Solid phase hybridization

General Aspects of Dendritic Polymers and Solid-phase Hybrid Polymers... 

This chapter will review some of the key applications presented by protein microarrays. The use of profein microarrays sfems from works on gene expression arrays described earlier. However, rmlike ifs predecessors whose process formats (mutation detection, polymorphism screening, gene expression analysis, etc.) are essentially based upon solid-phase hybridization of nucleic acid complementary strands, the protein array may play different roles and comprise a variety of formafs. 

T.T. Nikiforov, R.B. Rendle, M.L. Kotewicz, Y.H. Rogers, The Use of Phosphorothioate Primers and Exonuclease Hydrolysis for the Preparation of Single-stranded PCR Products and Their Detection by Solid-phase Hybridization , PCR Meth. Appl., 3(5), 285-291 (1994). 

The development of assay techniques that have convenience of solid-phase hybridization and are rapid and sensitive will have a significant impact on diagnostics and genomics [3]. In this respect, SPE genosensors have several advantages they are safe because they are disposable, they are reproducible, they are inexpensive, and the overall procedure is quite fast. In this respect, electrochemical adsorption (adsorption controlled by a positive potential) is an easy to perform and rapid way of immobilization. The method does not require special reagents or nucleic acid modifications. 

Solid-phase hybridization Multiple probe DNA immobilization SNPs detection... 

In Sect. 2 of this chapter, two types of DNA-conjugated polymers were prepared based on polyallylamine and PAA and tested for solid-phase hybridization. Both DNA-conjugated polymers show selective hybridization to fully matched target DNA. 

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. 

Ito, K., Hashimoto, K., and Ishimori, Y., 1996. Quantitative analysis for solid-phase hybridization reaction and binding reaction of DNA binder to hybrids using a quartz crystal microbalance. Anal. Chim. Acta, 3TI, pp. 29-35. 

Although many enzymes used in nucleic acid probes do not follow simple Michaelis-Menten kinetics, some important conclusions can still be drawn for enzymatic activity in solid phase hybridization (i) a higher diffusion constant of the substrate will decrease the of the reaction (ii) the accumulation or depletion of ionic species (e.g., protons) Influences the reaction rate. Shaking of the solid phase during the enzyme reaction may be advantageous, but for comparison care should be taken to agitate each reaction vessel equally. 

Solid phase hybridization is in most cases achieved on membranes. Target nucleic acid is immobilized and subsequently detected by a probe. This approach forms the basis of slot/dot blot hybridization, Northern and Southern hybridization and colony or plaque hybridization. Dot/slot blot hybridization (Kafatos et al., 1979) demonstrates the presence of target sequences but not their size. Although solid phase hybridization is convenient for hybrid/free probe separation, it has the disadvantages that nucleic acid is most often bound noncovalently and that targets are immobilized at fre-... 

Solid phase hybridization is convenient and popular but some limitations should be noted. Background due to nonspecific adsorption of the probe to the solid phase can be considerable without the proper precautions. Both charged and hydrophobic surfaces can yield non-... 

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... 

The reverse kinetic constant may be obtained by appealing to thermodynamic stability requirements of the dissociation kinetics representing the solid-phase hybridization reaction. Thermodynamic stability of the target probe complex is governed by Gibbs free energy of binding as... 

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