Probes production nucleic acid

Acceptable bridging molecule systems have been developed which have also simplified the utilization of different detection systems. To illustrate this point, a researcher who has developed a unique monoclonal antibody (a primary antibody) in the mouse may select from a variety of commercially available products consisting of different detection systems (e.g. fluorescein, alkaline phosphatase, colloidal gold) attached to an immunoglobulin that will specifically bind to mouse antibodies (a secondary antibody). In this way the researcher may readily obtain and test a number of detection methods for visualizing target-probe interactions without having to directly label the monoclonal antibody probe. For nucleic acid probes, which in themselves are not readily immunodetectable, it is useful to incorporate or attach detectable moieties to the nucleotides. 

Nobel-laureate Richard Feynman once said that the principles of physics do not preclude the possibility of maneuvering things atom by atom (260). Recent developments in the fields of physics, chemistry, and biology (briefly described in the previous sections) bear those words out. The invention and development of scanning probe microscopy has enabled the isolation and manipulation of individual atoms and molecules. Research in protein and nucleic acid stmcture have given rise to powerful tools in the estabUshment of rational synthetic protocols for the production of new medicinal dmgs, sensing elements, catalysts, and electronic materials. 

Chemiluminescence reactions are currently exploited mainly either for analyte concentration measurements or for immunoanalysis and nucleic acid detection. In the latter case, a compound involved in the light emitting reaction is used as a label for immunoassays or for nucleic acid probes. In the former case, the analyte of interest directly participates in a chemiluminescence reaction or undergoes a chemical or an enzymatic transformation in such a way that one of the reaction products is a coreactant of a chemiluminescence reaction. In this respect, chemiluminescent systems that require H2O2 for the light emission are of particular interest in biochemical analysis. Hydrogen peroxide is in fact a product of several enzymatic reactions, which can be then coupled to a chemiluminescent detection. 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Finally, along this line, an interesting development has been recently reported by Heckl et al. (68). They synthesized an Imaging Probe consisting of a Gd-complex, a PNA (Peptide Nucleic Acid) sequence, and a transmembrane carrier peptide. Although the system enters any type of cell, it accumulates only in tumor cells because of the specific binding of the PNA moiety of the c-myc mRNA whose production is upregulated in those cells. 

Stereochemical probes of the specificity of substrates, products, and effectors in enzyme-catalyzed reactions, receptor-ligand interactions, nucleic acid-ligand interactions, etc. Most chirality probe studies attempt to address the stereospecificity of the substrates or ligands or even allosteric effectors. However, upon use of specific kinetic probes, isotopic labeling of achiral centers, chronfium-or cobalt-nucleotide complexes, etc., other stereospecific characteristics can be identified, aU of which will assist in the delineation of the kinetic mechanism as well as the active-site topology. A few examples of chirality probes include ... 

Fig. 30. Detection of mRNA on a membrane or in situ with labeled gene probes. A Detection of mRNA with a fluorescein-labeled single stranded nucleic acid probe, using POD-conjugated anti-fluorescein antibody. B Use of two gene probes labeled with different molecules (fluorescein and digoxigenin) and detected with specific antibodies, both coupled to AP and using two substrates, leading to differently colored products. This in situ hybridization scheme allows the simultaneous detection of two mRNA species in a tissue or cell preparation. C Amplification systems involving more than one antibody can be used to increase specificity and signal intensity.

Nucleic acids - [NUCLEIC ACIDS] (Vol 17) -in cell culture products [CELL CULTURE TECHNOLOGY] (Vol 5) -coordination compounds as probes [COORDINATION COMPOUNDS] (Vol 7) -electrophoresis of [ELECTROSEPARATIONS - ELECTROPHORESIS] (Vol 9) -phosphorus m [MINERALNUTRIENTS] (Vol 16) -role m sterilization [DISINFECTANTS AND ANTISEPTICS] (Vol 8) -ruthenium cmpds as probes [PLATINUM-GROUP METALS, COMPOUNDS] (Vol 19)... 

The assay for transgene expression involves the transduction of tissue culture cells or animals, and testing for the presence of the transgene protein product. Reagents (antibodies, enzymatic substrates, nucleic acid probes) specific for the transgene product should be made in large quantities, so the assay can be repeated several times. 

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