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3'-BBQ-650 CPG II

3'-BBQ-650 CPG II

CPG used to incorporate the BBQ-650 quencher moiety at the 3' end of an oligonucleotide.
  • Dark quencher with broad absorbance centred around 650 nm
  • More rapidly cleaved from the solid support than its predecessor BL 2010.
  • A key advantage of the BBQ-650® is its stability to ammonia and oxidation conditions.
  • 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers)
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Product information

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.

Ref:

  1. Genotyping SNPs With Molecular Beacons, Marras, S. A. E.; Kramer, F. R.; Tyagi, S. Methods in Molecular Biology 2003, 212, 111-128.
  2. 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.

Applicable Products

LK1378 BBQ-650®N-hydroxysuccinimide ester
LK2427 3'-BBQ-650® CPG II
LK2550 BBQ-650®-(DMT)-CE-Phosphoramidite

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.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

LK1378 C36H39N7O9 713.74 559.67 - - -
LK2427 - - 638.57 - - -
LK2550 C59H67N8O10P 1079.18 638.57 2.32 4.63 9.27

Oligo Synthesis with LK2427 and LK2550

Dissolution

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.

Coupling

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

LK2550 -

  1. 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.
  2. Cleavage and deprotection are achieved in the same step by treating the support bound oligonucleotide with AMA, 10min, 65oC.

LK2427 -

  1. 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.
  2. Cleavage and deprotection are achieved in the same step by treating the support bound oligonucleotide with AMA, 10mins, 65oC.

Post-Synthetic Labelling with 1378

Solubility

DMF to 5-10mgmL-1 or 7-14µmolmL-1

Post synthetic labelling protocol

  1. Dissolve the amino functionalised biomolecule in 50mM carbonate buffer (pH 9.3).
  2. Dissolve the NHS ester in DMF.
  3. Add the solution from step 2 to the solution from step 1 such that the v:v of 1:2 is 2:1.
  4. Incubate overnight at 30oC.
  5. Quench the reaction with Tris-HCl buffer pH 7.2
  6. 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.

Spectral Data

ε (M-1cm-1) 40667 15077
λ 598nm 260nm
Solvent Methanol

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.

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