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Dyes & Fluorescence detection chemistry in qPCR


New! Nucleic Acid Detection  (PDF 2.2MB)  is a concise guide to all of our ultrasensitive
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Nucleic Acid Detection

Short summary of detection formats in real-time PCR
(including references)
http://medgen.ugent.be/rtprimerdb/techniques.php#intercalating

Public real-time PCR primer and probe database

which tends to be a repository for primer and probe sequences for different organisms 
and detection chemistries http://www.realtimeprimerdatabase.ht.st 


SYBR® Green based Quantitative PCR
 
SYBR Green I, a commonly used fluorescent DNA binding dye, binds all double–stranded DNA and detection is monitored by measuring the increase in fluorescence throughout the cycle. SYBR Green I has an excitation and emission maxima of 494 nm and 521 nm, respectively. Specificity of Sigma's SYBR based QPCR detection is greatly enhanced by the incorporation of a hot–start mediated taq polymerase, JumpStart Taq.

Convenient – Delivers the benefits of antibody–inactivated hot–start PCR with SYBR Green detection in a ReadyMix ideal for high throughput applications; only primers and template are required.
Specific – JumpStart Taq antibody prevents non–specific product formation through its hot–start mechanism.
Flexible – SYBR Green JumpStart Taq ReadyMixes for SYBR based QPCR are formulated with MgCl2 or packaged with a separate vial for ease of optimization. Additionally, our ReadyMixes are compatible with tube- and plate- based instruments.

Probe based Quantitative PCR
 
Probe based QPCR relies on the sequence–specific detection of a desired PCR product. Unlike SYBR based QPCR methods that detect all double–stranded DNA, probe based QPCR utilizes a fluorescent–labeled target-specific probe resulting in increased specificity and sensitivity. Additionally, a variety of fluorescent dyes are available so that multiple primers can be used to simultaneously amplify many sequences. 

Ideal for high throughput. ReadyMixes contain all necessary components for QPCR you simply add the fluorescent detection chemistry, primers, and template.
Specific – JumpStart Taq antibody prevents non–specific product formation through it hot–start mechanism.
Maximum flexibility in detection method, since no detection method has been incorporated into formulation.
Optimized formulations with the addition of dUTP to facilitate carry–over prevention from previous PCR.


http://www.sigmaaldrich.com


Real-time Reporters:

SYBR® Green, TaqMan®, and Molecular Beacons

by  Ambion TechNotes 8(1) - February 2001

http://www.ambion.com/techlib/tn/81/813.html

All real-time PCR systems rely upon the detection and quantitation of a fluorescent reporter, the signal of which  increases in direct proportion to the amount of PCR product in a reaction. In the simplest and most economical  format, that reporter is the double-strand DNA-specific dye SYBR® Green (Molecular Probes). SYBR Green binds double-stranded DNA, and upon excitation emits light. Thus, as a PCR product accumulates, fluorescence  increases. 

The advantages of SYBR Green are that it's inexpensive, easy to use, and sensitive. The disadvantage is that  SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which results in an overestimation of the target concentration. For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles. 

The two most popular alternatives to SYBR Green are TaqMan® and molecular beacons, both of which are  hybridization probes relying on fluorescence resonance energy transfer (FRET) for quantitation. 

TaqMan Probes are oligonucleotides that contain a fluorescent dye, typically on the 5' base, and a quenching  dye, typically located on the 3' base. When irradiated, the excited fluorescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing, resulting in a nonfluorescent substrate. TaqMan probes are  designed to hybridize to an internal region of a PCR product. During PCR, when the polymerase replicates a  template on which a TaqMan probe is bound, the 5' exonuclease activity of the polymerase cleaves the probe.  This separates the fluorescent and quenching dyes and FRET no longer occurs. Fluorescence increases in each  cycle, proportional to the rate of probe cleavage. 

Molecular beacons also contain fluorescent and quenching dyes, but FRET only occurs when the quenching dye is directly adjacent to the fluorescent dye. Molecular beacons are designed to adopt a hairpin structure while free in solution, bringing the fluorescent dye and quencher in close proximity. When a molecular beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Unlike TaqMan probes, molecular beacons are designed to remain intact during the  amplification reaction, and must rebind to target in every cycle for signal measurement. 


Quantification on the LightCycler Instrument

Randy Rasmussen Ph.D.  (Idaho Technology, Chief Operating Officer)
http://www.idahotec.com/lightcycler_u/lectures/quantification-on-lc.htm
 
SYBR Green I

SYBR Green I is dsDNA-binding dye.  It is thought to bind in the minor groove of dsDNA and upon binding increases in fluorescence over a hundred fold (Figure 8a).  It is compatible with PCR up to a point, at very high concentrations it starts to inhibit the PCR reaction.  In the LightCycler Instrument, SYBR is monitored in channel F1. The biggest advantage of SYBR is that it binds to any dsDNA; there is no designing and optimizing of probes required.  If you have a PCR that works, you can have a real-time quantitative assay working in about a day. The biggest disadvantage of SYBR is that it binds to any dsDNA; the specific product, non-specific products and primer dimers are detected equally well.  There are a number of ways to handle this problem.  Careful optimization of the PCR reaction can usually reduce primer dimers to a level that is only important for very low copy detection.  Hot start techniques like TaqStart antibody can be helpful in reducing primer dimer.  The LightCycler Instrument allows melting curve analysis of the reaction.  This can help to determine the fraction of the signal coming from the desired product and the fraction coming from primer dimer.  Once the melting point of the product has been determined the LightCycler Instrument's flexible programming allows the user to acquire fluorescence above the melting temperature of the primer dimers, but below the melting temperature of the product. 

Hybridization Probes

If sequence specific recognition is required, the HybProbe system allows detection of only the specific product.  Two probes are designed that hybridize side by side on the PCR product (Figure 8c).  The 3’ end of the upstream probe is labeled with fluorescein, which acts as a fluorescence resonance energy transfer (FRET) donor.  The 5’ end of the downstream probe is labeled with an acceptor dye, either LC Red 640, or LC Red 705.  The FRET signal is seen only when two specific hybridization events occur.  In the LightCycler Instrument, LC Red 640 is monitored in channel F2, LC Red 705 in channel F3.  There may sometimes be an advantage to monitoring the ration of the acceptor channel (where the signal goes up with increasing PCR product) and the signal from fluorescein in F1 (which goes down with increasing PCR product.
  TaqMan® Probes
TaqMan probes derive their fluorescence signal from the hydrolysis of the probe by Taq’s 5’ to 3’ exonuclease activity (Figure 8c).  The hydrolysis separates fluorescein from a quenching dye and results in an increased fluorescein signal. These probes can be used in the LightCycler Instrument and are monitored in F1 or F1/F2.

 


DNA Detection with SYBR Green I Dye

  Product Summary
Nucleic Acid Detection (PDF 2.2MB)    Nucleic Acid Detection
The fluorescent dye SYBR Green I binds to the minor groove of the DNA double helix. In solution, the unbound dye exhibits very little fluorescence, however, fluorescence is greatly enhanced upon DNA-binding. Since SYBR Green I dye is very stable (only 6% of the activity is lost during 30 amplification cycles) and the LightCycler instrument's optical filter set matches the wavelengths of excitation and emission, it is the reagent of choice when measuring total DNA. The principle is outlined in the following figures.

At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal which is subtracted during computer analysis.

After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules to emit light upon excitation.

During elongation, more and more dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in real-time. Upon denaturation of the DNA for the next heating cycle, the dye molecules are released and the fluorescence signal falls.

Fluorescence measurement at the end of the elongation step of every PCR cycle is performed to monitor the increasing amount of amplified DNA. Together with a melting curve analysis performed subsequently to the PCR, the SYBR Green I format provides an excellent tool for specific product identification and quantification.


Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR

Steven Giglio*, Paul T. Monis and Christopher P. Saint
Australian Water Quality Centre, South Australian Water Corporation, Salisbury, South Australia 5108, Australia

Nucleic Acids Research, 2003, Vol. 31, No. 22 e136



SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-speciÆc and additional testing, such as DNA melting curve analysis, is required to conÆrm the generation of a speciÆc amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (Tm) of the speciÆc amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the Tm values are suffiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by Tm analysis but multiple products as assessed by agarose gel electrophoresis. The detected Tm corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.



A new minor groove binding asymmetric cyanine reporter dye for real-time PCR

Martin Bengtsson, H. Jonas Karlsson, Gunnar Westman and Mikael Kubista*
Department of Chemistry and Bioscience, Chalmers University of Technology 41296 Go»teborg and TATAA
Biocenter, Medicinaregatan 9E, 413 90 Goteborg, Sweden
Nucleic Acids Research, 2003, Vol. 31, No. 8 e45



The minor groove binding asymmetric cyanine dye 4-[(3-methyl-6- (benzothiazol-2-yl)- 2,3-dihydro- (benzo-1,3-thiazole) -2-methylidene)]- 1-methyl-pyridinium iodide (BEBO) is tested as sequence nonspeciÆc label in real-time PCR. The Fluorescence intensity of BEBO increases upon binding to double-stranded DNA allowing emission to be measured at the end of the elongation phase in the PCR cycle. BEBO concentrations between 0.1 and 0.4 mM generated sufÆcient Øuorescence signal
without inhibiting the PCR. A comparison with the commonly used reporter dye SYBR Green I shows that the two dyes behave similarly in all important aspects.

BEBO for qPCR and HRM

TATAA Biocenter AB,  Göteborg,  Sweden

BEBO is an unsymmetric cyanine dye developed by TATAA Biocenter for use in qPCR applications.

The dye has absorbance and emission wavelengths that can be detected on the FAM channel on most common real-time PCR platforms, and shows a strong fluorescence increase when bound to dsDNA. BEBO can be used as an unspecific dye for real-time PCR applications or other applications where staining of dsDNA is wanted.

Combining sequence-specific probes and DNA binding dyes in real-time PCR for
specific nucleic acid quantification and melting curve analysis.

Lind K, Stahlberg A, Zoric N, Kubista M.
Biotechniques. 2006 Mar;40(3):315-9.
Chalmers University of Technology, Gothenburg, Sweden.


Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has the advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplification, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the same reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dimer formation and the presence of any other anomalous products.



BOXTO as a real-time thermal cycling reporter dye

ASHRAF I AHMAD
Journal of Biosciences, Volume 32, Number 2 / March, 2007



The unsymmetrical cyanine dyes BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro- (benzo-1,3-thiazole)-2-methylidene)]- 1-methyl-quinolinium chloride) and its positive divalent derivative BOXTO-PRO (4-[3-methyl-6-(benzoxazole-2-yl)- 2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]- 1-(3-trimethylammonium-propyl)- quinolinium dibromide) were studied as real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I (SG) (2-[N-(3-dimethylaminopropyl)-N-propylamino]- 4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]- 1-phenylquinolinium). Unmodified BOXTO showed no inhibitory effects on real-time PCR, while BOXTO-PRO showed complete inhibition, Sufficient fluorescent signal was acquired when 0.5–1.0 µM BOXTO was used with RotorGene and iCycler platforms. Statistical analysis showed that there is no significant difference between the efficiency and dynamic range of BOXTO and SG. BOXTO stock solution (1.5 mM) was stable at −20°C for more than one year and 40 µM BOXTO solution was more stable than 5x SG when both were stored at 4°C for 45 days.

Applied Biosystems Model 7700 sequence detection system
(the ABI TaqMan Probes)

Real-time systems for PCR were improved by probe-based, rather than intercalator-based, PCR product detection. The principal drawback to intercalator-based detection of PCR product accumulation is that both specific and nonspecific products generate signal. An alternative method, the 5' nuclease assay, provides a real-time method for detecting only specific amplification products. During amplification, annealing of the probe to its target sequence generates a substrate that is cleaved by the 5' nuclease activity of Taq DNA polymerase when the enzyme extends from an upstream primer into the region of the probe. This dependence on polymerization ensures that cleavage of the probe occurs only if the target sequence is being amplified.

The development of fluorogenic probes made it possible to eliminate post-PCR processing for the analysis of probe degradation. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by Förster resonance energy transfer (FRET) through space. Probe design and synthesis has been simplified by the finding that adequate quenching is observed for probes with the reporter at the 5' end and the quencher at the 3' end.

Figure 1 diagrams what happens to a fluorogenic probe during the extension phase of PCR. If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5' nuclease activity of Taq DNA polymerase as this primer is extended. This cleavage of the probe separates the reporter dye from quencher dye, increasing the reporter dye signal. Cleavage removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process. Additional reporter dye molecules are cleaved from their respective probes with each cycle, effecting an increase in fluorescence intensity proportional to the amount of amplicon produced.

The advantage of fluorogenic probes over DNA binding dyes is that specific hybridization between probe and target is required to generate fluorescent signal. Thus, with fluorogenic probes, non-specific amplification due to mis-priming or primer-dimer artifact does not generate signal. Another advantage of fluorogenic probes is that they can be labeled with different, distinguishable reporter dyes. By using probes labeled with different reporters, amplification of two distinct sequences can be detected in a single PCR reaction. The disadvantage of fluorogenic probes is that different probes must be synthesized to detect different sequences.


LightCycler Hybridisation Probes

The detection principle of LC™ Hybridization Probes (HybProbes) is Fluorescence Resonance Energy Transfer (FRET), the phenomenon of energy transfer from a donor to an acceptor fluorophor. If the donor and the acceptor fluorophor are in close proximity to each other, excitation of the donor by blue light results in energy transfer to the acceptor, which can then emit light of longer wavelength. This fact forms the basis for Roche’s real-time online LightCycler™ PCR System. It allows formation of PCR products to be monitored by using two sequence specific, fluorescent labeled oligonucleotide probes, called Hybridization Probes, in addition to the PCR primers. 

For this LC™ real-time PCR detection format the following are the major steps:

 
a) Denaturation
PCR template, primers and HybProbes are single-stranded. One HybProbe is labeled with the fluorescent donor dye Fluorescein, the other one is labeled with one of the two available acceptor dyes (LCRed 640 or LCRed 705). The donor dye is excited by blue light of 470 nm and emits green light of 530 nm.
b) Annealing
After reaching the annealing temperature, PCR primers and HybProbes hybridise to their specific target regions. The donor dye now comes into close proximity to the acceptor dye. Energy emitted from the donor dye excites the acceptor dye, which now emits red light of 640 or 705 nm.
c) Elongation
After annealing to their target sites, the primers are elongated by thermostable DNA polymerase. Due to the increased temperature of 72°C during elongation, most HybProbes have already melted off. Probes that are still annealed to their target sequence are displaced by the protruding DNA polymerase.
d) Completion
The amount of template DNA has doubled and the DNA is double-stranded HybProbes are displaced from their target site. The next cycle of PCR is ready to start again at step a).

HybProbes are designed as a pair of which one probe is labeled with the donor (3´Fluo) and one with the acceptor (5´ LCRed 640 or LCRed 705) dye. As FRET decreases with the sixth power of distance, HybProbes have to be designed to hybridise to adjacent regions of the template DNA (separated by 1-5 nucleotides). If both probes hybridise, the two dyes are brought close together and FRET to the acceptor dye results in a signal measurable by the built-in fluorimeter of the LightCycler™. 

The fluorescence signal disappears by increasing temperature above the melting temperature of the oligos because the probes melt away from the template strand which significantly increases the distance between the dyes. 

Mismatches between the probes and the target decrease the melting temperature of the respective probe compared to a perfectly matched probe. This effect can also be used to detect SNPs by melting curve analysis.

 

Principles & Applications: Parameter-specific Applications for the LightCycler® System

PCR Monitoring with Hybridization Probes

The Hybridization Probe format is used for DNA detection and quantification and provides a maximal specificity for product identification. In addition to the reaction components used for conventional PCR, two specially designed, sequence specific oligonucleotides labeled with fluorescent dyes are applied for this detection method. This allows highly specific detection of the amplification product as described below. 


The top figure shows the three essential components for using fluorescence-labeled oligonucleotides as Hybridization Probes: two different oligonucleotides (labeled) and the amplification product. Oligo 1 carries a fluorescein label at its 3' end whereas oligo 2 carries another label (LC Red 640) at its 5' end.

The sequences of the two oligonucleotides are selected such that they hybridize to the amplified DNA fragment in a head to tail arrangement. Why is this design important? When the oligonucleotides hybridize in this orientation, the two fluorescence dyes are positioned in close proximity to each other.

The first dye (fluorescein) is excited by the LightCycler's LED (Light Emitting Diode) filtered light source, and emits green fluorescent light at a slightly longer wavelength (middle figure). When the two dyes are in close proximity (as shown in the lower figure), the emitted energy excites the LC Red 640 attached to the second hybridization probe that subsequently emits red fluorescent light at an even longer wavelength. This energy transfer, referred to as FRET (Fluorescence Resonance Energy Transfer) is highly dependent on the spacing between the two dye molecules. Only if the molecules are in close proximity (a distance between 1–5 nucleotides) is the energy transferred at high efficiency. Choosing the appropriate detection channel, the intensity of the light emitted by the LightCycler – Red 640 is filtered and measured by the LightCycler instrument's optics.

The increasing amount of measured fluorescence is proportional to the increasing amount of DNA generated during the ongoing PCR process. Since LC Red 640 only emits a signal when both oligonucleotides are hybridized, the fluorescence measurement is performed after the annealing step. Hybridization probes can be labeled with LightCycler – Red 640 and with LightCycler – Red 705.  



The Universal ProbeLibrary - A New Concept for qPCR Assays

The unique combination of online available assay design software and only 165 prevalidated, real-time PCR probes allows to quantify virtually any transcript in the transcriptomes of a large number of organisms. Universal ProbeLibrary probes are fully compatible with commonly used PCR conditions and the hydrolysis probe detection format. They are labeled at the 5' end with fluorescein (FAM) and at the 3' end with a dark quencher dye.

Flexibility, specificity, convenience - all in one with the Universal ProbeLibrary
 
The Universal ProbeLibrary combines the flexibility, availability and covenience of SYBR Green I assays with the specificity of Hydrolysis Probe assays. Just 165 prevalidated probes, that can easily be stored in your freezer are sufficient to quantify virtually any transcript from the transcriptomes of a large number of organisms. Target specific intron-spanning qPCR assays are designed online with the ProbeFinder software, freely available at the Universal ProbeLibrary Assay Design Center. The complete assay information, including the sequence of specific primer pairs, and the appropriate Universal ProbeLibrary probe, probe location, amplification product, is displayed on the result page.

more info here  => 
Universal ProbeLibrary 



Molecular Beacons
Hybridization Probes for the Detection of Nucleic Acids in Homogeneous Solutions

Department of Molecular Genetics, Public Health Research Institute 
225 Warren Street, Newark, NJ 07103, USA.

http://www.molecular-beacons.org/

Table of contents:
 
1 Protocol on the synthesis and characterization of molecular beacons
2 Papers on molecular beacons
3 Licensing molecular beacons technology
4 Companies licensed to sell custom molecular beacon probes

              Molecular Beacons are oligonucleotide probes that emit fluorescence when hybridised to
              a target sequence of DNA or RNA. 
              These probes undergo a conformational change when they hybridise to their target. The
              stem and loop structure is made up by a loop structure which is a complementary
              sequence to the target sequence being detected, and the stem is formed by the annealing
              of complementary arm sequences that are on the end of the probe sequence.

              On the end of one arm, a fluorescent moiety is covalently attached, whilst at the end of the
              other arm is a quenching moiety also covalently attached. Due to the stem structure both
              moieties are kept in close proximity and the fluorescence is quenched by energy transfer.
              When the probe encounters it's target sequence a probe-hybrid is formed, which is longer
              and more stable than the stem-hybrid.
              This conformational change forces the arm sequences apart, leading to an increase in
              fluorescence.


 

When You Wish Upon A Star: Molecular Beacons:  Real Time in a Twinkle

   by Deborah Wilkinson

http://www.the-scientist.com/yr1999/mar/profile3_990301.html
 

Prime and Shine While Saving Time: Intergen's Amplifluor allows Direct Detection of PCR Products

by Deborah Wilkinson

http://www.the-scientist.com/yr1999/august/tools2_990830.html
 

<>   Table of Licensed Providers of Molecular Beacons and Kits 


http://www.synthegen.com/   Specializing in Modified Oligonucleotides

      SYNTHEGEN specializes exclusively in modified oligonucleotides.
      Our world-class manufacturing facility is specifically tailored to
      synthesizing, labeling and purifying modified oligonucleotides. This focus
      means you get high quality, reliable modified oligonucleotides backed by
      expert customer service.


Using molecular beacons for spectral genotyping

Differently-colored molecular probes specific for the wild-type and mutant alleles are designed. DNA amplified from homozygous wild-type individuals binds only to the fluorescein-labeled molecular beacons (left). DNA from homozygous mutants binds only the tetramethylrhodamine-labeled molecular beacons (right). Both types of molecular probes will bind to amplicons generated from the DNA of heterozygous individuals (center). 

by  L.G. Kostrikis et al. (1998)
Spectral genotyping of human alleles, Science 279,1228-9122.


Scorpions

Scorpions Info - Click Here   by  http://www.dxsgenotyping.com/ 

Our genotyping process is based on Scorpions Technology - a homogeneous or closed tube method with a simple mix and glow operation. A DNA sample is added to a Scorpions test and an increase in fluorescence indicates the genotype. There is no post-PCR manipulation and the use of two fluorescent dyes gives single tube SNP analysis

Scorpions is a class leading PCR detection technology with significant benefits 
over comparable approaches. These include:

stronger signals 
lower backgrounds
faster reactions
simplified design
improved SNP tests
extended multiplexes
ideal for sample pooling
straightforward manufacture

Typical Scorpions reaction

How Scorpions Works
Scorpions are bi-functional molecules containing a PCR primer element  covalently linked to a probe element. The molecules also contain a fluorophore  that can interact with a quencher to reduce fluorescence. When the molecules  are used in a PCR reaction the fluorophore and the quencher are separated  which leads to an increase in light output from the reaction tube. 

Elements of a Scorpions primer

The benefits of Scorpions derive from the fact that the probe element is physically coupled to the primer element - this means that the reaction leading to signal generation is a uni-molecular rearrangement. This contrasts to the bi-molecular collisions required by other technologies such as Taqman or Molecular Beacons.

The benefits of a uni-molecular rearrangement are significant - as the reaction is effectively instantaneous it occurs prior to any competing or side reactions such as target amplicon re-annealing or inappropriate target folding. This leads to stronger signals, more reliable probe design, shorter reaction times and better discrimination. 

The Scorpions reaction
 


Scorpions

The presence of the blocker group is an essential element of the Scorpions invention. Without such a blocker the Taq DNA polymerase would be able to read through the Scorpions primer and copy the probe region. This would generate signal but not in a target specific fashion. Copying the tail in this way would completely negate the benefits of the Scorpions reaction as any inappropriate side-reactions, including the formation of primer dimers, would also generate a signal. 

Scorpions are PCR primers with a " Stem-Loop " tail containing a fluorophore and a quencher (figue1).
The Stem-Loop tail is separated from the PCR primer sequence by a " PCR stopper ", a chemical modification that prevents the PCR from copying the stem-loop sequence of the Scorpions primer. During PCR, the Scorpions primers are extended to form PCR products. At the appropriate stage in the PCR cycle (the annealing phase), the probe sequence in the Scorpion tail curls back to hybridize to the target sequence in the PCR product (figure 2). As the tail of the scorpion and the PCR product are now part of the same strand of DNA, the interaction is intermolecular. The target sequence is typically chosen to be within 3 bases of the 3'end of the Scorpion primer.
A Scorpion consists of a specific probe sequence that is held in a hairpin loop configuration by complementary stem sequence on either end. A fluorophore is attached to the 5' end giving a fluorescent signal that is quenched in the hairpin loop configuration by a moeity joined to the 3'end.  The haipin loop is linked to the 5' end of a primer.

After extension of the Scorpion primer, during amplification, the specific probe sequence is able to bind to its complement within the same strand of DNA.  This hybridization event opens the hairpin loop so that fluorescence is not longer quenched and an increase in signal is observed. A PCR stopper between the primer and the stem sequence prevents read-though of the hairpin loop, which could lead to the opening of the hairpin loop in the absence of the specific target sequence. The unimolecular nature of the hybridization event gives rise to significant advantages over homogeneous probe systems.  Unlike Molecular Beacon and Double-Dye Oligonucleotides assays (for which Scorpions can be used as an aternative technology), Scorpion assays do not require a separate probe. 


Detection of PCR products using self-probing amplicons and flourescence
Whitcombe, D., Theaker J., Guy, S.P., Brown, T., Little, S. (1999)
Nature 17, 804-807

Molecular diagnostics is progressing from low-throughput, heterogeneous, mostly manual technologies to higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays, and a number of techniques rely on energy transfer between a fluorophore and a proximal quencher molecule. In these methods, dual-labeled probes hybridize to an amplicon and changes in the quenching of the fluorophore are detected. We describe a new technology that is simple to use, gives highly specific information, and avoids the major difficulties of the alternative methods. It uses a primer with an integral tail that is used to probe an extension product of the primer. The probing of a target sequence is thereby converted into a unimolecular event, which has substantial benefits in terms of kinetics, thermodynamics, assay design, and probe reliability.



Design considerations and effects of LNA in PCR primers
David Latorra1, Khalil Arar*, J. Michael Hurley2
Proligo LLC, 6200 Lookout Road, Boulder, CO 80301, USA
Molecular and Cellular Probes 17 (2003) 253–259

The effects of comprehensive LNA substitution in PCR primers for amplification of human genomic DNA targets are presented in this report. Previous research with LNA in other applications has shown interesting properties for molecular hybridization including enhanced specificity in allele-specific PCR. Here we systematically modified PCR primers and conditions for the human genomic DNA targets APOB and PAH, along with a b-globin amplification control, to study whether the number and position of LNA residues improves or diminishes amplification sensitivity and specificity. It was observed that the design rules for LNA substitution in PCR primers are complex and depend upon number, position and sequence context. Technical advantages were seen when compared to DNA controls for the best LNA primer designs, which were typically one to a few centrally located LNA residues. LNA advantages include increased maximum annealing temperature ðTmaxÞ and increased signal with limiting primer or Taq DNA polymerase. Several well-characterized designs exhibited different efficiencies with different brands of hot-start enzymes. Many shorter LNA primers were found to be functional compared to same-length non-functional native DNA controls. These results show that LNA-substituted PCR primers have potential for use in difficult PCR techniques, such as multiplex amplification at higher Tmax; once firm LNA primer design rules are established.



Displacing probes - A new class of homogeneous nucleic acid probes based on specific displacement
hybridization.

Li Q, Luan G, Guo Q, Liang J.
Nucleic Acids Res.  2002 Jan 15;30(2):E5.

The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of
Education, Xiamen 361005, Fujian, Chinas

We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but they become fluorescent upon displacement hybridization with the target. These probes display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. A comparison of double-stranded probes with corresponding linear probes confirms that the
presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant.


Real-time PCR genotyping using displacing probes.

Cheng J, Zhang Y, Li Q.
Nucleic Acids Res. 2004 Apr 15;32(7):e61.

The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of
Education, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian, China

Simple and reliable genotyping technology is a key to success for high-throughput genetic screening in the post-genome era. Here we have developed a new real-time PCR genotyping approach that uses displacement hybridization-based probes: displacing probes. The specificity of displacing probes could be simply assessed through denaturation analysis before genotyping was implemented, and the probes designed with maximal specificity also showed the greatest detection sensitivity. The ease in design, the simple single-dye labeling chemistry and the capability to adopt degenerated negative strands for point mutation genotyping make the displacing probes both cost effective and easy to use. The feasibility of this method was first tested by detecting the C282Y mutation in the human hemochromatosis gene. The robustness of this approach was then validated by simultaneous genotyping of five different types of mutation in the human beta-globin gene. Sixty-two human genomic DNA samples with nine known genotypes were accurately detected, 32 random clinical samples were successfully screened and 114 double-blind DNA samples were all correctly genotyped. The combined merits of reliability, flexibility and simplicity should make this method suitable for routine clinical testing and large-scale genetic screening.



Light-Up Probes

http://www.molbiotech.chalmers.se/research/mk/lightup/lightup.htm

Vanvik et al. developed light-up probes for sequence specific detection of nucleic acids in homogeneous solution. The probes are made of the nucleic acid analogue, PNA, and an assymmetric cyanine dye, which upon bind binding to nucleic acids becomes intesively fluorescent. Under optimum conditions the probe fluorescence increases 50-fold upon binding to target DNA and the fluorescence can be observed by the naked eye.

Negative
Positive


Main references: 



N. Svanvik, G. Westman, W. Dongyuan & M. Kubista. Light-up Probes Thiazole Orange Conjugated PNA for Detection of Nucleic Acid in Homogeneous Solution. Anal. Biochem. 281, 26-35 (2000).

N. Svanvik, A. Stålberg, U. Sehlstedt, R. Sjöback & M. Kubista. Detection of PCR Products in Real-time Using Light-up Probes. Anal. Biochem. 287, 179-182 (2000).

N. Svanvik, J. Nygren, G. Westman & M. Kubista. Free-probe Fluorescence of Light-up probes. J. Amer. Chem. Soc., 123, 803-809 (2001).

For further information see:


BD QZyme™ Assays for Quantitative PCR

http://www.bdbiosciences.com

QZyme-Assay.pdf

Measure the expression level of any human gene

  • Highly sensitive—detect fewer than 10 copies of a DNA target
  • Easy multiplex analysis—quantify two or more genes in a single tube
  • Accurate over a broad range of target concentrations—detect differences of up to 5 orders of magnitude
  • Bright fluorescent signal—works on any qPCR instrument

Introducing the BD QZyme™ Assay for quantitative PCR (qPCR), a novel DNA amplification system for the realtime detection and quantification of specific cDNA and genomic DNA targets. Compatible with all real-time PCR instruments and readily adapted for use in single or multiplex analyses, BD QZyme Assays can accurately measure fewer than 10 copies of target DNA. The assays are easy to set up and require no optimization since they rely on a single set of PCR cycling parameters, which can be universally applied for the detection of any genomic DNA or mRNA target. The dynamic range, or ability of the assay to accurately measure differences in target concentration, is extraordinarily broad, typically extending over 5 orders of magnitude for high-abundance genes.



Figure: The BD QZyme™ Assay. The 5' Primer is comprised of a target-specific sequence joined to the inactive (antisense) strand of the DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of the DNAzyme. The accumulation of amplicons is accompanied by an increase in fluorescence, produced by the action of the DNAzyme on its fluorogenic substrate. Elements not drawn to scale. CS = Cleavage Site.


Amplifluor Quantitative PCR Detection system

Amplifluor™ Universal Detection System

http://www.genxpress.at/

The Amplifluor™ Universal Detection System is a proprietary technology platform that allows the simultaneous amplification and detection of nucleic acids within a closed reaction vessel. The method is based upon the incorporation of energy transfer-labeled hairpin primers into the amplification product. Amplifluor™ hairpin primers are designed so that a fluorescent signal is generated only when the primer is unfolded during its incorporation into an amplification product. The fluorescence signal produced directly correlates with the accumulation of PCR product at each cycle. Unincorporated Amplifluor™ primers have an extremely low fluorescence signal eliminating the need to purify the PCR reaction prior to quantitation; therefore, PCR and fluorescent signal detection can occur in a single reaction vessel. Signal is measured either during the reaction (real-time) or after the last cycle of the reaction (endpoint). Amplifluor™ primers also perform extremely well for in situ PCR applications using paraffin-embedded tissues (see References utilizing Amplifluor™ Technology).

 

Amplifluor Universal Detection System  (PDF)

Amplifluor Hous Keeping Direct Gene System  (PDF)

Amplifluor Apoptosis Gene System  (PDF)

Assay setup for    endpoint or real-time protocols

Assay setup for   real-time instruments



LUX™  Fluorogenic Primers
offer high-performance, cost-effective gene analysis

LUX  (Light Upon eXtension)   primers

This is a new detection system for real-time qPCR which does not require the use of a probe. Simply put,  the LUX system is composed of two primers, just one being label (FAM or JOE). The quenching of the fluorescence of the labeled primer is provided by the secondary structure of the primer (LOOP configuration thanks to the addition of a 5' tail) and the terminal dG-dC or dC-dG base pair when the dye is attached within four nucleotides from the 3´-end.

A brochure and a manual is available on this product
to enable you to have a better idea of the capacity of our product.

invitrogen-lux-brochure.pdf

invitrogen-luxprimers-manual.pdf
 

LUX primers have multiple advantages and among them:

- low price:  Just 1 labeled primer and a regular primer, no need for a quencher or a probe.
- easy to design: software freely available on Invitrogen website
- multiplexing possibility (advantage over SYBR green)
- melting curve possibility (advantage over TaqMan)


The iCycler iQ™  Detection System for multiplex real-time PCR Assays

Faye Boeckman, Keith Hamby, and Larissa Tan, Bio-Rad Laboratories,
2000 Alfred Nobel Drive, Hercules, CA 94547 USA




A novel real-time quantitative PCR method using attached universal template probe
Yuanli Zhang1,2, Dabing Zhang2,*, Wenquan Li3, Jianqun Chen1, Yufa Peng4 and
Wei Cao3
Nucleic Acids Research, 2003, Vol. 31, No. 20 e123


A novel real-time quantitative polymerase chain reaction (PCR) method using an attached universal template (UT) probe is described. The UT is an approximately 20 base attachment to the 5-prime end of a PCR primer, and it can hybridize with a complementary TaqMan probe. One of the advantages of this method is that different target DNA sequences can be detected employing the same UT probe, which substantially reduces the cost of real-time PCR setup. In addition, this method could be used for simultaneous detection using a 6-carboxy-fluoresceinlabeled UT probe for the target gene and a 5-hexachloro- fluorescein-labeled UT probe for the reference gene in a multiplex reaction. Moreover, the requirement of target DNA length for UT±PCR analysis is relatively flexible, and it could be as short as 56 bp in this report, suggesting the possibility of detecting target DNA from partially degraded samples. The UT±PCR system with degenerate primers could also be designed to screen homologous genes. Taken together, our results suggest that the UT±PCR technique is efficient, reliable, inexpensive and less labor-intensive for quantitative PCR analysis.


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