Target-probe hybridization interactions

The specificity and affinity of interactions between target molecules bound to the microarray substrate and probe molecules in solution largely determine the quality of microarray assays. The complementary base hybridization is the most efficient and reproducible target-probe interactions used in DNA microarray analysis. 

Arrays have been produced on filter supports, in microtiter plate wells and on glass slides coated and modified with one-, two- or 3-dimensional surface architectures as shown schematically in Figure 813 19. Glass offers a number of practical advantages, such as mechanical stability and low autofluorescence. Due to the non-porous character of glass chips, the volume of the hybridization solution can be kept to a minimum and probe-target interaction is not limited by diffusion into pores. However, three-... 

Oligonucleotide probes may be labeled with small fluorescent molecules for detection of hybridization by luminescence. Fluorescent probes are widely used in assay systems involving bio-specific interactions (Chapter 9). Receptors for ligands may be localized in tissues or cells by modification of the ligand with the appropriate fluorophore. Targeted molecules may be... 

Figure 3.24 Schematic representation of the analytical protocol (A) Capture of the ALP-loaded CNT tags to streptavidin-modified magnetic beads by a sandwich DNA hybridization (a) or Ab-Ag-Ab interaction (b). (B) Enzymatic reaction. (C) Electrochemical detection of the product of the enzymatic reaction at the CNT-modified glassy carbon electrode MB, Magnetic beads P, DNA probe 1 T, DNA target P2, DNA probe 2 Abl, first antibody Ag, antigen Ab2, secondary...

Sonicated and denatured salmon sperm DNA (or other anionic maCTomolecules) may be used to reduce nonspecific probe interaction and electrostatic forces. The latter also may be reduced with dextran sulfate. High-stringency (low-sodium) hybridization ensures that complete complementarity will characterize the probe-target hybrid. 

A typical DNA array fabrication and application process involves three major steps. First, nucleic acids (the capture sequences or probes) are immobilized at discrete positions on surface activated substrates. Secondly, the resulting array is hybridized with a complex mixture of fluorescently labelled nucleic acids (the target), and thirdly subsequent to hybridization, the fluorescent markers are detected using a high-resolution scanning laser that quantifies the interaction. This chapter focuses on the first of these processes and provides the reader with an overview of substrates, surface activation methods and dehvery systems available for nucleic acid immobilization. 

The phosphate backbone of DNA molecules often results in undesirable electrostatic interactions with the substrate. Although the electrostatic interactions of DNA can be utilized for physical adsorption of DNA to the surface, this process can also lead to the nonspecific physical adsorption of target DNA on the surface. Rather than sample DNA hybridizing to the probe, it can adsorb to the surface and lead to interferences with the final detection call. Nonspecific adsorption effects have primarily been examined by the microarray community. Blocking strategies have been developed to prevent these nonspecific interactions. Succinic anhydride (SA) and bovine serum albumin (BSA) are two common methods to prevent nonspecific adsorption on amine modified surfaces. Blocking strategies are desired to react with or pas-... 

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