Key featuresShow Hide
- Used to add a non-fluorescent quencher to the 3' end of an oligonucleotide.
- Maximal absorption in the 560 to 670 nm range.
- Glycolate linker for rapid cleavage of the oligonucleotide.
- 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers).
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Since their introduction to the DNA marketplace in 2000, the Black Hole Quenchers have become THE gold-standard quencher of choice used in qPCR probes and other FRET applications.
When fluorogenic dual-labeled probes were first introduced, quenchers were often a second reporter dye, typically TAMRA. The sensitivity of these probes, such as FAM-TAMRA, is limited because the fluorescence from TAMRA can leak into the channel meant to detect FAM fluorescence. Dark quenchers, which are dyes with no native fluorescence, offer a solution to this problem.
The BHQ dyes are true dark quenchers with no native emission due to their polyaromatic-azo backbone. Substituting electron-donating and withdrawing groups on the aromatic rings produces a complete series of quenchers with broad absorption curves that span the visible spectrum into the near IR region.
BHQ dyes work through a combination of FRET and static quenching to enable researchers to avoid the residual background signal common to fluorescing quenchers such as TAMRA, or low signal to noise ratio.
These quenchers can be paired with all common reporter dyes to construct efficiently quenched qPCR probes for multiplexing assays. In addition to quenching by FRET, BHQ dyes have also been shown to efficiently quench fluorescence through static quenching via formation of a ground state complex with the reporter dye.
BHQ quenchers have broad absorption spectra and can be paired with reporter dyes that emit in the following ranges:
BHQ-0: 430-520 nm
BHQ-1: 480-580 nm
BHQ-2: 560-670 nm
BHQ-3: 620-730 nm
BHQ-10: 480-550 nm Water Soluble (WS)
LGC, Biosearch Technologies offers all Black Hole Quencher products available for 3’, internal and 5’ modification of oligonucleotides with a variety of options. BHQ-1 and BHQ-2 are the more popular, either as the 5'-Phosphoramidites, the dT-Phosphoramidites or the 3'-CPGs.
Only considering the excitation and emission values suggests Cy™5/Cyanine-5 and Quasar 670 require BHQ-3 for efficient quenching, however BHQ-2 is recommended because it is less susceptible to degradation. BHQ-1 is typically used to quench in the range 480 - 580 nm and can be used in conjunction with the commonly used fluorophores; e.g. FAM, TET, JOE and HEX. BHQ-2 is used to quench in the range 550 – 650 nm and is most effective in quenching fluorophores such as TAMRA, ROX, Cyanine-3, Cy3, Cy3.5™ and Red 640.
We also offer Black Hole Quenchers for labelling peptides. All of our BHQ dyes are available as carboxylic acid or succinimidyl esters. Our BHQ-1 and BHQ-2 dyes are available as FMOC lysine conjugates. Our water soluble BHQ-10 is available as a carboxylic acid or succinimidyl ester.
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in dry, alcohol-free dichloromethane for LK2156 & LK2157, and dry acetonitrile (LK4050) for LK2154 & LK2155. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
The dT amidites (LK2156 & LK2157) are dissolved in anhydrous (alcohol-free) dichloromethane, and the 5’-amidites (LK2154 & LK2155) are dissolved in anhydrous acetonitrile, all to standard concentrations. Prepare these amidite solutions at least 10min before placing on the synthesiser.
LK2154, LK2155 & LK2156– A 10min coupling time is recommended.
LK2157 – A 15min coupling time is recommended.
The CPGs are used per standard solid supports following the synthesiser instructions. Non-nucleosidic CPG supports do not detritylate as rapidly as nucleosidic ones, therefore an additional detritylation step is recommended. It is therefore necessary to use a cycle that does not contain an initial capping step.
Cleavage & Deprotection
When selecting a deprotection method for oligos containing the BHQ™ phosphoramidites, use conditions suitable for any other modifications on the oligonucleotide. The BHQ™ component itself is stable to typical standard, UltraFAST or UltraMILD conditions. When using a base-sensitive fluorophore such as TAMRA, UltraMILD deprotection (0.05M potassium carbonate in methanol for 4h at room temperature) or deprotection with tbutylamine/water (1:3) for 2.5h at 70°C is recommended.
The CPGs have a glycolate linkage. This allows for rapid cleavage of the oligonucleotides and is labile enough for base-sensitive oligos. For cleavage use ammonium hydroxide for 20min at 25°C or AMA for 5min at 25°C; or, when using TAMRA, tbutylamine/methanol/water (1:1:2) for 45min at 25°C.
Complete the deprotection with conditions suitable for the other nucleobases or modifications as above.
Note that if analysing oligos after purification by MS, an additional peak at M-298 (BHQ-1) or M-300 (BHQ-2) is observed. This is attributed to fragmentation within the MS through the diazo bond.
Storage & Stability
Products are stored in light-protected containers in the freezer at –10 to –30°C. All phosphoramidites are susceptible to oxidation when left exposed to air and/or moisture. All phosphoramidites are stable in solution under argon for 2 days.