Real-time Reporters:

 SYBR® Green, TaqMan®, and Molecular Beacons

 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. 

 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

 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.

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.

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

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.

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). 

 Scorpions

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. 

 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 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. 

 Figue1

 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.