Introduction to qPCR

Fluorescence and fluorescent probes

Fluorescence is the process in which a molecule absorbs light photons that excite the molecule’s electrons to a higher energy state. When these electrons relax to the lowest vibrational point of their excited state, the fluorescent molecule emits photons. These emitted photons will have a longer wavelength, compared to the photons that exited the fluorescent molecule in the first place (1)

Fluorescent molecules are an invaluable tool in molecular biology to identify, analyze and sequence DNA and they are widely used to label primers and probes.

Metabion offers a variety of different fluorescent dyes to use as a modification for your oligonucleotides. It is important that you carefully choose the fluorescent dyes that fit your needs best. To do this, you might need to take the following factors into account:

• It is key that the excitation and emission spectra of the fluorescent dye of choice are compatible with the instrumentation you are using. For instance, 6-Fam can be successfully used with a 488nm light source (2).
• For experiments in which several dyes are used, like in multiplex PCR, a good rule is that the difference between the peak emission wavelengths of each fluorophore is of at least 15 nm (2).
• Guanine shows an intrinsic fluorescence-quenching effect. Therefore, it is best not to position your fluorescent modification next to a G (3).
• To choose the fluorescent dye that best fits your need, you can check the excitation and emission peak of each dye in our Dual-labelled probes portfolio. For any further question, do not hesitate to contact our Customer Service by email, telephone and chat!
• For more information about how to choose a quencher, you can jump to the section dedicated to quenchers by clicking here

Energy transfer and quenching

The electronic energy of an excited fluorescent moiety can be transferred to another molecule according to different mechanisms. Perhaps the two most relevant mechanisms concerning electronic energy transfer in fluorescent probes are Förster Resonance Energy Transfer (FRET) and contact quenching. In FRET, the excited fluorescent dye works as an electron donor and the absorbing dye works as an electron acceptor. The acceptor may or may not be a fluorophore. In the first case scenario, the acceptor will emit photons. Instead, if the acceptor is not a fluorescent molecule, the transferred energy will be dissipated as heat. In either case, the resulting detectable fluorescence of the donor is decreased (2).

For contact quenching to take place, molecular contact between fluorophore and quencher has to occur, such that the electron clouds of both molecules interact (4). Therefore, donor and acceptor need to be in close proximity, such that molecular contact between the two molecules occur and the fluorescence quenching takes place (4)

Fluorescent Probes

Fluorescent probes used in qPCR are a powerful tool to enhance the specificity of your amplification product. Two main categories of DNA probes use fluorescence to detect nucleic acids: single-labelled and dual labelled probes. An excellent example of single labelled probes is represented by Light Cycler™ Hybridization probes, which are typically used in pairs, are single stranded and bind two different but neighboring target sequences. The probe pair is formed by one probe bearing a donor fluorophore and one probe bearing an acceptor fluorophore. FRET occurs only when both probes are hybridized to their target sequences. For more information and an exhaustive review about Light Cycler™ Hybridization probes in qPCR, please read our article.

Dual Labelled Probes

Dual labelled Probes are labelled with both, a fluorophore and a quencher. Typically, albeit not necessarily, the fluorophore is positioned at the 5’- and the quencher at the 3’-end of the probe. However positioned, the quencher blocks fluorescence emission either by fluorescence resonance energy transfer (FRET), or by contact quenching, depending on the kind of probe. Fluorescence emission only occurs when the fluorescent dye is somehow released from the close proximity to the quencher (2).
Dual labelled Probes can be further classified into the following categories: 5'-nuclease probes, molecular beacon probes, and strand-displacement probes (2).

Taqman™dual labelled Probes

TaqMan™dual labelled probes are single-stranded oligonucleotides labelled with a dye and a quencher. In these probes, dye and quencher interact via FRET and no detectable fluorescence is emitted. A fluorescent signal is emitted when these probes are degraded, due to the 5’ → 3’ exonuclease activity of the Taq polymerase (5; 6).

Molecular Beacons probes

With molecular beacons probes, fluorescent dye and quencher are brought in close proximity due to the hairpin structure of these probes, whose 5’ and 3’ regions are complementary and form the hairpin-stem. Instead, the loop of the hairpin is complementary to the DNA target sequence that will be amplified. Fluorescent dye and quencher are in such close proximity that the quenching effect takes place by contact quenching (7). Fluorescence is emitted only when the molecular beacon anneals to the target nucleic acid and dye and quencher are brought apart. Amplifluor and Scorpion primers have similar characteristics (2; 8; 9).

Strand-displacement probes

Strand-displacement probes are two separate and complementary probes, one bearing a fluorescent dye, the other bearing a quencher. Given that the probes are complementary, contact quenching avoids fluorescence to be emitted. Fluorescent dye and quencher are brought apart when one of the two probes hybridizes with the target sequence to form a more stable DNA duplex. At this point, fluorescence emission takes place. Duplex scorpion primers function on a similar basis (2; 10).

Quenchers: the dark side of your probe

Depending on which kind of probe you are going to use, fluorescence quenching might take place via FRET or contact quenching. Therefore, how can you choose a good quencher?

The first criterion to consider is that the emission spectrum of your fluorescent dye should be as similar to absorption spectrum of the quencher, as possible. In the past, fluorophores were used as quenchers. For instance, TAMRA was commonly used as a quencher for FAM, with the complication that TAMRA exhibits its own fluorescence, with an emission peak around 579nm. This results in fluorescence background, which can instead be avoided by using dark quenchers (2; 11). metabion offers a variety of these and we can recommend the following for best performance:

Jump to our Dual labelled probes portfolio, for more information!

References

1. Albani, Jihad René. Principles and Applications of Fluorescence Spectroscopy. France : Laboratoire de Biophysique Moléculaire Université des Sciences et Technologies de Lille, 2007.

2. Johansson, Mary Katherine. Methods in Molecular Biology, vol. 335: Fluorescent Energy Transfer Nucleic Acid Probes: design and Protocols. Totowa, NJ : Humana Press Inc., 2006.

3. Seidel, C. A. M., Schulz, A., and Sauer, M. H. H. Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiences. 1996, Vol. 100, pp. 5541–5553.

4. Lakowicz, Joseph R. Principles of Fluorescence Spectroscopy. s.l. : Springer, 2006.

5. E. Navarro, G. Serrano-Heras, M.J. Castano, J. Solera. Real-time PCR detection chemistry. 2015, Vol. 439, pp. 231-250.

6. Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W., and Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4 1995, pp. 357–362.

7. Tyagi, S. and Kramer, F. R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 1996, 303–308, p. 14.

8. Nazarenko, I. A., Bhatnagar, S. K., and Hohman, R. J. A closed tube format for amplification and detection of DNA based on energy transfer. 1997, 2516–2521, p. 25.

9. Whitcombe, D., Theaker, J., Guy, S. P., Brown, T., and Little, S. Detection of PCR products using self-probing amplicons and fluorescence. 1999, 804–807, p. 17.

10. Li, Q., Luan, G., Guo, Q., and Liang, J. A new class of homogeneous nucleic acid probes based on specific displacement hybridization. 2002, e5, p. 30.

11. Marras S.A.E., Krame F.R., and Tyagi S. Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res. 30, 2002, Vol. 21, e122.

12. Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York : W. H. Freeman, 2000.

13. Nazarenko IA, Bhatnagar SK, Hohman RJ. A closed tube format for amplification and detection of DNA based on energy transfer. 1997, Vol. 25, pp. 2516-2521.

14. Sittampalam GS, Coussens NP, Nelson H, et al.,. Assay Guidance Manual [Internet]. s.l. : Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2004.