Solution-phase hybridization assay

This multiprobe RPA offers the advantages of its sensitivity and capacity to simultaneously quantitate several mRNA species in a single sample of total RNA. This allows comparative analysis of different mRNA species within samples, moreover, by incorporating probes for housekeeping gene transcripts, the levels of individual mRNA species can be compared between samples. Furthermore, the assay is highly specific and quantitative owing to the RNase sensitivity of mismatched base pairs and the use of solution-phase hybridization driven toward completion by excess probe. Finally, the multiprobe RPA can be performed on total RNA preparations derived by standard methods from either frozen tissues or cultured cells. 

When the nucleic acid target or probe is immobilized on a solid support, the kinetics of hybridization are even more complex. Many of the preceding observations stfil hold true, but the rate and extent of solid-phase hybridization are lower than with solution-phase hybridization. Depending on the concentrations of the reactants, solid-phase hybridization can be either nucleation-hmited or diffusion-limited. Optimal efficiency of solid-phase hybridization is achieved under conditions that facilitate diffusion of the probe to the support and that favor hybridization over strand-reassociation if double-stranded probes are used. This usually means a small volume of hybridization solution and relatively low probe concentrations. In practice, solid-phase hybridization assays are empirically designed. Time of hybridization and probe concentration are the two variables most frequently adjusted in the assay. Conditions that tend to maximize the extent of hybridization and minimize the background or nonspecific attachment of the probe are selected. 

The first, and simplest of these, exploits the fact that cationically charged microspheres (which can be magnetic particles) selectively bind hybridized AE probes. Thus, after a solution-phase hybridization between a DNA AE probe and a target ribosomal RNA, containing a sequence complementary to the probe, the hybridized probe can be removed in a 10-min separation step and quantitated in a luminometer (Gil, T6). Assays have been described for a number of bacterial... 

In addition to the solution-phase protein detection assays such as biobarcode assays, a surface-based protein detection method was developed by Niemeyer and Ceyhan (70). In this work, a biotin-labeled antibody was conjugated to DNA using streptavidin as a tinker molecule. This conjugate was hybridized to complementary DNA-AuNPs and antibody-functionalized AuNPs. These particles are allowed to form a sandwich structure with a target protein and a second antibody immobilized on a flat surface. This was followed by a silver enhancement step catalyzed by the AuNPs which resulted in a LOD of 50 fmol (200 pM). This method is conceptually similar to the corresponding surface-based scanometric detection of DNA. Further work demonstrated a similar protein detection scheme without the silver enhancement (71). Instead, multiple layers of secondary DNA-AuNPs were used to increase signal enhancement [LOD = 0.1 finol (2 pM)]. 

Nakagami et al. (1991) proposed a different universal probe system. In their procedure, phagemids are used to obtain strand-specific ss DNA molecules, one with the insert complementary to the target sequence and a second without the insert and with the opposite polarity (Fig. 7.23). The ss DNA without insert is labeled with any label (usually a secondary label) and this labeled DNA is hybridized with the complementary strand containing the insert. This leaves only the probe sequence ss whereas the tag sequence is ds. This method has several advantages (i) like the sandwich method it leaves the probe sequence single-stranded (ii) hybridization of the tag sequences in solution instead of on the solid phase as in a sandwich assay is much faster (iii) no fragmentation is required. 

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