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When a fluorescent dye is excited at a particular wavelength, it is promoted to an excited state. In the absence of a quencher, the excited dye emits light in returning to the ground state. When a quencher is present, the excited fluorophore can return to the ground state by transferring its energy to the quencher, without the emission of light, while the quencher is promoted to its excited state.
FRET quenching depends on the ability of the fluorophore to transfer energy to the quencher. In order for this to happen, the emission spectrum of the fluorphore must overlap with the absorption spectrum of the quencher. In order for a quencher to quench fluorescence from several different fluorophores, it must therefore have a wide absorption spectrum and a high extinction coefficient.
Standard fluorophores return from the excited state to the ground state with the emission of light. The overall process of excitation, transfer, and emission to a second fluorophore is called fluorescence resonance energy transfer (FRET). Dark quenchers return from the excited state to the ground state via non-radiative decay pathways, without the emission of light. This is also a FRET mechanism but there is no secondary emission of light.
The black hole quenchers (BHQs) are sold by Biosearch Technologies.
Table 1 ⎪ Black hole quenchers (BHQs)
| Name | λmax / nm (absorption) | Appox. quenching range / nm | E at λmax | Compatible fluorophores |
|---|---|---|---|---|
| BHQ0 | 493 | 430-520 | 34 000 | - |
| BHQ1 | 534 | 480-580 | 34 000 | FAM, TET, JOE, HEX, Oregon Green® |
| BHQ2 | 579 | 550-650 | 38 000 | TAMRA, ROX, Cy3, Cy3.5 |
| BHQ3 | 672 | 620-730 | 42 700 | Cy5, Cy5.5 |
Figure 1 | Structures of Black Hole Quenchers (BHQs)
Table 2 ⎪ Atto quenchers
| Name | λmax / nm (absorption) | Appox. quenching range / nm | E at λmax | Compatible fluorophores |
|---|---|---|---|---|
| ATTO 540Q | 542 | 530-560 | 105 000 | - |
| ATTO 580Q | 586 | 560-610 | 110 000 | - |
| ATTO 612Q | 615 | 600-630 | 115 000 | - |
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