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Oligo Modifications List | Oligo Modifications Reference Category
Modification : BBQ-650 NHS
Reference Catalog Number 26-6734
Category Quenchers
Modification Code BBQ-650 N
5 Prime Y
3 Prime Y
Internal Y
Molecular Weight (mw) 667.63
Technical Info (pdf) PS26-6734.pdf
Catalog NoScalePrice
26-6734-0550 nmol$210.00
26-6734-02200 nmol$210.00
26-6734-011 umol$385.00
26-6734-032 umol$576.00
Related Modifications
BHQ-1 (3')
BHQ-3 (5')
BHQ-1 (5')
BHQ-2 NHS
Dabcyl-3'
BHQ-1-dT
BHQ-3 (3')
BHQ-2 (3')
BHQ-1 NHS
BBQ-650-dT
BBQ-650 3'
BHQ-2-dT

NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

YIELD
NHS based modifications are post synthesis conjugation performed using a primary amino group. The yield is lower as compared to direct automated coupling of modifications that are available as amidites. Approximate yield for various scales are given below.

~2 nmol final yield for 50 nmol scale synthesis.
~5 nmol final yield for 200 nmol scale synthesis.
~16 nmol final yield for 1 umol scale synthesis

BlackBerry Quencher 650 (BBQ650) is classified as a dark quencher (a non-fluorescent chromophore). Dark quenchers are extensively used as the 3’-quencher moiety in a variety of Fluorescence Resonance Energy Transfer (FRET) DNA detection probes in which the fluorophore has a long wavelength (yellow to far red) emission maximum (e.g. Cy3, ROX, Cy5, Cy 5.5). Dark quenchers can serve in this role because they have long wavelength absorbance maxima. Dark quenchers are primarily used in nucleic acid assays, but also find a place in nucleic acid structural studies (1). Examples include TaqMan probes (2), Scorpion primers (3), and Molecular Beacons (4).

BBQ650 has an absorbance maximum of 650 nm, and an effective absorbance range of 550-750 nm (yellow to far red). It is chemically resistant to both oligonucleotide synthesis reagents (iodine, TCA) or deblocking solutions (ammonia, AMA). Consequently, for synthesis of longer oligos (> 50 bases), BBQ650 is the preferred quencher over BHQ-2 or BHQ-3, as the latter are chemically less stable, and degrade when exposed to oligo synthesis and deprotection conditions for long periods of time, such as when synthesizing or processing longer oligos.
A list of specific fluorescent dyes compatible with BBQ650 is found at this link.
Click here for complete list of quenchers
The emission spectra of this set of dyes sufficiently overlaps the absorbance spectrum of BBQ650 to allow the latter to quench the fluorescence of the former with a high degree of efficiency.

The advantages of using BBQ650 as a dark quencher in a FRET probe are (a) low background fluorescence (and thus better signal-to-noise ratio), (b) higher dynamic range, (c) amenability to multiplex assays (due to a dark quencher having no secondary fluorescence), and (d) ease of synthesis of FRET probes (due to BBQ650 being resistant to degradation during the oligo deprotection step).


Quencher Spectral Data

Quencher

Absorption Max, nm

Quenching Range, nm

Dabcyl 453 380-530
BHQ-0 495 430-520
BHQ1 534 480-580
BHQ2 579 550-650
BHQ3 672 620-730
BBQ-650 650 550-750
Click here for complete list of quenchers
**Black Hole Quencher License Agreement
"Black Hole Quencher", "BHQ-1", "BHQ-2" and "BHQ-3" are registered trademarks of Biosearch Technologies, Inc., Novato, CA. The BHQ technology is licensed and sold under agreement with Biosearch and these products are sold exclusively for R&D use by the purchaser. They may not be used for clinical or diagnostic purposes and they may not be resold, distributed or re-packaged.


References
1. Didenko, V.V. DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications. Biotechniques (2001), 31: 1106-1121.
2. Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W., Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. (1995), 4: 1-6.
3. Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. (2000), 28: 3752-3761.
4. Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. (1996), 14: 303-308.
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