Abstract
Publisher Summary The analysis of DNA by membrane hybridization techniques is important in the characterization of cloned genes, investigation of genetic diseases, detection of pathogens, forensic determinations, as well as many other areas of biology. The analysis of DNA by chemiluminescence is discussed in this chapter. Chemiluminescent organic reactions can be classified in several ways. The majority of these reactions require a critical step that involves oxidation of a substrate with molecular oxygen or its synthetic equivalent. The oxidation of luminol is a classic example of a chemiluminescent reaction in which the key oxidative step involves hydrogen peroxide and aminophthalhydrazide in the presence of suitable catalysts. The mechanism leading to AMPPD luminescence in the presence of alkaline phosphatase involves two steps. In the first step, dephosphorylation by alkaline phosphatase occurs, generating a moderately stable anion, AMP-D. The second step involves a further breakdown of AMP-D to adamantanone and the charge-transfer excited state of methyl m-oxybenzoate anion, which emits light. Chemiluminescence detection with AMPPD can be performed using various experimental formats, such as solution, bead, and membrane hybridizations.
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