The BiosearchTech Blog

Biosearch Technologies Expands Licensing of QIAGEN Scorpions® Patents

Biosearch Technologies today announces that it has entered into a new license relationship with QIAGEN (NASDAQ: QIA, Frankfurt Prime Standard) providing broad commercialisation rights to Scorpions Primers.  This agreement allows Biosearch the right to manufacture, catalog, and sell Scorpions primer assays into the research, applied, and infectious disease testing markets.

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

Taq Facts

A "Referral from the Doctor" Blog Article-

When the polymerase chain reaction (PCR) was first described, the Klenow fragment derived from the Escherichia coli DNA Polymerase I was the paramount enzyme for sequence extension. Due to its lack of stability at high temperature, it needs be replenished before each cycle. Upon the discovery of thermophilic bacteria which thrive at temperatures greater than 45 °C, heat-stable polymerases which function at higher temperatures were investigated in an effort to eliminate the need to replenish enzyme following each denaturation cycle.

Taq DNA Polymerase was originally isolated from thermophilic bacterium of the Deinococcus-Thermus group located near the Lower Geyser Basin of Yellowstone National Park by Thomas D. Brock and Hudson Freeze, in 1969. This thriving bacterium was named Thermus aquaticus (T. aquaticus). Several enzymes have been isolated from T. aquaticus, the most known of which is Taq DNA polymerase (Taq). Taq can be isolated either from its original source or from its cloned gene expressed in E. coli. While many similarities in sequence and structure exist between E. coli DNA polymerase I and Taq, differences in enzyme functionality, character and dependencies make Taq the ideal polymerase for use in qPCR-based gene expression analysis, with the exception of sensitive measures of certain bacterial genes. This caveat is explained later.

Characterization of Thermus aquaticus DNA Polymerase

Functionality: Utilizing the inherent 5’ to 3’ exonuclease activity of Taq, researchers are able to simultaneously achieve PCR amplification and signal release from a target-specific fluorogenic probe. The 5’ to 3’ exonuclease activity of Taq cleaves the 5’ terminus of a hybridized oligo probe to release both mono- and oligonucleotides. The probe is hydrolyzed concomitant with strand replication so that the accumulating fluorescent signal correlates with amplification.

Size and activity: While variable weights have been reported, the approximate size of Taq is 94 kd, with the activity of a DNA polymerase localized to the C-terminus and 5’ to 3’ exonuclease activity localized to the N-terminus. To date, no 3’ to 5’ exonuclease activity has been observed as in other polymerases, where the 3’-end mismatched base is excised during a “proofreading” process. The error rate has been reported to be as low as 10^-5 for base substitution errors and 10^-6 for frameshift errors.

Temperature dependency: Thermophiles are prevalent in nature and certain prokaryotic species thrive at temperatures above 45 ^°C. The temperature-dependency of Taq makes it optimum at 80 ^°C, where its catalytic activity is more than ten times that typically observed at 37 ^°C. It is possible that the decrease in activity above 80 ^°C is actually due to the denaturation of double-stranded DNA at these temperatures.

Monovalent and divalent cation dependencies: Salt concentrations required for optimum performance are considered to be 40 mM NaCl and 60 mM KCl. Concentrations greater than 100 mM for these monovalent cations are prohibitive to catalytic activity. This is in contrast to the salt-insensitivity of the E. coli Polymerase I enzyme. In addition, Taq may depend — like other polymerases — upon the presence of divalent cations, namely MgCl₂ or MnCl₂, with optimum concentrations depending on the experimental design. Higher concentrations of manganese can lead to an increased error rate of nucleoside incorporation. Activity with other divalent ions may be significantly decreased or absent, as is the case with greater than 0.25 mM Ca⁺². Taq requires the presence of all four species of deoxyribonucleoside triphosphates and DNA for optimum catalytic activity.

pH dependency: The pH optimum for the enzyme is within the range of 7-8 pH units when at 80 ^°C, and will vary depending on the buffer system used. In a 25 mM Tris-hydrochloride buffer for example, the alkalinity optimum is 7.8 pH units.

The great caveat: uninvited guests

On occasion, cloned Taq polymerase has been shown to have contaminating bacterial DNA that is possibly carried over from the expression vector system or other sources used during polymerase manufacture. This residual contamination may limit the use of cloned Taq in the detection of dilute bacterial DNA in certain samples. Trace contamination may be impossible to completely remove, and indeed certain estimates of contamination counts in commercially available Taq have claimed as many as 1000 genome equivalents of bacterial DNA per unit of enzyme.

Several methods for removing bacterial DNA from Taq polymerase have been tested and appear in the literature.  Methods such as exposure to ultraviolet light below 320 nm (UVB or UVC) has the effect of making DNA resistant to amplification; however it also affects the integrity of the Taq polymerase, reducing the efficiency of nucleoside incorporation. Ultra-filtration of the Taq polymerase, while often able to eliminate false-positives, does so with the unwanted effect of decreasing the assay sensitivity. UVA-activated 8-methoxypsoralen treatment to intercalate contaminating DNA into double-stranded DNA is difficult to optimize and inhibits the PCR reaction.  In addition to the above methods, restriction endonucleases and DNAse I treatment to digest DNA in Taq preparations may introduce contaminants or become the contaminants themselves.

A recent methodology under interrogation is the serial dilution of the Taq polymerase (up to 32-fold) to effectively dilute-out the contaminating bacterial DNA while maintaining Taq activity and sensitivity. This technique has the consequence of reaction plateauing at lower cycle numbers. This effect generates a lower signal in end-point analysis, with minimal consequence to quantification based on a threshold during the exponential phase.

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

Selected Citations:

Detection of specific polymerase chain reaction product utilizing the 5’ – 3’ exonuclease activity of Thermus aquaticus DNA polymerase. 1991. Holland, P. M., Abramson, R.D., Watsohn, R., Gelfand, D.H. Proc. Natl. Acad. Sci. 88: 7276-7280

Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. 1976. Chien, A., Edgar, D.B., Trela, J.M. Journal of Bacteriology. 127 (3): 1550-1557

Characterization of the 5’ to 3’ exonuclease associated with Thermus aquaticus DNA polymerase. 1990. Longley, M.J., Bennett, S.E., Mosbaugh, D.W. Nucleic Acid Research 18(24):7317-7322

Characterization of contaminating DNA in Taq polymerase which occurs during amplification with a primer set for Legionella 5S ribosomal RNA. 1994. Maiwald, M., Ditton, H.J., SonnTaq, H.G., von Knebel Doeberitz, M. Molecular and Cellular Probes 8(1):11-14

Optimizing Taq polymerase concentration for improved signal-to-noise in the broad range detection of low abundance bacteria. 2009. Spangler, R., Goddard, N.L., Thaler, D.S. PLoS ONE 4(9):e7010

High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. 1990. Eckert, K.A. and Kunkel, T.A. Nucleic Acids Research 18:3739-3744

Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time pcr. 2002. Klaschik, S., Lehmann, L.E., Raadts, A., Hoeft, A., Stuber, F. Molecular Biology 22(3):231-242

Optimization of real-time PCR assay for rapid and sensitive detection of eubacterial 16S ribosomal DNA in platelet concentrates. 2003. Mohammadi,T., Reesink, H.W., Vandenbroucke-Grauls, C.M.J.E., Savelkoul, P.H.M. Journal of Clinical Microbiology 41(10):4796–4798

Pipetting for qPCR

A "Referral from the Doctor" Blog Article-

Research trends in laboratories today increasingly steer towards gene expression analysis and genetic testing, often in the form of qPCR. As reproducibility is essential to genetic research it is imperative that scientists know the fundamentals of micro-volume pipetting.

Forward and Reverse Pipetting: This discussion is limited to the use of manual pipettors. Electronic pipettors are capable of other pipetting techniques such as dispensing, sequential dispensing and diluting which are not discussed here.

• Forward pipetting is used for aqueous solutions such as water, buffers, diluted saline, diluted acid or base. Appropriate aqueous solutions may also contain low concentrations of proteins or detergents. This technique is appropriate for milliliter and microliter volumes. In forward pipetting, aspiration involves compression of the key to the first stop followed by the slow release of the key, creating a vacuum within the barrel and aspirating the solution volume desired. Expulsion involves dispensing the solution by pressing the key down to and beyond the first stop to "blow out" the entirety of the aspirated volume.

• Reverse pipetting is used for viscous solutions, solutions with high vapor pressure or extremely small microliter volumes. In reverse pipetting, aspiration involves compression of the key to the second stop followed by the slow release of the key, creating a vacuum within the barrel and the aspiration of a volume greater than that selected. Expulsion involves pressing the key down to the first stop only, thus dispensing only the desired volume.

Pipetting micro-volumes:

• Pipette with smooth and deliberate action.
• Hold the pipette vertically at all times. This is best accomplished by using your index finger to dispense and aspirate instead of your thumb. 
• Immerse the pipette tip only slightly to avoid coating the outside of the tip with excess liquid that may be inadvertently transferred during dispensing.
• Pipette the initial volume directly to the bottom of the receiving container while lifting the pipette upward slowly so as not to introduce bubbles to the dispensed solution. Add additional volumes to the initial volume using the same technique.
  

Tips to improve accuracy:

• Pre-wet the tip. When pipetting greater than 10 microliters, it is good practice to do an initial aspiration and expulsion to decrease the amount of volume that will "stick" to the inside of the tip during dispensing. Pre-wetting will coat the inside of the tip and decrease the amount of volume lost by sample manipulation.
• Take sample temperature into consideration. When pipetting cold samples, the first aspiration is larger than all subsequent pipetting using the same tip. The opposite occurs when pipetting solutions warmer than ambient temperature wherein the first aspiration has a decreased volume than subsequent volumes using the same tip. To avoid these temperature related issues, always pre-wet the pipette tip before dispensing. 
• Consider the ambient temperature for your pipette. Pipettes are calibrated at room temperature. Using a pipette in a cold room will result in variable volumes being dispensed. 
• Use an appropriate pipette for the desired dispensed volume. Check the range of volumes allowed for your pipette and do not go over or below that recommended volume. For example, use a P2 pipette to dispense a 1 microliter volume as its range in linear between 0.2 through 2 microliters. Do not use a p20 which has a linear range between 2 through 20 microliters, for dispensing a 1 microliter volume. 
• Use the largest volume possible to make your dilutions. It is easier to maintain accuracy when pipetting a larger volume than a smaller volume. 
• Wait until the total volume has aspired before moving forward. It takes time to aspire and dispense volumes. One second should be allowed for the liquid to fill the tip.
• Account for the angle of inclination. The pipette should be held vertically at all times. During aspiration the hydrostatic pressure of the liquid decreases as the angle of inclination increases resulting in the over-aspiration or too much volume. The technique of pipetting at a 45 to 60 degree angle is appropriate for sterile technique pipetting associated most commonly with cell culture. Small deviations in the volumes associated with cell culture are not critical to the success of cell growth making the sterile technique preferable in that situation.
  

Posture:

• Maintains the natural curve of the spine. Prior to pipetting, stand or sit with your shoulders back, slightly, and parallel to the floor. Hold your head in the midline position and level so that your ears are directly over your shoulders and your eyes facing front. When standing, the pelvis should be shifted forward to align the hips directly over the ankles.
• Keep all objects close to the body and placed within an easy reach between usages. Minimize twisting and bending motions at the hip. 
• Lift objects that need to remain level or are awkward or heavy, with your back straight and using your legs. 
• Stretch your hands and arms frequently. Grip the pipette gently and use only the minimum force needed to activate the plunger. Alternate hands used to pipette, if possible. Purchase pipette tips that do not require high insertion force.
  

Pipette Maintenance:

• Recalibrate every 6-12 months, depending on usage. Service should include recalibration, replacement of seals and greasing of all moving parts.
• Inspect the pipette for damage or discoloration before each use. Any compromise to the pipette condition may have significant repercussions in pipetting reproducibility. Have the pipette serviced immediately to avoid wasted time and money on failed experiments due to pipetting errors. 
• Clean the pipette before each daily use. Wipe down the barrel, handle, aspirator key and plunger release key with a nucleotide-free 0.1 N HCl solution using laboratory grade tissue paper, such as Kimwipes, to destroy any RNA or DNA on the surface of the pipette. Follow that wash with a nucleotide-free 70% Ethanol solution to precipitate and remove the lingering nucleotide fragments. This same procedure is excellent for cleaning your work station and associated equipment.
• Store pipettes in an upright position using a pipette stand or hanging pipette holder. Pipettes have a hollow barrel and so may easily become contaminated with liquid remaining in the tip if placed on their sides. By keeping pipettes upright and using filtered pipette tips, the pipette barrel is less likely to become contaminated.
• Use tips designed for the pipette. Each manufacturer has a recommended tip to be used with the pipette. Only use tips that fit the specifications listed in the brochure or user manual for your specific pipette brand and type.

By following the guidelines suggested above individual scientists may improve their pipetting accuracy and consequentially the reproducibility of data. For more information on pipetting technique go to www.rainin.com or www.eppendorf.com.

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

Easier Online Ordering through RealTimeDesign™ Software

RealTimeDesign (RTD^TM) Software from Biosearch Technologies is an easy to use, yet powerful assay design application for real-time qPCR. This user-friendly software is available free of charge through our website and requires NO installation. We have recently updated RealTimeDesign software with more options and improvements to the ordering process:

• Option to rename sequences
• Chance to review sequences before purchase
• Purification selection for primers
• Larger selection of synthesis scales
• Improved transition from design to order

 If you're not already familiar with RealTimeDesign software, this powerful design application puts you a few clicks away from:

• Designing assays for Multiplex qPCR, SNP Genotyping, and Gene Expression
• Designing BHQ Probes, BHQplus^TM Probes, Amplifluor® Primers, and matching optimal primer pairs
• Designing anywhere from single assays to high-throughput batches
• Saving a list of your custom designs in your account "Design Run History"
• Choosing from Biosearch's wide selection of dyes including our very own Black Hole Quencher® (BHQ®) dye

Designing Dual-Labeled BHQ® Probes has never been easier. To start using RealTimeDesign Software, visit www.biosearchtech.com/realtimedesign and create an account today!

See Also:

• RealTimeDesign Quickstart Guide
• RealTimeDesign Brochure
  

Buy One qPCR Probe & Get One FREE

Buy one CAL Fluor® or Quasar® -BHQ® Probe and get a FREE ValuProbe^TM BHQ Probe* and calibration standard ($190 in savings!). Simply enter promo code CFQVP upon checkout to receive this offer. Take advantage of this promotion before it expires on June 30, 2010.

Biosearch Technologies offers CAL Fluor and Quasar dyes that span the spectrum, for 2-plex, 3-plex, 4-plex, 5-plex, and even 6-plex PCR. They perfectly complement the Black Hole Quencher dyes, a true dark quencher that extinguishes signal from any fluorophore.

This promotion is for one-time use only and limit one free probe per customer. Multiple promotions and discounts may not be combined. 25 nmol synthesis scale probes excluded.

Visit our Dual-Labeled BHQ Probes webpage for more details about our products and this current promotion.

New Product Line! ValuPanel™ Reagents

We are pleased to introduce ValuPanel Reagents, a new product line of mission-critical probe and primer sets that discriminate different strains of pathogens. This product line culminated from our successful collaboration with Centers for Disease Control and Prevention* (CDC) in response to the 2009 H1N1 pandemic. As of now, ValuPanel Reagents are available for both 2009 H1N1 and seasonal Influenza A subtyping.

*CDC does not endorse products or services.

The Details:
Probes are labeled with FAM and BHQ® dyes and all probes and primers are HPLC purified. ValuPanel Reagents are available with same day shipping via FedEx Priority for all orders placed before 2 PM (PST). ValuPanel Reagents are for Research Use Only (RUO) and currently resolve genetic signatures specific to the following viruses and subtypes:

2009 H1N1

• InfA for the universal detection of influenza A
• swInfA for the specific detection of swine influenza A
• swH1 for the specific detection of swine influenza H1
• RNase P for a positive control

Influenza A Subtyping

• InfA - for the universal detection of influenza A
• H1 subtype of influenza A
• H3 subtype of influenza A
• H5a subtype of influenza A
• H5b subtype of influenza A
• InfB - for the universal detection of influenza B
• RNase P for a positive control

Probe pricing for ValuPanel Reagents start from $135.00 and provide 5 nmol delivered. Forward and reverse primers are each available for $14.00 and provide 20 nmol delivered.

Learn more about Biosearch's NEW ValuPanel Reagents:

• 2009 H1N1 ValuPanel Reagents
• Influenza A Subtyping ValuPanel Reagents
  

Black Hole Scorpion® Primers - Killer probes for qPCR

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.

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)

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).

• 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

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.

 

Molecular Beacons - Lights in the storm

A "Referral from the Doctor" Blog Article-

Molecular Beacons are a special type of dual-labeled oligonucleotide probe. Beacons are hairpin loop structures with a 5'-fluorophore and a 3'-quencher dye. The stem region is a short sequence of 5-7 complementary bases. The loop sequence is complementary to the target sequence. In the absence of target or prior to amplification, the stem anneals to form a closed hairpin conformation which holds the reporter and quencher close together to enable efficient FRET quenching and to promote contact or static quenching. The beacon is engineered such that the probe-target hybrid is more stable than the closed hairpin conformation, which is respectively more stable than a probe-mismatch hybrid. Therefore it is only in the event of a perfect-match hybridization that signal occurs, allowing beacons to discriminate mismatches as small as a single nucleotide polymorphism (SNP).

Structure

Q: What parts are similar to dual-labeled Black Hole Quencher® (BHQ) probes?
A: A 5'-fluorophore and a 3'-BHQ dye covalently bound to the oligonucleotide termini.

Q: What parts are unique?
A: A target-binding region comprised of a stem-loop structure resembling a "closed hairpin".

Q: What is its native conformation at melting and annealing temperatures?
A: 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?
A: 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: What happens with each consecutive PCR cycle?
A: 1. At high temperatures target DNA duplexes and Molecular Beacons are melted and maintain random coil conformations.
2. As the reaction cools to temperatures appropriate for the binding of primers and targets, the beacon will reach an equilibrium between the hairpin and hybridized states. This equilibrium depends on the availability of a perfect-match target, whose binding by the probe is thermodynamically favored, by design, over the hairpin conformation. This ensures that the molecular beacon can hybridize when appropriate target is available. In the hybridized conformation the reporter and quencher dyes are separated, releasing fluorescence.
3. During polymerization, the beacon is dislodged from the target sequence, refolds into and maintains the hairpin conformation until the next cycle of amplification. The increase in fluorescence intensity with repeated PCR cycles indicates the accumulation of product and allows for accurate quantification of template.

Q: What if the available target is a mismatch?
A: 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).

• 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 - Molecular Beacons 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; allelic discrimination; SNP analysis; DNA microarray-immobilized probes and biosensors and; as nuclease-resistant antisense probes for detection RNA in vivo.

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

Tyagi, S. and Kramer, F.R. "Molecular beacons: probes that fluoresce upon hybridization". Nature Biotechnology 14 (1996): 303-308.
Bonnet, G., Tyagi, S., Libchaber, A., Kramer, F.R. "Thermodynamic basis of the enhanced specificity of structured DNA probes". Proc Natl Acad Sci. 96 (1999): 6171-6176.
Marras, S.A.E., Kramer, F.R., Tyagi, S. "Multiplex detection of single-nucleotide variations using molecular beacons". Genet Anal, 14 (1999): 151-156.
Marras, S.A.E., Kramer F.R., and Tyagi, S. "Genotyping single nucleotide polymorphisms with molecular beacons". Single nucleotide polymorphisms: methods and protocols (v. 212). Ed. P.Y. Kwok. Totowa, NJ: The Humana Press, 2003. 111-128.
Vet, J.A.M. and Marras, S.A.E. "Design and optimization of molecular beacon real-time polymerase chain reaction assays". Oligonucleotide synthesis: Methods and Applications (v. 288). Ed. P. Herdewijn. Totowa, NJ: Humana Press, 2004. 273-290.
Bustin, S.A. A-Z of Quantitative PCR. (IUL Biotechnology Series). La Jolla, California: International University Line, 2004.
Broude, Natalia E. "Molecular Beacons and Other Hairpin Probes". Encyclopedia of Diagnostic Genomics and Proteomics. (2005): DOI: 10.1081/E-EDGP 120020717. 846-850
Public Health Research Institute. "Designing Molecular Beacons". www.molecular-beacons.org

 

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.

.

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

This Year's MVP (Most Valuable Practice): Touchdown PCR

A "Referral from the Doctor" Blog Article-  

The aim of the Polymerase Chain Reaction (PCR) method is to amplify or increase the number of copies of a target sequence. Most often that sequence is part of a gene of interest and the total copy number or relative copy number of that transcript will impart some knowledge to the researcher regarding the impact of a drug, disease status of a sample or may represent a control condition. In some situations, the copy numbers of a particular transcript are very low and it may be necessary to nudge the PCR reaction in favor of amplifying the exact splice variant, family member or a construct the researcher wishes to examine.

 

 

In PCR, the temperature at which primers anneal during a cycle determines the specificity of annealing. The melting point (T_m) of the coolest primer sets the upper limit on annealing temperature. At temperatures just below the T_m, only very specific base pairing between the primer and the template will occur. As the temperature decreases, primer binding becomes less specific. Non-specific primer binding results in the amplification of undesired products and may mask the actual copy number of the gene of interest.

Touchdown PCR (Step-down PCR) is a variant of qPCR that reduces nonspecific target amplifications by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5 °C) above the T_m of the primers used. This ensures high specificity binding of the primers and is least-permissive of non-specific binding. As the PCR continues, each cycle occurs at lower and lower temperatures, decreasing by 0.2 °C per cycle. At the later cycles, it is a few degrees (3-5 °C) below the primer T_m. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles. Thus, the first sequence amplified is the one between the regions of greatest primer specificity and will be the most abundant product after cycling is complete.

This method is effective for amplification of one or only a few gene transcripts at a time. For larger comparative studies, standard PCR thermocycling conditions are recommended.

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

Hecker K, Roux K (1996). "High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR". Biotechniques 20 (3): 478-85. PMID 8679209