HRP is remarkably stable, resists denaturation under the conditions used for hybrid formation, and is relatively inexpensive. Direct fluorescence detection of DNA hybrids has several advantages over enzymatic techniques. Fluorescent molecules can be subjected to the rigorous conditions used in DNA synthesis and in performing hybridization reactions, without substantial losses in detectability. Thus, they are detectable with high sensitivity. Fluorescent oligonucleotides are applicable in situations such as DNA sequencing, which is difficult to perform with direct enzymatic labels.
Fluorescent labels provide for exceptionally sensitive detection.
Background fluorescence can be substantially reduced by using fluorophores that emit toward the red end of the visible light spectrum. Direct fluorescent labels are useful in several applications. One of the most useful applications of fluorescence quenching, however, is the development of the Taqman system sold by Perkin-Elmer for quantifying PCR products. The assay employs a fluorogenic probe, which consists of an oligonucleotide with both a reporter and a quencher dye attached. During PCR, a fluorogenic probe anneals specifically between the forward and reverse primer sites if, and only if, the target sequence is present.