Figure 1. Illustration of how probe-based qPCR works. In the figure R - reporter dye, Q - quencher. Green line illustrates the probe with the fluorescent reporter dye and quencher. Blue lines represent the forward and reverse primers.
The good thing about this method is that you can be sure that the signal comes from the intended target, because you can only get a signal if the primers and the probe bind to the correct sequence. This is provided that the probes and primers are designed well and don’t bind non-specifically to somewhere else. We will talk more in detail about the correct design in the next section.
In addition to the higher specificity (than with the dye-based qPCR), probe-based qPCR also saves time from analysing the qPCR results after the reaction has finished, since you don’t need to figure out whether you are looking at the correct target amplification or not [1]. Because of the specificity of the probes, they are also useful if you don’t have a lot of target material or if you want to detect small changes in the target like SNPs (single nucleotide polymorphisms) [4].
As previously mentioned, probe-based qPCR can be used for multiplexing. It means that it is possible to detect several sequences in a single reaction by using differently labelled probes that carry different reporter dyes. Since different fluorophores are detected in different channels, it is later possible to distinguish which one of the targets was amplified [1][3]. This can make experiments faster and less expensive than using singleplexing. Nevertheless, it is important to remember to add more reaction components to the mix or use a designated reagent designed for multiplexing, so that there wouldn’t be a competition, which can limit the amplification of one or more targets.
If you use multiplexing, you should get a result similar to this (Figure 2):
Figure 2. Multiplexing results with SolisFAST® Probe qPCR Mix with UNG. Fourplex qPCR amplification with four tenfold serial dilutions of human gDNA (40 ng – 40 pg, three replicates at each concentration). qPCR was performed on a QuantStudio™ 6 Flex qPCR cycler (Applied BioSystems™) with SolisFAST® Probe qPCR Mix (ROX) using ROX dye for normalisation. Thermal conditions: activation 3 min at 95 °C, cycling 5 sec at 95 °C, 20 sec at 60 °C.
How to design a probe?
The most difficult part of probe-based qPCR is probably designing the probes. Once that is done everything else is really not that different from dye-based qPCR (which we will talk about in our future blogposts).
When starting to design a probe it would be wise to also keep the primers in mind to avoid self-complementary and to ensure their specificity for the target. Running a BLAST alignment can most likely help with that. You can also try other softwares like SnapGene, Benchling etc. Probes should be close to the primers in terms of location (about 50 bp), but not overlap. Medium amount (~50%) of GC content should be targeted to allow complexity while maintaining a unique sequence [5][6]. Also the probe should not be very long (~30 bp), otherwise it will be difficult to properly quench the fluorescence [5][6]. Probes should have a melting temperature a bit higher than the primers (8-10°C) to ensure better sensitivity [6]. In the cycling programme the annealing temperature should be set maximum 5°C below the lower primer melting temperature to allow specific binding [6]. These are the recommendations that most probe design programs will also tell you to keep in mind, but depending on the experiment optimisation might be required.
When planning a multiplex experiment it is important to design probes with reporter fluorophores that have different enough excitation and emission wavelengths from each other, so that the qPCR machine can differentiate between them [6]. The quenchers must also match the reporters [6]. In addition, make sure that your qPCR machine is actually capable of reading all the fluorophore signals and whether you have a no ROX, high ROX or low ROX machine.
In the end you should know that there are different probe variants for qPCR and these variants also have variants and these variants have modifications, so there are really a lot of options from which to choose from. The most popular ones are hydrolysis probes (e.g. TaqMan®) that are good for quantification and multiplexing [3][5][6]. Then there are also molecular beacons, which are considered more specific and can be used for example for SNP detection [1]. There are of course many others about which you can find out more by yourself depending on the experiment you are planning [1][5][6][7]. But if there is a specific type of probe you would like to see a blogpost about, then let us know and we will make it happen.
Solis BioDyne options
Once you have decided on which probes you are going to use, there is another important choice you need to make and that is choosing the right qPCR mix for your experiment. Luckily Solis BioDyne has an option for your every need. Whether you want to go fast or slow, multiplex or not, deal with difficult sequences or regular ones, we have something for you.
SolisFAST® Probe qPCR Mix - extremely fast, inhibitor tolerant and highly sensitive probe-based qPCR Mix that has been optimised for detection and quantification of up to five targets simultaneously.
SolisFAST® Probe qPCR Mix with UNG - extremely fast and highly sensitive probe-based qPCR Mix. For extra convenience the mix includes dUTP and UNG enzymes that enable UNG treatment to prevent carryover contamination. Has been optimised for detection and quantification of up to five targets simultaneously.
HOT FIREPol® Probe qPCR Mix Plus - cost-effective real-time qPCR master mix for probe-based qPCR assays. This master mix has been developed for TaqMan® probes, but is suitable for other hydrolysis probe types as well.
HOT FIREPol® Probe Universal qPCR Mix - high performing, precisely-optimised probe-based qPCR Mix for AT-rich, regular and GC-rich templates. This master mix has been developed for TaqMan® probes, but is also suitable for other hydrolysis probe types.
HOT FIREPol® Multiplex qPCR Mix - probe-based qPCR master mix that has been optimised for highly sensitive and accurate quantification of up to 4 targets in a single reaction. This master mix was developed for TaqMan® probes, but is also suitable for other hydrolysis probe types.
For more information
We have previously written about multiplexing, different PCR options and primer design. Have a look at those blogpost for more details.
In case you are looking for help with your qPCR troubleshooting check out our troubleshooting guide or write to our customer support support@solisbiodyne.com or contact us through the chat on our website.
References:
[1] Marras, S. A. E., Tyagi, S., & Kramer, F. R. (2006). Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes. Clinica Chimica Acta, 363(1-2), 48–60. doi:10.1016/j.cccn.2005.04.037[2] Wong, W., Farr, R., Joglekar, M., Januszewski, A., & Hardikar, A. (2015). Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs. Journal of Visualized Experiments, (98).doi:10.3791/52586 [3] Hulley, E. N., Tharmalingam, S., Zarnke, A., & Boreham, D. R. (2019). Development and validation of probe-based multiplex real-time PCR assays for the rapid and accurate detection of freshwater fish species. PLOS ONE, 14(1), e0210165.doi:10.1371/journal.pone.0210165 [4] Lefever, S., Rihani, A., Van der Meulen, J., Pattyn, F., Van Maerken, T., Van Dorpe, J., … Vandesompele, J. (2019). Cost-effective and robust genotyping using double-mismatch allele-specific quantitative PCR. Scientific Reports, 9(1), 2150. doi:10.1038/s41598-019-38581-z [5] De Muro, M. A. (2008). Probe Design, Production, and Applications. Molecular Biomethods Handbook, 41–53. doi:10.1007/978-1-60327-375-6_4[6] Rodríguez, A., Rodríguez, M., Córdoba, J. J., & Andrade, M. J. (2015). Design of Primers and Probes for Quantitative Real-Time PCR Methods. PCR Primer Design, 31–56. doi:10.1007/978-1-4939-2365-6_3 [7] Murray, J. L., Hu, P., & Shafer, D. A. (2014). Seven Novel Probe Systems for Real-Time PCR Provide Absolute Single-Base Discrimination, Higher Signaling, and Generic Components. The Journal of Molecular Diagnostics, 16(6), 627–638. doi:10.1016/j.jmoldx.2014.06.00
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