There are two general types of fluorescent reporters that are used in real-time PCR assays:
Double-stranded DNA-binding Dyes
Double-stranded DNA-binding dyes are dyes that bind to the minor groove of double-stranded DNA (dsDNA). The free dye has a very low fluorescence, whereas the bound dye has a high fluorescence. The more double-stranded PCR product that is produced, the greater the fluorescence signal will be. BRYT Green® Dye is an example of a dsDNA-binding dye. These dyes allow you to use standard PCR primers, so there is no need to order special labeled primers. The disadvantage is that the signal is not sequence-specific, so you will need to verify that the reaction is producing the desired product.
Labeled Primer or Probe
The second type of reporter method uses a labeled primer or probe to detect the amplification product. There are many different ways to modify reporter signal generation. Real-time PCR using labeled oligonucleotide primers or probes employs two different fluorescent reporters and relies on energy transfer from one reporter (the energy donor) to a second reporter (the energy acceptor) when the reporters are in close proximity. The second reporter can be either a quencher or a fluor. If the second reporter is a quencher, the energy from the first reporter is absorbed but re-emitted as heat rather than light, leading to a decrease in fluorescent signal. Alternatively, if the second reporter is a fluor, the energy can be absorbed and re-emitted at another wavelength through fluorescent resonance energy transfer (FRET, reviewed in Didenko, 2001), and the progress of the reaction can be monitored by the decrease in fluorescence of the energy donor or the increase in fluorescence of the energy acceptor.
Some qPCR strategies employ complementary nucleic acid probes to quantify the DNA target. These probes also can be used to detect single nucleotide polymorphisms (1–2). There are many different types of real-time PCR probes, including hydrolysis, hairpin and simple hybridization probes. Probes contain a complementary sequence that allows the probe to anneal to the accumulating PCR product, but can differ in the number and location of the fluorescent reporters. The use of simple hybridization probes involves two labeled probes or, alternatively, one labeled probe and a labeled PCR primer. In the first approach, the energy emitted by the fluor on one probe is absorbed by a fluor on the second probe, which hybridizes nearby. In a second approach, the emitted energy is absorbed by a second fluor that is incorporated into the PCR product as part of the primer. Both of these approaches result in increased fluorescence of the energy acceptor and decreased fluorescence of the energy donor. The TaqMan® Assay is an example of a method that uses a labeled probe. The big advantage that label-based methods offer is the ability to multiplex two or more targets in a single reaction. This would allow you to measure an experimental gene and a control gene for normalization in the same reaction.