What are the widely used detection methods in quantitative PCR (Q-PCR)
Posted August 29, 2023
One detection method of qPCR is dye-based detection via the incorporation of a DNA binding dye in the PCR. The dyes are non-specific and are able to bind to any dsDNA produced during amplification resulting in emission of enhanced fluorescence. This permits the initial DNA concentration to be calculated with reference to a standard sample. Fluorescent signal intensity is directly related to the quantity of amplicon present. SYBR I dye is commonly used in Q-PCR experiments. Another widely used method is sequence-specific DNA probes consisting of oligonucleotides. These probes are labeled with a fluorescent reporter which allows detection only following hybridization of the probe with its complementary sequence. A third oligonucleotide and sometimes a fourth are conjugated to a reporter dye and/or a quencher moiety; dual-labeled probes are used in the 5’ nuclease assay. Another detection method is the quencher. Dark quenchers (e.g. Black Hole Quencher) are excellent alternatives to dye molecules, as they quench over a wide range of wavelengths and provide the possibility of multiplex reactions containing a high number of target/probe combinations.