Hybridization Assays—Examples

Leclerc, Ho, and co-workers have developed many sensing systems for different targets using cationic PT and exploiting the differences in emission and color between the extended conjugation form and the coiled form. The basis of these assays is that the polymer microstructure and conformation change as it interacts with the target. For example, in DNA hybridization assays, imidazolium-PT (see... 

DNA probing can be done not only on a membrane, but also on solid supports. An example is the classical sandwich hybridization assay, which uses a capture... 

Homogeneous hybridization assays are analyses for the detection of a given DNA/RNA sequence by hybridization without separation processes (B/F separation). These assays form a basis of real-time PCR as described below. Furthermore, sufficiently sensitive homogeneous hybridization assays could be done without PCR and enable, for example, direct detections of certain mRNA forms expressed in cells. This type of experiment has been actually performed with some organic fluorophores (Peng et al., 2005 Santangelo et al., 2006). 

Fig. 14. Examples of homogeneous hybridization assay methods (F luminophore, Q quencher, D donor, A acceptor). Thick lines represent DNA strands. Open circles on DNA strands indicate a SNP/mutation site for Molecular Beacon and insertion/deletion sites for dual FRET probe and dual FRET Molecular Beacon when these methods are applied to SNP/mutation typing or deletion/insertion detection. The solid circle on die strand indicates the complementary site.

The Cytostar-T microplate from Amersham Biosciences Inc. is specifically designed for cell-based proximity assays. Scintillant is incorporated into the well bottom of the microplate. As an example, Cytostar-T plates have been used to detect mRNA transcripts in a high-volume in situ hybridization assay... 

Fig. 8.6. Sensitivity and detectability depend on various factors. In example I (Thompson and Gillespie, 1987), the sensitivity is 1. Increasing the probe concentration does not improve sensitivity but deteriorates the detectability. In another example (van Gijlswijk et al., 1992), the sensitivity in a hybridization assay, using POase-catalyzed luminol reaction, was similar in the one-step and three-step methods but the detectability improved for the latter. Similarly, in reverse capture assays detectability improves after one or a few cycles while sensitivity decreases only slightly. In example II (Oser and Valet, 1988), simple adjustments in the (time-resolved fluorescence) procedure improved the detectability somewhat but the sensitivity increased about 100-fold for the well-strip method.

It would seem that this has little to do with SM-protein interaction profiling, and that would be true were it not for the amazing versatility of the approach. To look at two examples of how modified yeast two-hybrid approaches can potentially be used for SM screening, let us consider SM-induced protein-protein interaction inhibition (PPII) and SM-protein binding. To assess the first situation, a modified two-hybrid assay involves a lethal reporter gene and a protein pair that is known to interact (Fig. 2-3). Now, an array of SMs potentially causing PPII... 

The n-hybrid assay can also be used for directed evolution. For example,... 

Fig. 5 An example of TR-ERET decay change upon binding. Hybridization assay of target (related to cebac disease) is accomplished with dual-label flexible probe, which upon hybridization is extended resulting in prolonged decay time as shown in the measured values. Redrawn from [43]...

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