(BHQ™) Dyes - Custom Oligos
(BHQ™) Dyes - Custom Oligos
(BHQ™) Dyes - Custom Oligos
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Significantly Improved Signal-to-Noise Ratios using<br />
Black Hole Quencher (BHQ) <strong>Dyes</strong><br />
Features:<br />
A NEW class of high-efficiency dark quenchers<br />
Optimized for FRET – by design<br />
<strong>Dyes</strong> that really go the distance<br />
DABCYL eclipsed by BHQ dyes<br />
TRUE dark quenchers – NO native fluorescence<br />
Access visible into near-IR spectrum for reporting – 480 to<br />
730 nm<br />
Enable wider choice of reporter dyes for multiplexing<br />
Wide variety of probe formulations<br />
A NEW Class of High-Efficiency Dark Quenchers<br />
<strong>Custom</strong>-synthesized hybridization probes incorporate spectrally paired<br />
fluorophores and quenchers, each covalently linked to minimize interference<br />
with probe-target hybridization. The BHQ dyes are a new class of highefficiency<br />
dark quenchers that prevent fluorescence until a hybridization<br />
event occurs. These powerful and highly specific dyes enable the identification<br />
and quantification of a variety of biomolecules.<br />
Figure 1. Hybridization event turns on fluorescence. BHQ dyes are compatible with linear<br />
probes and beacons on any real-time Q-PCR platform or end-point fluorescent plate reader.<br />
Optimized for FRET – by Design<br />
Hybridization probes are designed to take advantage of quenching by<br />
fluorescence resonance energy transfer (FRET) to detect and report binding<br />
to target molecules.<br />
Figure 2. The FRET Process<br />
FRET is a highly distance-dependent interaction between a reporter dye in<br />
an excited state and a quencher in its ground state. Energy is transferred<br />
from one molecule (the fluorophore) to the other (the quencher) without<br />
the emission of a photon.<br />
In order for efficient FRET quenching to take place: a) the fluorophore and<br />
quencher molecules must be close to each other (10-100 Å) and, b) the<br />
absorption spectrum of the quencher must overlap with the emission<br />
spectrum of the fluorophore.<br />
Normalized Intensity<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
BHQ-2 Absorption<br />
400 500 600 700 800<br />
Wavelength (nm)<br />
Figure 3. Spectral overlap of BHQ-2 dye with TAMRA and ROX.<br />
Each member of the family of BHQ dyes was conceived and<br />
designed to maximize spectral overlap, thus increasing the efficiency<br />
of quenching.<br />
<strong>Dyes</strong> that really go the Distance<br />
FRET quenching efficiency depends on 1/r 6 where r is the dye-quencher<br />
distance. In solution, unhybridized FRET probes are thought to exist as<br />
random coils allowing the reporter and quencher dyes to remain in close<br />
proximity favoring FRET quenching. Upon hybridization to a complementary<br />
target, the probe is stretched out of its random coil configuration. Thus, the<br />
reporter and quencher are separated and increased fluorescence results.<br />
The efficiency of FRET is given by:<br />
6 6 6 E = R0 /(R0 + r ) where R0 = Förster distance<br />
r = donor-quencher distance<br />
6 R0 ∝QD and J where QD = donor fluorescence efficiency<br />
J = overlap integral
DABCYL Eclipsed by BHQ <strong>Dyes</strong><br />
Quenching efficiency increases as spectral overlap J of the dye emission and<br />
quencher absorption profile increases. Historically, DABCYL has been routinely<br />
used as a general-purpose dark quencher for many commonly used fluorophores<br />
including FAM, TET, and JOE. As shown in Figure 4, however,<br />
DABCYL’s absorption maximum of 474 nm places it below the maxima of the<br />
shown fluorophores, limiting its efficiency to quench via FRET. The BHQ-1 dye<br />
(with an absorption maximum of 534 nm) is absorbed at higher wavelengths<br />
than DABCYL and directly superimposable with emission maxima of FAM, TET<br />
and JOE, providing a significant increase in FRET quenching efficiency.<br />
Abs (DABCYL-T9)<br />
RFU (JOE, TET, FAM)<br />
Abs (BHQ-1-T9)<br />
RFU (JOE, TET, FAM)<br />
250 300 350 400 450 500 550 600 650 700 750<br />
Wavelength (nm)<br />
250 300 350 400 450 500 550<br />
Wavelength (nm)<br />
Figure 4. Absorption spectra of DABCYL-T9 and BHQ-1-T9 with the emission spectra of FAM,<br />
TET and JOE. DABCYL and BHQ-1 dye were normalized at the poly-T absorbance<br />
(i.e. 260 nm) to better demonstrate the larger extinction coefficient of the BHQ-1 dye.<br />
RFU=relative fluorescence units.<br />
True Dark Quenchers – NO Native Fluorescence<br />
The two most commonly used quenchers, DABCYL and TAMRA, both limit<br />
the ultimate sensitivity and flexibility of FRET assays. DABCYL has an inadequate<br />
absorption footprint that overlaps very poorly with fluorophores<br />
emitting above 480 nm. TAMRA is not a dark quencher and contributes to<br />
an overall increase in background because of its own native fluorescence.<br />
As shown in Figure 5, each of the BHQ dye probes have much higher<br />
signal-to-noise ratios when compared to the corresponding DABCYL and<br />
TAMRA probes.<br />
Figure 5. Signal-to-noise (S:N) ratios were calculated by dividing the fluorescence signal of a<br />
25-mer in the presence of a five-fold excess of an exactly complementary target sequence by<br />
the fluorescence intensity of the probe alone. Each probe was formulated with a 5' reporter<br />
group (FAM, Cy3, Cy5) and a quencher (TAMRA, DABCYL, BHQ-1, BHQ-2 or BHQ-3).<br />
Access the Visible Spectrum and Near-IR for<br />
Reporting – 480 to 730 nm<br />
The BHQ family of quenchers were developed to provide excellent spectral<br />
overlap over the entire range of commonly used reporter dyes. As shown in<br />
Figure 6, the BHQ dyes cover the spectrum from 480 nm into the near IR<br />
making it possible to utilize reporter dyes that emit anywhere in this range.<br />
Abs<br />
250 300 350 400 450 500 550 600 650 700 750<br />
Wavelength (nm)<br />
Figure 6. Absorption spectra of the three BHQ dyes (conjugated to T-9 and normalized to the<br />
poly-T absorbance of 260 nm) with the emission maxima of many commonly used reporter<br />
groups indicated.<br />
Enable Wider Choice of Reporter <strong>Dyes</strong><br />
for Multiplexing<br />
As detection instrumentation has become more sophisticated, the ability to<br />
multiplex several fluorophores in a single reaction tube has gained much<br />
interest. One of the major drawbacks in any FRET-based multiplexing assay is<br />
cross-talk between the fluorophores, which hampers the ability to distinguish<br />
between dyes. Historically, this drawback has resulted from an absence of<br />
quenchers able to effectively quench throughout the visible and near-IR<br />
spectra. As shown in Figure 7, the BHQ family readily permits single-tube<br />
multiplexing due to the increased variety of reporter dyes that can be<br />
effectively quenched with little or no cross-talk between reporters. The<br />
broader spectral coverage of the BHQ dyes provides the scientist with a<br />
larger pool of distinct, spectrally resolved reporter dyes which simplifies the<br />
design, implementation and interpretation of multiplexed hybridization<br />
probe assays.
Normalized Emission<br />
Normalized Emission<br />
Figure 7. The unique characteristics of the BHQ class of quenchers permits flexibility in the<br />
choice of spectrally well-resolved fluorophores enabling single-tube multiplexing with little<br />
or no cross-talk.<br />
U.S. Pricing and Availability<br />
Wide Variety of Probe Formulations<br />
Sigma-Genosys can incorporate BHQ dyes into almost any probe formulation:<br />
from linear DNA probes to Molecular Beacons. BHQ dyes function as efficient<br />
dark quenchers over the entire visible spectrum and into the near-IR, reemitting<br />
their energy as heat rather than light. Probes made with BHQ dyes<br />
exhibit extremely low background fluorescence, enabling enhanced detection<br />
sensitivity. We would be pleased to work with you to design BHQ probes<br />
specific for your application!<br />
BHQ Dye Absorption Maxima and<br />
Quenching Range<br />
Quencher Abs max Quenching Range (nm)<br />
BHQ-1 534 480 – 580<br />
BHQ-2 579 550 – 650<br />
BHQ-3 672 620 – 730<br />
BHQ Dye Reporter Combinations<br />
Quencher Suggested Fluorophores<br />
BHQ-1 FAM, TET, JOE, HEX<br />
BHQ-2 TAMRA, ROX, Cy3, Cy3.5<br />
BHQ-3 Cy5, Cy5.5<br />
References<br />
Haugland, R. P., and Yguerabide, J., Stryer, L. Proc.Natl. Acad. Sci.USA, 1969, 63: 23-30.<br />
Parkhurst ,K.M., and Parkhurst, L. J., Biochemistry, 1995, 34: 293-300.<br />
Parkhurst, K. M., and Parkhurst, L. J., Biochemistry, 1995, 34: 285-292.<br />
Tyagi, S., Bratu, D. P. and Kramer, F. R., Nature Biotechnology, 1998, 16: 49-53.<br />
For Research use only. Not for use in Diagnostic Procedure<br />
”Black Hole Quencher”, ”BHQ“ is a trademark of Biosearch Technologies, Inc. BHQ technology is<br />
covered by patents applied for by Biosearch Technologies, Inc. All rights reserved. Information subject<br />
to change without notice. Cy is a trademark of Amersham Biosciences Ltd.<br />
Typical Yield<br />
Scale OD 260<br />
0.025 µmole 0.5–1.0<br />
0.05 µmole 1.0–3.0<br />
0.2 µmole 3.0–5.0<br />
1.0 µmole 5.0–10.0<br />
Yields will vary based upon oligo length and base composition.<br />
Please contact us if you have specific yield requirements.<br />
5’-end 3’-end<br />
Reporter Quencher 0.025 µmole 0.05 µmole 0.2 µmole 1.0 µmole<br />
6-FAM BHQ-1 $110 $225 $275 $475<br />
Fluorescein BHQ-1 $110 $225 $275 $475<br />
HEX BHQ-1 $110 $225 $275 $475<br />
TET BHQ-1 $110 $225 $275 $475<br />
Inquire for alternate dye and quencher combinations.<br />
Ordering Information<br />
450 500 550 600 650 700 750<br />
Wavelength (nm)<br />
Wavelength (nm)<br />
FAM<br />
TAM<br />
ROX<br />
Cy5<br />
450 500 550 600 650 700 750<br />
Black Hole Quencher Probes from Sigma-Genosys<br />
Sigma-Genosys is pleased to offer dual-labeled probes for use in applications<br />
such as real-time PCR, multiplexing, SNP analysis, allelic discrimination and<br />
spectral genotyping. We are a licensed supplier of Molecular Beacons and<br />
Black Hole Quenchers.<br />
Guaranteed Quality<br />
<strong>Oligos</strong> are routinely checked for length, purity and composition. Our global<br />
quality control capabilities include MALDI-TOF mass spectrometry, electrospray<br />
ionization mass spectrometry (ESI-MS), capillary electrophoresis (CE),<br />
and polyacrylamide gel electrophoresis (PAGE). In addition, the signal-tobackground<br />
ratio is measured against a target sequence for all Molecular<br />
Beacons. If you are dissatisfied and report an oligo problem within 30 days<br />
of order receipt, we will gladly remake your oligo free of charge or provide<br />
a credit for the purchase amount.<br />
Internet: www.sigma-genosys.com E-mail: genosysoligos@sial.com Fax: (800) 515-2155 or (281) 363-2212
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