Real-time PCR uses fluorescent dyes, such as SYBR Green, or fluorescence-containing DNA probes, such as TaqMan, to measure the amount of amplified product as the amplification progresses. There are two main methods for detection of the real-time PCR products: non-specific fluorescent dyes and sequence-specific DNA probes.
Non-specific dyes
These are dyes that bind to all pieces of double-stranded DNA and result in fluorescence. SYBR Green is an example of a non-specific dye that is commonly used in real-time PCR. SYBR Green is an asymmetrical cyanine dye used to stain nucleic acids. It preferentially binds to double-stranded DNA and will also bind to single-stranded DNA as well as RNA but with lower performance. The resulting DNA-dye complex emits green light providing a fluorescent signal to measure amplification.
Because these dyes bind to all double-stranded DNA sequences, the fluorescence intensity increases after each cycle and allows for detection and quantitation. Since there is no associated unit of measure, only a fraction or ratio to a standard dilution can be determined. One problem with this method is that the dyes are non-specific and will even bind with primer dimers. This can potentially cause inaccurate quantification of the intended target sequence.
Sequence-specific DNA probes
This method of detection is more specific than the above as probes are designed to bind only to certain DNA sequences and are labeled with a reporter molecule that permits detection only after hybridization. The use of these sequence-specific probes allows for the detection of only the specific DNA product. Because different probes have different signals, multiple targets can be detected within a single reaction mixture. This process is known as multiplex detection.
TaqMan is an example of a sequence-specific DNA probe technique that is a widely used detection method. It uses Taq polymerase and a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR cycles.
A specific example of how a sequence-specific probe works is detailed with a fluorescence resonance energy transfer (FRET) probe. In this process, fluorescence is emitted after hybridization takes place. Each probe consists of both a donor and a reporter fluorophore with the donor being on the 3' end and the reporter on the 5' end. The proximity of the two fluorophores to each other allows for the donor to reduce the fluorescent state of the reporter. When the probe attaches to the single-stranded piece of DNA, the fluorescence is still suppressed. As the polymerase takes effect and begins to synthesize the new complementary strand of DNA, the reporter and donor fluorophores are separated. This separation allows for the reporter to emit its energy, which is detected by the instrument. The more amplification that takes place, the higher the fluorescence.
