Key featuresShow Hide
- Dark quencher with broad absorbance centred around 650 nm
- A key advantage of the BBQ-650® is its stability to ammonia and oxidation conditions
- Ester for post-synthetic 5' modification of oligonucleotides
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LGC, Biosearch Technologies offers our BlackBerry Quencher 650 (BBQ-650) as a synthesis-stable dark quencher of long wavelength fluorescence. An 8-alkoxyjulolidine moiety was found to be a powerful π-electron donor, affording a surprising bathochromicshift when compared to related compounds. The broad absorbance centred around 650 nm effectively overlaps the emission maxima of popular long-wavelength fluorophores such as Cy™ 3, TAMRA, Texas Red, ROX, Cy™ 5, and Cy5.5, allowing efficient quenching.
Molecular beacon probes bearing various 5'-fluorophores (FAM, Cy™ 3, Texas Red, Cy™ 5, Cy™ 5.5) have been synthesised using 3'-BBQ-650 CPG. Signal-to background ratios upon binding to fully complementary target were noted to be excellent, e.g., >90 with Cy5 and >88 with Cy5.5. The probes are known to be successful in typing C to T transitions at positions 627 and 630 of the human chemokine receptor 5 gene,(1) and produced excellent results in real-time PCR studies.
BlackBerry Quenchers are known to be excellent FRET-mode quenchers. Pairs of complementary strands were designed that would bring a 5'-Cy5.5 fluorophore to within 5 or 10 base pairs (20-40 Å) of a 3'-BBQ-650 upon hybridisation,(2) where quenching efficiencies of ≥98.3 and ≥98.9%, respectively, were observed. Melting temperatures of these hybrids were unchanged from non-labelled hybrids, showing that these quenching efficiencies were due to FRET quenching and not contact quenching.
BlackBerry Quenchers may be installed at the 3’ terminus, internally, or at the 5’ position, using the variety of products available.
- Genotyping SNPs With Molecular Beacons, Marras, S. A. E.; Kramer, F. R.; Tyagi, S. Methods in Molecular Biology 2003, 212, 111-128.
- Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. , Marras, S. A. E.; Kramer, F. R.; Tyagi, S. Nucleic Acids Res. 2002, 30, e122.
|LK2427||3'-BBQ-650® CPG II|
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions for LK2550. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
Oligo Synthesis with LK2427 and LK2550
LK2550 - This is sparingly soluble in acetonitrile therefore it is recommended that the amidite is solubilised in anhydrous DCM, AF (7 parts) and once completely dissolved (~10-15min) add anhydrous acetonitrile (3 parts). Do not premix the diluents. 0.1M concentration recommended.
LK2550 – A 15min coupling time is recommended. Fast deprotection amidites are recommended.
After synthesis; it is recommended to flush the lines with DCM followed by MeCN to remove the dye immediately after use. This will prevent any precipitation or cross contamination.
LK2427 is 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. Fast deprotection amidites are recommended.
Cleavage & Deprotection
- If the amidite is added to the 5’-end or the 3’-end on universal support. After synthesis, treat the support bound oligonucleotide with 20% DEA in acetonitrile (LK4028) to remove the cyanoethyl protection from the phosphate linkages. This will prevent any loss of the quencher during deprotection.
- Cleavage and deprotection are achieved in the same step by treating the support bound oligonucleotide with AMA, 10min, 65oC.
- After synthesis, treat the support bound oligonucleotide with 20% DEA in acetonitrile (LK4028) to remove the cyanoethyl protection from the phosphate linkages. This will prevent any loss of the quencher during deprotection.
- Cleavage and deprotection are achieved in the same step by treating the support bound oligonucleotide with AMA, 10mins, 65oC.
Post-Synthetic Labelling with 1378
DMF to 5-10mgmL-1 or 7-14µmolmL-1
Post synthetic labelling protocol
- Dissolve the amino functionalised biomolecule in 50mM carbonate buffer (pH 9.3).
- Dissolve the NHS ester in DMF.
- Add the solution from step 2 to the solution from step 1 such that the v:v of 1:2 is 2:1.
- Incubate overnight at 30oC.
- Quench the reaction with Tris-HCl buffer pH 7.2
- The conjugated product is now ready for purification.
Reference: A. Valanne, P. Malmi, H. Appelblom, P. Niemelä and T. Soukka, Anal. Biochem., 375, 71-81, 2008.
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.