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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

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 the concentrations to be determined. 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. However, if the specificity is not required, this is a more cost-effective method than the sequence-specific DNA probe method.

Sequence-specific DNA probes

As the name implies, this method of detection is more specific than non-specific dyes. Probes can be designed to bind only to certain DNA sequences and are labeled with a fluorescence reporter that permits detection only after hybridization. The use of these sequence-specific probes allows for the detection of only the specific DNA product and utilizes fluorescence resonance energy transfer (FRET).

These probes are also used for multiplexing. Multiplexing is the process of assaying several different genes in the same reaction. In this process, each probe has a different color reporter, which allows for the quantification of several different sequence products at the same time.