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The page below is a sample from the LabCE course Real-Time PCR. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Detection Methods

Real-time PCR uses fluorescent dyes, such asSybr 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. 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 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 detcted within a single reaction mixture. This process is known as multiplex detection and is the subject of another course.
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 being 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 detetced by the instrument. The more amplification that takes place, the higher the fluorescence.