Probes, nucleic acid

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

If an antibody to the protein of interest is available, it is sometimes possible to use vector sequences, eg, the beta-galactosidase promoter sequence, to direct the transcription of the passenger DNA into messenger RNA and the translation of that mRNA into protein which can be recognized by the antibody. Although this method is somewhat less reHable than the use of nucleic acid probes, specialized vectors are available for this purpose. 

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. 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Hongmanee P., Stender H., Rasmussen O.F. Evaluation of a fluorescence in situ hybridization assay for differentiation between tuberculous and nontuberculous Mycobacterium species in smears of Low-enstein-Jensen and mycobacteria growth indicator tube cultures using peptide nucleic acid probes. J. Clin. Microbiol. 2001 39 1032-1035. 

Normal cells also were found to contain src-like genes when their DNA was hybridized with labeled nucleic acid probes from the v-src gene. As with other cellular genes, the c-onc genes were interrupted with introns. v-Onc genes lack introns. Consequently, in the distant past c-onc genes must have been transferred to the retroviruses. If the transfer had been from the virus to the cell, c-onc genes probably would not contain introns. 

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. 

Psoralen, or derivatives of 9-methoxy-7H-furo[3,2-g]chromen-7-one tricyclic ring structures, are used as photoreactive groups in crosslinkers, biotinylation compounds, and nucleic acid probes. Psoralens have been used for many years as photochemotherapy agents for treatment of psoriasis and vitiligo (Smith and Barker, 2006). Psoralens react when exposed to UV light... 

Didenko, V.V. (1993) Biotinylation of DNA on membrane supports A procedure for preparation and easy control of labeling of nonradioactive single-stranded nucleic acid probes. Anal. Biochem. 213, 75-78. 

Hopman, A.H.N., Wiegant, J., Tesser, G.I., and Van Duijn, R (1986) A nonradioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten hgands. Nucleic Acids Res. 14, 6471-6488. 

J. Wang, M. Jiang, A. Fortes, and B. Mukherjee, New label-free DNA recognition based on doping nucleic-acid probes within conducting polymer films. Anal. Chim. Acta 402, 7-12 (1999). 

An array or a matrix of nucleic acid probes immobilized at discrete locations on a silicon or glass surface provides a convenient means to simultaneously probe a sample for the presence of many different target sequences. Microarray biochip scanning devices, mostly based on fluorescent labels, are now currently available, and could also be used with CL labels to take advantage of the higher sensitivity of this detection principle. 

A Brief Practical Example for the Immobilization of Nucleic Acid Probes... 

Nucleation sites, in ferrosilicon, 22 516 Nucleation track membranes, 15 802 Nucleic acid bases, recognition of, 16 794 Nucleic Acid Database (NDB), 17 606 Nucleic acid probe assays, 16 380. See also DNA analysis Nucleic acid probes, 14 153 Nucleic acids, 17 602-643 20 444-447. See also Deoxyribonucleic acid (DNA) Ribonucleic acid (RNA)... 

Didenko, V.V. and Hornsby, P.J. (1996) A quantitative luminescence assay for nonradioactive nucleic acid probes. Journal of the Histochemistry Society, 44 (6), 657-660. 

Raymond FR, Ho H-A, Peytavi R, Bissonnette L, Boissinot M, Picard FJ, Leclerc M, Bergeron MG (2005) Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support. BMC Biotechnol 5 10... 

Gaylord BS, Massie MR, Feinstein SC, Bazan GC (2005) SNP detection using peptide nucleic acid probes and conjugated polymers applications in neurodegenerative disease identification. Proc Natl Acad Sci USA 102 34—39... 

Gaylord BS, Heeger AJ, Bazan GC (2002) DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes. Proc Natl Acad Sci USA 99 10954—10957... 

Sutherland G, Mulley J, Symons RH (1989) In nucleic acid probes. CRC, Gainesville, EL... 

Receptor autoradiography and immunohistochemistry are used to demonstrate the final location of receptor proteins. In the nervous system, these receptor proteins are synthesized within soma, but are generally transported to dendrites or axons, distant from cell bodies where they are originated. Thus, demonstration of a receptor population by receptor autoradiography or immunohistochemistry is not sufficient to directly discriminate which perikarya synthesize the receptors (Kuhar et al, 1986 Quirion et al., 1993 Chabot et al., 1999). Knowledge of the sequence of the mRNA coding receptor proteins has made the development of nucleic acid probes possible, which can be used in combination with the... 

Nucleic acid-based technologies Nucleic acid probe Polymerase chain reaction, DNA amplication 16S rRNA sequencing techniques automated riboprinting... 

In situ hybridization (ISH) permits the detection and iocaiization of DNA and RNA in a cytoiogicai preparation affixed to a microscope siide. Such detection and iocaiization is made possibie by hybridization of cellular DNA and/or RNA targets with nucleic acid probes tagged with a signal generating system such as a fluorochrome or enzyme. 

Cellular autoradiography techniques using radioactive nucleic acid probes have several features in common with nucleic acid immunocytochemistry. The method is based on the hybridization of radioactive probes to cellular targets and the subsequent exposure of photographic emulsion, which, when developed, reveals blackened (exposed) silver grains close to the site of hybridiza-hon. Hence, cellular autoradiography techniques permit excellent specihcity and localizahon of the hybridized probe—to 1 qm when tritium is the label used in the autoradiography-based method (9). 

Hockfield, S., Carlson, S., Evans, C., Levitt, P., Pintar, J., Silberstein, L. (1994) Combining immunocytochemisti y with in situ hybridization, in Selected Methods for Antibody and Nucleic Acid Probes. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 261,262. 

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