To understand many things in the qPCR world and to do the right strategic choice, it is better to know few little things about the fantastic world of...fluorophores.
As previously said, PCR systems need primers and if used a probe. I will take a double hybridized probe TaqMan type as an example for this topic.
These probes are grafted on both extremities by a reporter and a quencher (explained in a previous post)
If using only one probe in the PCR master mix (a simplex), most used fluorophore is then FAM (6-carboyfluorescien or 6-FAM) and the most used quencher is TAMRA (6-carboxy-tetramethyl-rhodamine)
FAM is also one of the most intense molecule considering fluorescence answer to excitation and TAMRA is just the most used for a while but having the drawback to have its own residual fluorescence, able to cause interferences to PCR reaction with small target quantities. TAMRA is replaced more and more by non fluorescent quencher like BHQ (Black Hole Quencher), DDQ (Dark Quencher) or MGB from Applied Biosystem-Life Technologies (comprising a Non fluorescent Quencher).
Hereunder, normalised emission spectra from few fluorophores used in qPCR
It can be observed that FAM and TAMRA spectra are well dissociated and allow a good signal distinction.
In the following table, many more fluorophores are described with their emission and excitation wavelength. Quenchers are also reported on the table side with their use range.
If the table is not clearly visible, here are few links to find any answer about fluorophores association
- IDT report very interesting : http://www.idtdna.com/pages/docs/technical-reports/fluorescence-and-fluorescence-applications.pdf
- Gateway table : http://www.sciencegateway.org/resources/fae1.htm
- Eurogentec guide : http://www.eurogentec.com/uploads/qPCR-guide.pdf
These references are good ones, especially Eurogentec guide. To be honest, I avoid in this blog to repeat one more time things that are described already elsewhere, but sometimes it has to be made.
We can setup probes having FAM-BHQ1 but not FAM-BHQ3 considering the aborption spectra from the quencher. We can also setup a Cy5-BHQ3 probes but not with BHQ1
Fluorophores choice is essential for a good PCR. It is much more essential when PCR strategy needs a duplex or duplex amplification because chosen ones have to be efficient enough but without having a big wavelength overlapping or too much different fluorescence intensity.
All of this will be part of part 2.
Have a good PCR.
