot that visualises the accumulation of DNA over the duration of the qPCR. The lines show fluorescent signal from the dye normalised to the signal of the passive reference dye (for example ROX) minus the baseline generated by the qPCR machine [2][3]. It should look something like this: 

Amplification plot of our SolisFAST® SolisGreen® qPCR Mix. 

What are the pros and cons of dye-based qPCR?  

There are many reasons why dye-based qPCR is so popular among scientists. Here are some of them: 
  1. Cost (just 2 primers) - a more budget-friendly option than probe-based qPCR.
  2. Easier to design than probe-based qPCR. Again, you only need 2 primers for the former. The latter requires 2 primers and a very specifically placed probe.
  3. Possible to detect mistakes in addition to the gene of interest, unlike with probe-based qPCR. So, it is possible to optimise the experiment and have better results next time.    
    Unfortunately, like with every method, dye-based qPCR is not ideal for every experimental design. Here are some cons: 
    1. It’s not possible to do multiplexing. 
    2. It has low specificity. 

    What are the dye options for dye-based qPCR? 

    The most important part of dye-based qPCR is of course the dye. Choosing the right one might be even more difficult than choosing a new look for yourself at the hair salon. Unfortunately, we can’t help with hairstyles, but we do have some recommendations for selecting the right dye for your qPCR experiment. 
    The classical and probably most often used dye is SYBR® Green [7]. There are, however, better options, that may not be as popular yet, but are worth considering. One of them is EvaGreen®, which is spectrally similar to SYBR® Green, so there is no need to change any optical settings when using it [8]. EvaGreen® is very stable at room temperature and also under PCR condition [8]. It is less inhibiting concentration wise – it’s possible to increase the dye’s concentration to increase fluorescence without messing up the reaction [8. This additionally enables better high-resolution melt analysis [8]. Furthermore, EvaGreedye has less background than SYBR® Green I due to its novel “release-on-demand” DNA-binding mechanism [9]. On top of that, EvaGreen® is also considered less toxic and more environmentally safe than SYBR® Green [9]. 
    Another option is SolisGreen® that also shares spectral properties with other commonly used qPCR dyes. qPCR mixes based on SolisGreen® dye have high sensitivity and increased performance (brighter fluorescence) with low target concentration demonstrating improved precision and less variance between technical replicates. 

    There are of course multiple other dyes to choose from, but all Solis BioDyne products are based either on EvaGreen® or SolisGreen® to ensure good product stability, efficiency and sustainability. 
    EvaGreen® options: 
    SolisGreen® options: 
    [1] Higuchi, R., Fockler, C., Dollinger, G. et al. Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions. Nat Biotechnol 11, 1026–1030 (1993). https://doi.org/10.1038/nbt0993-1026 
    [2] Schefe, J.H., Lehmann, K.E., Buschmann, I.R. et al. Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s C T difference” formula. J Mol Med 84, 901–910 (2006). https://doi.org/10.1007/s00109-006-0097-6 
    [3] Article 5: qPCR data analysis – Amplification plots, Cq and normalisation. (2009). Retrieved 1 November 2021, from https://www.europeanpharmaceuticalreview.com/article/725/article-5-qpcr-data-analysis-amplification-plots-cq-and-normalisation/ 
    [4] Udvardi, M. K., Czechowski, T., & Scheible, W. R. (2008). Eleven golden rules of quantitative RT-PCR. The Plant cell, 20(7), 1736–1737. https://doi.org/10.1105/tpc.108.061143 
    [5] Raso, A., & Biassoni, R. (2014). Twenty Years of qPCR: A Mature Technology? Quantitative Real-Time PCR, 1–3.doi:10.1007/978-1-4939-0733-5_1  
    [6] Ririe, K. M., Rasmussen, R. P., & Wittwer, C. T. (1997). Product Differentiation by Analysis of DNA Melting Curves during the Polymerase Chain Reaction. Analytical Biochemistry, 245(2), 154–160.doi:10.1006/abio.1996.9916  
    [7] Zipper, H., Brunner, H., Bernhagen, J., & Vitzthum, F. (2004). Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic acids research, 32(12), e103. https://doi.org/10.1093/nar/gnh101 
    [8] Mao, F., Leung, WY. & Xin, X. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol 7, 76 (2007). https://doi.org/10.1186/1472-6750-7-76 
    [9] Selvaraj V., Maheshwari Y., Hajeri S., Yokomi R. (2019) Droplet Digital PCR for Absolute Quantification of Plant Pathogens. In: Khurana S., Gaur R. (eds) Plant Biotechnology: Progress in Genomic Era. Springer, Singapore. https://doi.org/10.1007/978-981-13-8499-8_25