Nucleic Acid Hybridization Assays

In this structure, the bases on opposite strands pair up with each other in a very specific way adenine (A) pairs with thymine (T), and guanine (G) pairs with 

FIGURE 3.3 The form of the DNA double helix, showing major and minor grooves. 

Double-stranded DNA molecules with one or more mispairs are more easily disrupted or denatured than are properly and fully paired double helices the more mispairing, the less the stability of the molecule. If a DNA molecule, with or without mispairs, is too unstable, then the two strands will dissociate from one another to 

FIG U RE 3.4 Watson-Crick base pairing in DNA. Adenine is complementary to thymine, and guanine is complementary to cytosine. 

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In the application of alkaline-phosphatase-sensitive, triggerable 1,2-dioxetanes, the nucleic-acid hybridization assay is nowadays quite popular . Such techniques include viral load assays for hepatitis B and C and for human immunodeficiency viruses (HBV,... 

To understand how these modem methods work, it is necessary first to review some general laboratory techniques ubiquitous in genetic engineering. Among the most important are gel electrophoresis of nucleic acids, nucleic acid hybridization assays, and the polymerase chain reaction. 

Fluorescence-Based Nucleic Acid Hybridization Assays. 245... 

Solution-based nucleic acid hybridization assays represent a class of analytical methodologies that provide for detection of target-probe hybridization events. Solution-based assays offer several advantages in comparison to configurations that use surface-bound probes, primarily from the standpoint of negating the requirement for immobilization of a probe sequence to a solid surface. Therefore, thermodynamic, kinetic and adsorptive effects that are relevant in terms of consideration of hybridization at interfaces are not encountered. Solution-based hybridization assays provide a simple way of detecting hybridization events in real-time. 

Morrison, L., Fluorescence in nucleic acid hybridization assays, in Topics in Fluorescence Spectroscopy, vol. 7, J. R. Lakowicz, Ed. New York Kluwer Academic Publishers/Plenum Press, 2003, pp. 69-103. 

Enzyme DNA hybridization assays with electrochemical detection can offer enhanced sensitivity and reduced instrumentation costs in comparison with their optical counterparts. Efforts to prevent non-specific binding of the codissolved enzyme and to avoid fouling problems by selecting conditions suitable to amplify the electrode response have been reported by Heller and co-workers [107]. A disposable electrochemical sensor based on an ion-exchange film-coated screen-printed electrode was described by Limoges and co-workers for an enzyme nucleic acid hybridization assay using alkaline phosphatase [108] or horseradish peroxidase [109]. In another methodology to improve sensitivity, a carbon paste electrode with an immobilized nucleotide on the electrode surface and methylene blue as hybridization indicator was coupled, by Mascini and co-workers [110], with PGR amplification of DNA extracted from human blood for the electrochemical detection of virus. 

H10. Hunsakcr, W. R., Badri, H., Lombardo, M., and Collins, M. L., Nucleic acid hybridization assays employing dA-tailed capture probes. Anal. Biochem. 181, 360-370 (1989). 

Microfluidic DNA hybridization assays Nucleic acid hybridization assays On-chip DNA hybridization analysis... 

Erikson, G. H. Daksis, J. I. Improving the signal/ noise ratio of nucleic acid hybridization assays by preincubation of primer and target with nucleic acid binding agents. U.S. Pat. Appl. Publ. US 2004180345, 2004 Chem. Abstr. 2004, 141, 255469. 

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