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.
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
Black Hole Quencher-2 (BHQ-2) is classified as a dark quencher (a non-fluorescent chromophore), and is extensively used as the 3’-quencher moiety in a variety of Fluorescence Resonance Energy Transfer (FRET) DNA detection probes. Such probes 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).
BHQ-2 has an absorbance maximum of 579 nm, and an effective absorbance range of 550-650 nm. It is the preferred quencher for pairing with fluorescent dyes that emit in the yellow-orange part of the visible range (557-617 nm). The emission spectra of this set of dyes sufficiently overlaps the absorbance spectrum of BHQ-2 to allow the latter to quench the fluorescence of the former with a high degree of efficiency.
The advantages of using 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 with a dark quencher (due to dark quenchers being resistant to degradation during the oligo deprotection step) (5).
Quencher Spectral Data
Absorption Max, nm
Quenching Range, nm
Click here for complete list of quenchers
**Black Hole Quencher License Agreement
Black Hole Quencher License Agreement.
""Black Hole Quencher®, BHQ®, CAL Fluor® and Quasar® are registered trademarks of
Biosearch Technologies, Inc., Petaluma, California.
The BHQ, CAL Fluor and Quasar dye technologies are protected by U.S. and world-wide patents either issued or in application. Compounds
incorporating these dyes are made and sold under agreement with Biosearch Technologies, Inc. for end-user's non-commercial research and development use only.
Their use in commercial applications is encouraged but requires a separate Commercial Use License granted by Biosearch Technologies, Inc."
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.
5. Yeung, A.T., Holloway, B.P., Adams, P.S., Shipley, G.L. Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR. Biotechniques. (2004), 36: 266-270, 272, 274-275.