User:Rdbrady/Dual Labeled Probes

Dual Labeled Probes (DLPs) are hybridization probes which have been covalently bonded to a fluorophore and a FRET quencher. In general, DLPs begin in a quenched state with the fluorophore and quencher in close proximity. The specific type of probe determines how fluorescence is released. Dual labeled probes have uses in SNP genotyping, real time PCR, and other genomics applications.

Hydrolysis Probes
Hydrolysis probes consist of a fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3’-end. They are designed such that they anneal within a DNA region amplified by a specific set of primers. As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5' to 3' exonuclease activity of the polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the real-time PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR. A typical hydrolysis probe can be divided in 3 parts: 5' fluorophore Probe sequence: A sequence of nucleotides which specifically anneals to the target site. Quencher: The quencher dye is covalently attached either to the 3' end or to a thymadine base approximately 15 bases from the 3' end. If an internal quencher is used, a spacer is required. Spacer (optional): If a quencher is not present on the 3' end, a short carbon chain spacer must be added to prevent the probe from being extended by polymerase. Hydrolysis probes are sometimes referred to simply as Dual Labeled Probes, Linear Probes, Dual Labeled BHQ® Probes (after the quencher typically used) or TaqMan®.

Molecular Beacons
Molecular beacons are hairpin shaped molecules with an internally quenched fluorophore. A typical molecular beacon probe is 25 nucleotides long. The middle 15 nucleotides are complementary to the target DNA and do not base pair with one another, and the five nucleotides at each end are complementary to each other and not to the target DNA. A typical molecular Beacon Structure can be divided in 4 parts: Loop: This is the 18-30 base pair region of the molecular beacon which is complementary to the target sequence. Stem: The beacon stem sequence lies on both the ends of the loop. It is typically 5-7 bp long at the sequences at both the ends are complementary to each other. 5' fluorophore: Towards the 3' end of the molecular beacon, is attached a dye that fluoresces in presence of a complementary target. 3' quencher (non fluorescent): The quencher dye is covalently attached to the 3' end of the molecular beacon and when the beacon is in closed loop shape, prevents the fluorophore from emitting light.