Hydrolysis probes

Hydrolysis Probes. A fluorophore-labeled probe can be synthesized with a quencher located in a position that allows... 

Figure 37-24 Common probes and dyes for real-time PCR. f/J Double-stranded DNA dyes show a significant increase in fluorescence when bound to DNA (hv = excitation light). (2) Adjacent hybridization probes. Fluorescence resonance energy transfer (FRET) is illustrated between a donor and acceptor fluorophore.The x indicates phosphorylation of the 3 terminus of the probe to prevent polymerase extension. (3) FRET between a labeled primer and a single hybridization probe. (4) Hydrolysis probes are cleaved between the reporter and quencher, resulting in increased fluorescence. (5) Hairpin probes are quenched in the native conformation, but increase in fluorescence when hybridized. (6) Hairpin primers retain their native, quenched conformation until they are incorporated into a double-stranded product (Modified with permission of the publisher from Pritham GH, Wittwer CT Continuous f/uorescent monitoring of PCR.J Clin Ug Assay 1998, 21 404-412. 1998 Clinical Ligand Assay Society, Inc.)...

A similar technology relies on hydrolysis probes, namely, Taqman chemistry. Two probes are designed, one a perfect match to the normal allele and one to the mutant or variant allele. In Taqman chemistry, probes are released from quenching when the Taq polymerase degrades the probe, releasing the fluorophore into solution. This technology was developed from Applied Biosystems (Foster City, California) and is compatible with the 7900 real-time instrument (Applied Biosystems). Taqman chemistry also is developed easily by individual laboratories [7]. 

T. Kaasgaard, J.H. Ipsen, O.G. Mouritsen, and K. Jorgensen. In situ atomic force microscope imaging of phospholipase A2 lipid bilayer hydrolysis. /. Probe Microsc., 2001, 2, 169-175. 

Bioluminescent Detection ofRNA Hydrolysis Probes in DMA Testing... 

Hydrolysis probes (e.g.,TaqMan probes) are the most widely used and pubhshed in detection chemistry hterature for probe-based real-time PCR apphcations. In addition to the primers, it includes a third oligonucleotide, 20 to 26 bases in length, in the reaction known as the probe. A fluorescent reporter dye, most frequendy... 

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