Nucleic acid labeling with enzymes

Another common enzyme used m these procedures is alkaline phosphatase. Alkaline phosphatase will cleave phosphates off of a donor molecule, which then in turn acts as a mediator of a color change involving a third molecule. This system is often used because alkaline phosphatase can create more of the color-producing molecules per enzyme molecule than can peroxidase, resulting in better sensitivity. Alkaline phosphatase systems are especially sensitive for examining protein or nucleic acid blots with enzyme labels. The problem when examining tissue is the presence of the endogenous enzyme m the tissues examined. 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Firefly luciferase, together with luciferases from other organisms, can be used as the labeling enzyme in immunoassays and nucleic acid assays [25], Recently, a highly sensitive BL ELISA using firefly luciferase was applied to thyreo-... 

Dioxetanes, labeled with triggers sensitive to the alkaline-phosphatase enzyme, serve as highly sensitive chemiluminescent probes in numerous bioassays. Current applications include immunoassays, membrane-based detection of proteins and nucleic acids, and microplate-based and array-based nucleic-acid detection. ... 

Fig. 28. Synthesis of labeled DNA probes. A Labeled DNA can be generated using different enzymes (Klenow fragment of DNA polymerase or a terminal transferase) to incorporate labeled nucleotides into specific DNA sequences. Probes can be labeled using radioactive nucleotides or nucleotides labeled with an immunogenic molecule such as biotin. B The labeled probe is then hybridized to the target nucleic acid, which is either bound to a membrane or in a tissue section or cell. An antibody is then used to detect the non-radioactively-labeled probe. C The antibody may be conjugated to a fluorescent or chemiluminescent dye, or an enzyme that produces a color reaction. The target nucleic acid is thus visualized.

Similar techniques can be used to devise avidin—biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind their complementary DNA target through the use of avidin-labeled complexes (Bugawanefrz/., 1990 Lloyd etal., 1990). Direct detection of hybridized probes can be accomplished, in a manner similar to that for LAB, by incubating with an avidin-enzyme conjugate followed by substrate development. BRAB-like and ABC-like assays also can be utilized to further enhance a DNA probe signal (Chapter 17, Section 2.3). 

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