HYBRIDIZATION WITH NUCLEIC ACID PROBES solid phase

DNA arrays have been categorized into different formats based upon what is immobilized to the surface (also known as the solid phase, substrate, or chip) and what is captured from the sample solution. Definitions change depending upon the format. For the classic Southern dot blot, the sample was first spotted down on the surface, cross-linked, and then bathed with a radio-labeled oligonucleotide under hybridization (complementary nucleic acid strand base-pairing) conditions to detect the presence of a parhcular sequence within the sample. This was called probing. The oligonucleohde... 

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 main difference between the capture and the sandwich assays is how the label probe-target complex is immobilized on a solid phase. In capture assays, hybridization with the immobilized capture probe determines the kinetics and specificity/detectability/sensitivity characteristics of the assay. In sandwich assays, an immobilized molecule, which is not a nucleic acid (e.g., streptavidin, antibodies), serves to capture the hapten probe-target-label probe ternary complex (thus both probes need to be modified). The affinity matrix-hapten interaction thus determines the kinetics (usually 3-10 times faster than solid phase hybridization), the sensitivity and detectability... 

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