The BiosearchTech Blog

Dual-labeled BHQ probes - Performance to “dye” for

A "Referral from the Doctor" Blog Article-  

A BHQ® probe is a dual-labeled oligonucleotide covalently labeled with a fluorophore and a Black Hole Quencher® (BHQ) dye.

Q. What are the main components of a dual-labeled Black Hole Quencher® (BHQ) probe?

• An oligonucleotide, typically 30 bases long
• A 3' BHQ dye
• A 5'-fluorophore (reporter) dye

Q. What is its native conformation at melting and annealing temperatures?
Melted - random coil conformation
Unhybridized - unrestricted hairpin, FRET-quenched (no signal)
Hybridized- stable double helix, probe-target hybrid (signal)

Q. How does it yield signal?
When a complementary sequence is available, the probe hybridizes to the complementary sequence. Conformational changes associated with hybridization separate the fluorophore and quencher, decreasing FRET quenching and releasing fluorescence.
Q. What happens with each consecutive PCR cycle?
1. Heat melts or denatures the probe, sense and antisense strands of a DNA duplex.
2. As temperatures cool, hydrophobicity and electrostatics promote dye-dye attractions and enhance fluorescent quenching.
3. At annealing temperatures, the primers and the BHQ probe anneal to their complementary sequences within the target DNA. Conformational changes during hybridization separate the dyes which decreases FRET quenching thus releasing fluorescence.
4. During elongation, the DNA polymerase incorporates nucleotides complementary to the strand as it progresses in a 5' to 3' direction from the primer. When the polymerase encounters the 5'-end of the probe, it cleaves off the nucleotide, or a flap of nucleotides, with the bound reporter dye, thereby permanently separating the reporter and quencher dyes.

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Dual-labeled BHQ probes have replaced earlier reporter-quencher dye pairings, such as FAM-TAMRA or FAM-DABCYL. In such sub-optimal probes, the quencher has inherent limitations such as auto-fluorescence or insufficient quenching at certain wavelengths which limit the choice of quenchable fluorophores. In contrast, the BHQ dyes:

• are highly efficient dark quenchers;
• have broad absorption spectra;
• and yield high signal to noise ratios.

BHQ dyes can be paired with all common reporter dyes emitting between the ultraviolet and infrared wavelengths, thereby making multiplexed hybridization assays easy to design and interpret.
Applications: Single and multiplex, quantitative and qualitative, real-time and endpoint PCR analyses; allelic discrimination; and SNP detection.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist

Didenko, V.V. "DNA probes using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications". BioTechniques 31 (2001): 1106-1121. (Review)
Bustin, S.A. A-Z of Quantitative PCR. (IUL Biotechnology Series). La Jolla, California: International University Line, 2004.
Ranasinghe, R.T., Brown, T. "Fluorescence based strategies for genetic analysis". Chem. Commun. (2005): 5487-5502.
Johansson, M.K. "Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers." Methods in Molecular Biology (v. 335, ch. 2). Ed. V.V. Didenko. Totowa, NJ: Humana Press, 2004. 17-29.
Marras, S.A.E. "Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols" Methods in Molecular Biology (v. 335, ch. 7). Ed. V.V. Didenko. Totowa, NJ: Humana Press, 2004. 3-16.
Biosearch Technologies website. Black Hole Quencher Dyes, www.biosearchtech.com/support/applications/dyes-from-biosearch-technologies
Biosearch Technologies website. Genotyping-qPCR, www.biosearchtech.com/support/applications/genotyping-qpcr

The Grand Ballet: Wine and Biotechnology

At the third qPCR Symposium, held November 9th -12th in Millbrae, CA, ETS laboratories (ETS) scientist Rich DeScenzo gave a compelling presentation describing the inherent difficulties associated with wine production and quality control. Biosearch Technologies, Inc. is proud of its collaborative efforts with ETS to produce the world's first wine spoilage test kit. Biosearch manufactures the biotechnology used in these test kits, namely Scorpions® Primers, designed by ETS scientists for the identification and quantification of microbes at each stage of fermentation in the maturing wine product. Before we present the technology and methods used in wine spoilage testing, we want to present an overview of what role genetic testing has in the development of aromas and flavors in our favorite dinner libation. The Grand Ballet is an extraction taken from the presentation of Dr. DeScenzo, paraphrased and presented with a bit of artistic license, with permission from ETS. We hope you enjoy it.

The Grand Ballet

The biological conversion of sugars to alcohol and malic acid to lactic acid are backdrops to the grand ballet of yeast and bacteria - the white hat microbe and the dark sinister microbe rising and falling with the tides of aerobic and anaerobic conditions. Wine spoilage and the preemptive detection of microbes and yeast that cause spoilage is an art in and of itself. Spoilage comes in two flavors, if you'll excuse the pun: overt and smelly or covert and disappointing. The cast of characters in this drama are either yeast bi-products including:

ethyl acetate - the fruity taste;
sulfides - the complex and misunderstood;
aldehydes - the cider aroma;
4-ethyl phenols with their "wet dog" odor;

or bacterial bi-products which confer the following properties on the unsuspecting libation:

acetic acid, - the spoiler, aka vinegar taste;
biogenic amines - the aroma thieves;
vermin - lysine degradation leaving a "mousy" aftertaste.

Whether a microbe is a "spoiler" or not is dependent upon their timing during the fermentation and bottling process. As the backdrop of chemistries change from filling the barrel to opening the bottle, the wine detective must use available tools to detect the spoiler, identify it, and deter its continued growth. In the detective's kit are all the tools he needs to solve the case and bring this ballet to a refined and tasty wine delivery.

The process of wine production is described as "managing a biological process". The key is to manage without draconian interaction, or "minimal touch" as Dr. DeScenzo puts it. It is the detection of these spoiler characters, yeast or bacteria, in all stages of wine development that enables the wine maker to preemptively curtail their uncontrolled or undesirable growth. The ETS Scorpions® Wine Spoilage Detection Kit contains the primary components necessary to provide the winery with high quality diagnosis and quantification of yeast or bacteria. Scorpions Primers were uniquely designed to detect specific members of the yeast and bacterial families. Multiplex hybridization assays of up to 4 Scorpions Primers are done at various stages of the fermentation process. The Scorpions Primers are labeled with CAL Fluor®, Quasar® and BHQ® dyes, which provide:

• Specific and sensitive detection
• Large dynamic range of detection (10 through 10 million cells)
• Multiplexing capability
• High signal to noise ratio
• Rapid intramolecular signaling

Despite the capabilities of the Scorpions Primers, there are a few challenges which accompany multiplexing experiments, including: intermolecular interactions, the consideration of emission spectral overlap upon dye selection, and the need for easily interpretable data of biological relevance.

Other non-chemistry related issues that confound the analysis and detection of spoilers include variability within the sampling process. Collecting representative samples of the wine is a truly difficult hurdle for wineries to overcome. The difficulty is due to varying collectors, stratification of the wine over time, and the necessity to homogenize the wine before testing. In addition to the process of sample collection, there is the very nature of wine matrix diversity - the change from grape juice to wine, as well as the different types of varietals and wine styles to consider. Examples include: light, sterile-filtered white wines, which are easy to test; and heavy, unfiltered red wines, which are difficult to test. Each wine category has different levels of inhibitors that can interfere with cell lysis or the PCR reaction. The Scorpions diagnostic assay must be able to generate accurate results at each phase of production and with different wine varietals and styles.

Target number variability is another issue further complicating the use of qPCR in the industry. High target number increases the potential for cross contamination between wells on the PCR plate. When emission spectra overlap and interfere in individual channel analysis, cross-talk between channels of a PCR machine occurs and makes data interpretation more difficult. Finally, the diagnostic kit must be compatible across multiple platforms so the world can readily accept the kit upon market launch. It is therefore essential that the kit provides universally usable reagents that make analysis and data interpretation platform-independent and easy to conduct. Currently the Scorpions Wine Spoilage Kit is used in the US, Europe, and Australia as a preemptive screening tool.

ETS Laboratories is the first lab to adapt genetic testing to the wine industry. Over the past seven years, ETS has worked closely with Biosearch Technologies on the design and development of Scorpions primers for qPCR-based wine spoilage kits. The advancement of this technology will yield tremendous benefits to wineries by improving the speed and accuracy of their wine analysis. Genetic testing has the power to detect small populations of target organisms and in conjunction with other testing techniques can be implemented as an effective diagnostic tool in the wine production industry.

For more on ETS laboratories, visit their website at http://www.etslabs.com/. To learn more about Scorpions Primers and the qPCR methodology, visit our website at http://www.biosearchtech.com.

*Scorpions Primers are a product of DxS.

Micronics Acquires License to Biosearch Technologies’ Nucleic Acid Assay Chemistries

 

 

Micronics, Inc., a leading developer of point-of-care molecular diagnostic products, announced today that it has entered into a license agreement with Biosearch Technologies, Inc. for the right to make, have made, use and sell Micronics' products that incorporate Biosearch's proprietary fluorophores and quencher dyes for nucleic acid assays. Under the terms of the worldwide license, Biosearch has granted Micronics a royalty-free use of certain of its chemistries for inclusion in Micronics' infectious disease tests for sale in underdeveloped countries.

To read the entire story, read the full press release.

Labeling Oligos with Internal BHQ Dyes

When to use an internal Black Hole Quencher (BHQ) label in a fluorescence-quenched probe for real-time qPCR.

 

When the sequence length of your probe is 30 bases or shorter, we recommend selecting a conventional BHQ label for the 3' end of your probe. We have noticed, however, that probe lengths longer than 30 bases exhibit high background noise and are frequently nonfunctional. They are too long for efficient FRET quenching, a mechanism that is highly dependent upon the distance between the fluorophore and quencher. A shorter average distance between fluorophore and quencher is optimal for a FRET quenching environment, which is common in real-time qPCR experiments.

 

To resolve the problematic issues that could potentially arise when designing a longer probe, we recommend using an internal BHQ label, and putting a Spacer 3 (C3) modification at the 3' end to block polymerase extension. Without this Spacer 3 modification, the probe would instead behave as a primer and produce non-specific amplicons, which may be observed as a false positive. Placing your quencher internally can significantly reduce background noise because the quencher is positioned in closer proximity to the 5' fluorophore.

 

Biosearch Technologies offers T-BHQ-1, an internal BHQ dye attached to a thymine (T) base.  The internal BHQ label should be positioned no closer than 10 bases from the 5’ end to minimize interference.  Visit our dual-labeled probes product page to order probes internally labeled with a BHQ dye.  Simply select FAM from the 5’ Fluorophore drop down menu and an Internal Quencher drop down menu will appear with the option to select T-BHQ-1.

Biosearch Technologies is adding more COLOR to qPCR

Since its introduction in 2005, the ValuProbe BHQ Probe has become Biosearch Technologies' most popular product sold to the real-time qPCR industry.  Many molecular biologists choose the ValuProbe BHQ Probe because they are quality, superior performing fluorescence-quenched probes that work great for validating and optimizing real-time qPCR assays.

Always offered at the low price of $95, Biosearch is adding more colors to the ValuProbe product line for a limited time.  In addition to FAM, ValuProbe BHQ Probes will also be available with CAL Fluor Orange 560, CAL Fluor Red 610, and Quasar 670 dyes.  These new promotional fluorophore options are available as 5 nmol delivered and are HPLC purified.  ValuProbe BHQ Probes labeled with FAM, however, will continue being sold as 10 nmol delivered, also HPLC purified.  

With emission spectra that span the entire visible spectrum, Biosearch's CAL Fluor and Quasar dyes are compatible with the optics of many popular thermal cyclers and were developed specifically to complement the BHQ dyes.  Not only are these specifications highly advantageous for real-time qPCR assays, but they are especially functional when used in multiplexed qPCR assays. 

Take advantage of this promotion before it's too late!  Visit www.biosearchtech.com/valuprobe to learn how to order ValuProbe BHQ Probes online today!