Posted on Wed, Mar 24, 2010 @ 10:57 AM
A "Referral from the Doctor" Blog Article-
Scorpions®Primers are dual-labeled FRET probes that combine a Molecular Beacon®-like probe structure and a PCR primer element in a single oligonucleotide, allowing for specific target amplification and advanced target detection through a unimolecular or single oligonucleotide driven mechanism.
Structure
Q. What parts are similar to dual-labeled Black Hole Quencher® (BHQ) probes?
A BHQ dye and a 5'-fluorophore covalently bound to the oligonucleotide termini.
Q. What parts are similar to Molecular Beacons?
A target-binding region, comprised of a stem-loop structure resembling a "closed hairpin".
Q. What parts are unique?
• A 3' template-binding region representing the primer region;
• A PCR blocker, often a hexethylene glycol modification linker sequence and;
• An internal thymidine labeled with a BHQ.
Q. What is its native conformation at melting and annealing temperatures?
Melted - random coil conformation
Annealed (perfect match) - stable double helix, probe-target hybrid (high signal to noise ratio)
Annealed (mismatch) - hairpin conformation (FRET quenched, no signal)
Mechanism
Q. How does it yield signal?
The target-binding region is located within the loop portion of the hairpin conformation and sets atop an annealed stem region. When a perfectly matched complementary sequence is available, the probe region hybridizes to the complementary sequence. Conformational changes associated with hybridization force the hairpin stem region open, separating the fluorophore and quencher, decreasing FRET quenching and releasing fluorescence.
Q. How does it amplify its own target?
During the first round of amplification, the template-binding region hybridizes to the complementary sequence of the template DNA and extends to form a primer extension product. The primer extension product contains the desired target sequence. The probe region remains in a closed hairpin conformation until the next cycle of amplification. The presence of a PCR blocker in the linker sequence prevents the polymerase-mediated duplication of the probe region.
Q. How does it detect the target it has amplified?
During the second round of amplification, the target-binding region of the probe sequence hybridizes to the complementary sequence within the primer extension product. Hybridization forces the hairpin open, releasing fluorescence.
Q. What happens with each consecutive PCR cycle?
The primer extension product and connected probe region, establishes a temperature-dependent equilibrium between the hybridized conformation and the random coil conformation. Therefore, each Scorpions Primer accounts for only one product amplification per experiment.
The unimolecular mechanism allows for the linear amplification of targets with initially high copy number. For the quantitative analysis of low copy number sequences, it is essential that the reverse primer be included in the PCR master mix to enable exponential amplification of the target sequence.
Q. What if the available target is a mismatch?
Mismatched hybrids are less stable than reformation of the hairpin stem. Therefore signal is only produced when the target binding region hybridizes to a perfect-match target sequence. This enables specificity of signal generation to be accurate at the level of single nucleotide polymorphisms (SNP).
Advantages of Scorpions Primers
• Rapid hybridization - the proximity of the probe region and the target sequence kinetically favors formation of the probe-template hybrid over template duplex re-annealing. The unimolecular event enables rapid signal generation during hybridization.
• High signal to noise ratio - the efficiency of FRET quenching, when in the unhybridized state, decreases errant fluorescence and enables a high signal to noise ratio with hybridization.
• High specificity - single base mismatches can be detected.
• Post-PCR melt curve analyses - Scorpions Primers do not require the enzymatic activity of a polymerase for fluorescent signal generation in qPCR and under non-hydrolytic conditions allow for post-PCR melt curve analyses.
Applications: Single and multiplexed, quantitative and qualitative, real-time and endpoint qPCR analyses; environmental analyses; gene quantification and allelic discrimination.
Written by: Christina Ferrell, Ph.D., Technical Applications Specialist
Recommended reading
Whitcombe, D.M., Theaker, J., Gibson, N.J., Little, S. "Methods for detecting target nucleic acid sequences". United States Patent 6270967. Aug. 07, 2001
Whitcombe, D., Theaker, J., Guy, S.P., Brown, T., Little, S. "Detection of PCR products using self-probing amplicons and fluorescence". Nature Biotechnology 17 (1999): 804-807.
Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T. "Mode of Action and application of Scorpion primers to mutation detection". Nucleic Acids Research 28(19) (2000): 3752-3761.
Solinas, A., Brown, L.J., McKeen, C., Mellor, J.M., Nicol, J.T.G., Thelwell, N., Brown, T. "Duplex Scorpion primers in SNP analysis and FRET applications". Nucleic Acids Research 29(20) (2001): e96.
Bustin, S.A. A-Z of Quantitative PCR (IUL Biotechnology Series). La Jolla, California: International University Line, 2004.
Posted on Wed, Jan 27, 2010 @ 01:15 PM
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 Challenges of Detection
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 and Biosearch Technologies
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.