Probe detection systems

Several proprietary fluorescence dye-based detection systems that characterize and quantify probe-bound nucleotide sequences have been developed and commercialized in recent years. For example, general and nonspecific DNA dyes can be used that bind with any double-stranded DNA (i.e., the probe-strand complex or otherwise) and are useful in gel electrophoresis. Much more sophisticated systems rely on oligonucleotide probes that incorporate fluorescent dyes that illuminate when a match between complementary strands are made. Included among the latter are TaqMan , molecular beacons, and Scorpion probes. 

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Fig. 11. Schematic of the picosecond pulse-probe detection system at NERL, University of Tokyo.

A luminescent probe detection system called the hybridization protection assay, or HP A, makes use of an acridinium ester-derivatized oligonucleotide that is hybridized to the amplified DNA (02). Unhybridized probe is preferentially... 

Reference PD B Electron Beam Figure 6. Schematic representation of the LEAF pulse-probe detection system. 

The example above of tire stopped-flow apparatus demonstrates some of tire requirements important for all fonns of transient spectroscopy. These are tire ability to provide a perturbation (pump) to tire physicochemical system under study on a time scale tliat is as fast or faster tlian tire time evolution of tire process to be studied, the ability to synclironize application of tire pump and tire probe on tliis time scale and tire ability of tire detection system to time resolve tire changes of interest. 

The author has a patent for a dynamic surge-detection system, using a boundary-layer probe, presently undergoing field tests. This system consists of specially mounted probes in the compressor to detect boundary-layer flow... 

Since there are a large number of different experimental laser and detection systems that can be used for time-resolved resonance Raman experiments, we shall only focus our attention here on two common types of methods that are typically used to investigate chemical reactions. We shall first describe typical nanosecond TR spectroscopy instrumentation that can obtain spectra of intermediates from several nanoseconds to millisecond time scales by employing electronic control of the pnmp and probe laser systems to vary the time-delay between the pnmp and probe pnlses. We then describe typical ultrafast TR spectroscopy instrumentation that can be used to examine intermediates from the picosecond to several nanosecond time scales by controlling the optical path length difference between the pump and probe laser pulses. In some reaction systems, it is useful to utilize both types of laser systems to study the chemical reaction and intermediates of interest from the picosecond to the microsecond or millisecond time-scales. 

These experiments also show the value of NEXAFS as a technique for following the kinetics of surface processes. We have shown that experiments can be tailored so a specific reaction can be studied, even if gas evolution is not involved. This represents an advantage over thermal desorption experiments, where several steps may be required in order to desorb the products to be detected. Another advantage of NEXAFS is that rates are measured isothermally, so the kinetic parameters can be determined with accuracy. Finally, NEXAFS is not a destructive technique, so we need not to worry about modifying the surface compounds while probing the system, as would be the case with other techniques such as Auger electron spectroscopy. 

On-line pervaporation, 18 518 On-line probes, for fermentation, 11 39 On-line vapor permeation, 18 518 On/off feedback controllers, 20 691 Onstream analyzers, 20 682 On-tank leak detection systems, 24 311 Onyx thermal printing process, 19 321 Oocytes, retroviral infection of, 12 457 Oolitic limestone, 15 28 Opacification, colorants for ceramics, 7 344-345... 

Otherwise, depolarization would also be a result of energy transfer between probe molecules. Because the transition moments of two interacting probes are unlikely to be parallel, this effect is indeed formally equivalent to a rotation. Moreover, artefacts may arise from scattering light that is not totally rejected in the detection system. 

N4. Nelson, N. C., and Kacian, D. L., Chemiluminescent DNA probes A comparison of the acridinium ester and dioxetane detection systems and their use in clinical diagnostic assays. Clin. Chim. Acta 194, 73-90 (1990). 

Calibration method. The measured fluorescence parameter should be independent of indicator concentration, geometry of sample, and sensitivity of detection system. Thus, an intensity-based method requires wavelength-ratiometric probes. Lifetime and anisotropy methods do not require wavelength-ratiometric probes, but the lifetime or anisotropy must be sensitive to analyte. 

The complexities of protocols for fluorescent and chromogenic in situ hybridization necessarily entail careful attention to controls. In particular, the possibility of native enzyme activity or the presence of endogenous biotin in the experimental tissue should be considered, though this can be addressed by exposing control tissue to the detection system in the absence of probe. The relative merits of digoxigenin versus biotin, and some of the technical problems associated with each, have been previously discussed (Chevalier et al., 1997 Luo and jackson, 1999). 

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