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Fig. 3 Analysis of SYBR Green 1 dissociation curve. The first derivative view with respect to temperature provides a clear view of the rate of SYBR Green 1 loss and the temperature range over which this occurs. The example shows a view of data between 72°C and 90oC. The small peak at 74.5°C is probably due to primer dimer product formation as this is the only peak to occur in the NTC sample. The main peaks occur around 85.55°C although there are some with a distinctly different profile and a peak at 86.55°C. These distinct profiles represent different products in the final PCR product.

Molecular beacons (MB) and Scorpions are based on stem-loop hairpin structures, and MB in particular have found wide-ranging application as diagnostic tools.[12] Molecular beacons consist of a hairpin loop structure, with the loop complementary to a target nucleic acid and the stem formed by the annealing of complementary termini (Fig. 6A). One end of the stem has a reporter fluorophore attached and the other a quencher. In solution, free MB adopt a hairpin structure and the stem keeps the arms in close proximity, resulting in efficient proximal quenching of the fluorophore. During the denaturation step, the MB assume a random-coil configuration and fluoresce. At the annealing temperature, MB bind to any target amplicons as the probe/target duplex is designed to be thermody-namically more stable than the hairpin structure at that temperature. Once the probe binds to its target the hairpin is opened out and the fluorophore and quencher are separated, resulting in fluorescence. A major strength of this technology is the high specificity of the MB in recognizing nucleotide sequence mismatches in DNA and RNA. The hairpin shape of the MB causes mismatched probe/target hybrids to easily dissociate at significantly lower temperatures than exactly complementary hybrids. This is because the thermodynamic properties of the MB favor the formation of a hairpin form rather than continued hybridization to a less than perfectly matched target sequence. When the temperature is raised to allow primer extension, the MB dissociate from their targets and do not interfere with polymerization. A new hybridization takes place in the annealing step of every cycle, and the intensity of the resulting fluorescence indicates the amount of accumulated amplicon. Again, as this is a reversible process, melt curves can be used to analyze the dynamics of the reaction and determine the best temperature for fluorescent acquisition (Fig. 6B). There is some evidence to suggest that the measured signal ratios

Fig. 4 Fluorophore-labeled probes. (A) The original design used a primer with a hairpin structure at its 5'-end that contained a fluorophore and quencher at opposite ends of the hairpin. During the first cycle of PCR the primers are extended and become templates during each subsequent cycle. This linearizes the hairpin, separates the donor and acceptor moieties, and results in fluorescence emission from the fluorophore. (B) LUX™ primers. One primer contains a fluorophore, the other one is unlabeled. The fluorogenic primer has a short sequence tail of 4-6 nucleotides on the 5'-end that is complementary to the 3'-end of the primer. The resulting hairpin secondary structure provides optimal quenching of the attached fluorophore. When the primer is incorporated into the double-stranded PCR product, the fluorophore is dequenched and a signal is reported.

Fig. 4 Fluorophore-labeled probes. (A) The original design used a primer with a hairpin structure at its 5'-end that contained a fluorophore and quencher at opposite ends of the hairpin. During the first cycle of PCR the primers are extended and become templates during each subsequent cycle. This linearizes the hairpin, separates the donor and acceptor moieties, and results in fluorescence emission from the fluorophore. (B) LUX™ primers. One primer contains a fluorophore, the other one is unlabeled. The fluorogenic primer has a short sequence tail of 4-6 nucleotides on the 5'-end that is complementary to the 3'-end of the primer. The resulting hairpin secondary structure provides optimal quenching of the attached fluorophore. When the primer is incorporated into the double-stranded PCR product, the fluorophore is dequenched and a signal is reported.

Fig. 5 Scatter plot for real-time SNP analysis using MB. Fluorescence is reported during each annealing step when the MB is bound to its complementary target and the Ct value for each dye in the sample is used to determine the genotype of the samples. A Ct value equal to the final cycle of the PCR reaction (typically 40 or 45) indicates the absence of a specific allele. Each plotted point represents the coordinates of the Ct values for the two dyes in a single well. Here the x axis corresponds to FAM Ct whereas the y axis corresponds to HEX Ct and the plotted points (x,y) correspond to the coordinates describing the two Ct values determined for a given well. The position of the data point for a given well on the scatter plot indicates the presence or absence of each allele, providing a rapid method for clustering the samples that are homozygous for either of the two alleles or are heterozygous. If a highly quantitative measurement of copy numbers is not necessary, it is also possible to measure fluorescence when cycling is complete.

with MB assays are proportional to the amount of the minor allele over a wider range than with the TaqMan assay.[13] Multiplexing of MB is enhanced by the use of wavelength-shifting MB (Fig. 6C).[14] These extend the range of fluorophore/quencher pairs that will function at a given wavelength and contain a generic harvester fluorophore, a probe-specific emitter fluorophore, and a quencher. Scorpions were originally made up of a single oligonucleotide containing a 5' fluorophore, a stem-loop structure containing the probe, a quencher, and a PCR blocker to prevent read-through by DNA polymerase and primer. In the presence of a target, the specific probe sequence folds back on itself to bind its complement within the same DNA strand, opening up the hairpin loop and separating the fluorophore and quencher. This unwieldy structure was replaced by the second-generation Scorpions that are made up of two oligonucleotides. One contains the fluorophore-coupled probe, the other a quencher-coupled complementary sequence. For SNP analysis, the fluorescence is monitored above the Tm of the mismatch probe/target duplex and below the Tm of the fully complementary probe/target duplex. Under these conditions the mismatched probe reassociates with the quencher element to become nonfluorescent, whereas the hybridized wild-type probe is separated from the quencher element and is fluorescent. Because the hybridization of probe sequence to amplicon is intramolecular, Scorpion probes are more efficient than binary systems such as MB and as a result generate significantly greater signal intensities.1-15-1

There are many more probe chemistries available, all with their own advantages and disadvantages. These include Hybeacons, which require only a single fluo-rophore and make use of the quenching properties of DNA. This makes them easy to design and synthesize.[16] Light-up probes are composed of thiazole orange conjugated to peptide nucleic acid (PNA) (see below) and combine the excellent hybridization properties of PNA, which allows the use of shorter probes, with the extraordinary fluorescence enhancement of asymmetric cyanine dyes upon binding to nucleic acids.[17] Eclipse™ probes are linear probes that have a minor groove binder (MGB) and quencher on the 5'-end and the fluorophore on the 3'-end.[18] This is the other way round compared with hydrolysis probes, and the presence of the MGB at the 5'-end prevents cleavage of the Eclipse probe by Taq polymerase. Other chemistries are described in detail elsewhere.[19]

Real-time PCR assays generally use symmetric primers. However, this results in the reactions typically slowing down and entering the plateau phase in a stochastic manner, because reannealing of the template strands gradually outcompetes primer and probe binding to the template strands. This is a particular problem when the aim is to detect specific DNA targets down to alleles of single-copy genes in single cells. Asymmetric PCR potentially circumvents the problem of amplicon strand reannealing by using unequal primer concentrations. However, asymmetric amplification is much less efficient and requires extensive optimization to identify the proper primer ratios, the amounts of starting material, and the number of amplification cycles that can generate reasonable amounts of product for individual template/target combinations. A recent innovation termed linear-after-the-exponential-PCR (LATE-PCR) uses unequal primer concentrations but takes into account the effect of the actual primer concentrations on primer Tm.[20] It corrects for the fact that the Tm of the limiting primer is often several degrees below the Tm of excess primer and allows the asymmetric PCR to proceed as efficiently as symmetric PCR. Furthermore, ss amplicons are generated with predictable kinetics for many cycles beyond the exponential phase. This permits uncoupling of primer annealing from product detection. As a result, the Tm of the probe no

Fig. 6 Molecular beacons and melting curves. (A) Structured probes are better at discriminating single base-pair mismatches than linear ones such as TaqMan™. Only one MB is shown, which hybridizes to its perfect complement and emits fluorescence but remains closed and unbound to a target with a single nucleotide mismatch. (B) Molecular beacon melting profile for allelic discrimination. Three melting curves are visible. One for MB alone (a), a second one for MB and its perfectly complementary single-stranded oligonucleotide target (b), and a third one for MB plus a single-strand target that produces a probe/target hybrid containing a single mismatched base pair (c). The two vertical bars indicate the optimal annealing temperature range in which the perfectly matched MB will have greater fluorescence than the mismatched MB, with background fluorescence still low. (C) Wavelength shifting MB. The MB has two fluorophores on one end, a ''harvester'' and an ''emitter,'' and a quencher on the other end. In the hairpin loop structure, the quencher forms a nonfluorescent complex with the harvester. Upon hybridization of the MB to a complementary sequence, quenching of the harvester fluorophore is relieved, and it transfers energy via FRET to the emitter, which emits fluorescence.

Fig. 6 Molecular beacons and melting curves. (A) Structured probes are better at discriminating single base-pair mismatches than linear ones such as TaqMan™. Only one MB is shown, which hybridizes to its perfect complement and emits fluorescence but remains closed and unbound to a target with a single nucleotide mismatch. (B) Molecular beacon melting profile for allelic discrimination. Three melting curves are visible. One for MB alone (a), a second one for MB and its perfectly complementary single-stranded oligonucleotide target (b), and a third one for MB plus a single-strand target that produces a probe/target hybrid containing a single mismatched base pair (c). The two vertical bars indicate the optimal annealing temperature range in which the perfectly matched MB will have greater fluorescence than the mismatched MB, with background fluorescence still low. (C) Wavelength shifting MB. The MB has two fluorophores on one end, a ''harvester'' and an ''emitter,'' and a quencher on the other end. In the hairpin loop structure, the quencher forms a nonfluorescent complex with the harvester. Upon hybridization of the MB to a complementary sequence, quenching of the harvester fluorophore is relieved, and it transfers energy via FRET to the emitter, which emits fluorescence.

longer needs to be higher than the Tm of either primer. This permits the use of low-Tm probes, which are inherently more allele-discriminating, generate lower background, and can be used at saturating concentrations without interfering with the efficiency of amplification.

Another important advantage of using probe-based chemistries is that it is possible to multiplex, i.e., amplify multiple targets in a single tube, as fluorescent dyes with different emission spectra may be attached to the different probes (Fig. 7). Probes afford a level of discrimination

Fig. 7 Multiplex PCR. Four TaqMan™ probes were labeled with FAM, HEX, ROX, or Cy5, and a PCR assay was performed using the Stratagene ''Brilliant1 Multiplex QPCR'' master mix on the MX4000 real-time PCR system. Each sample was analyzed in triplicate. The four horizontal lines indicate the four thresholds calculated for the individual fluorophores. The four targets differ by 14 Cts, which translates into a 1.6 x 104-fold difference in target abundance.

Fig. 7 Multiplex PCR. Four TaqMan™ probes were labeled with FAM, HEX, ROX, or Cy5, and a PCR assay was performed using the Stratagene ''Brilliant1 Multiplex QPCR'' master mix on the MX4000 real-time PCR system. Each sample was analyzed in triplicate. The four horizontal lines indicate the four thresholds calculated for the individual fluorophores. The four targets differ by 14 Cts, which translates into a 1.6 x 104-fold difference in target abundance.

impossible to obtain with SYBR Green, as they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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