HYBRIDIZATION WITH NUCLEIC ACID PROBES labeling

Fig. 1. Dual nucleic acid detection. The target DNAs (A and B) are denatured and hybridized with complementary DNA probes labeled with biotin (Bio ), or digoxigenm (Dig ). Biotin and digoxigenm residues are detected, respectively, with avidin peroxidase (red) and antibody to digoxigenin labeled with alkaline phosphatase (blue).

Schmitt-John, T Palmisano, R. Plessow, R. Brockhinke, A. Weidner, J. Detection of a nucleic acid by hybridization with pairs of probes labeled with dyes that interact by FRET. Ger. Offen. DE 10133308, 2003 Chem. Abstr. 2003, 138, 118424. 

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...

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. 

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. 

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). 

DNA arrays have been categorized into different formats based upon what is immobilized to the surface (also known as the solid phase, substrate, or chip) and what is captured from the sample solution. Definitions change depending upon the format. For the classic Southern dot blot, the sample was first spotted down on the surface, cross-linked, and then bathed with a radio-labeled oligonucleotide under hybridization (complementary nucleic acid strand base-pairing) conditions to detect the presence of a parhcular sequence within the sample. This was called probing. The oligonucleohde... 

A useful approach to monitor microbial populations in the biotreatment of hazardous wastes involves the detection of specific sequences of nucleic acids by hybridization with complementary oligonucleotide probes. Radioactive labels, fluorescent labels, and other kinds of labels are attached to the probes to increase sensitivity and simplicity of the hybridization... 

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.

For in situ hybridization, a tissue sample is incubated with a labeled nucleic acid probe, excess probe is washed away and the location of hybridized probe is examined. The technique enables the spatial localization of gene expression to be determined as well as the location of individual genes on chromosomes. 

Sandwich hybridization, using affinity-based hybrid collection, is based on two nonoverlapping nucleic acid probes (one is labeled, the other can be collected by the affinity matrix) (Syvanen et al., 1986 Jalava et al., 1990). The principles are shown in Fig. 8.3. Target nucleic acid thus mediates binding of labeled probe to the matrix. The detectability is about 10 molecules with a linear range to at least 10 molecules with radioisotopes as labels. In contrast to capture hybridization assays, the immobilization of the complex is at 22-37°C (leaching is then usually less important). 

In another approach, the colony hybridization technique (Figure 18D, bacteria are screened by using a radioactively labeled nucleic acid probe, an RNA molecule or a single-stranded DNA molecule with a sequence complementary to that of a specific sequence within the recombinant DNA. Bacterial cells are plated out onto solid media in petri dishes and allowed to grow into colonies. Each plate is then blotted with a nitrocellulose filter. (Most of the original colonies remain on the petri dishes.) The cells on the nitrocellulose filter are lysed, and the released DNA is treated so that hybridization with the probe can occur. Once nonhybridized probe molecules have been washed away, autoradiography (Biochemical Methods 2.1) is used to identify the colonies on the master plate that possess the recombinant DNA. 

To detect a nucleic acid of a specific sequence, a nucleic-acid probe having a sequence complementary to the sequence of the sought-after nucleic acid is prepared. The probe may be either radioactive or chemically tagged with a fluorescent or chemiluminescent label. The blot is incubated with the probe under conditions that allow hybridization so that the probe reassociates with its complementary sequence on the blot and becomes bound to the blot. After sufficient time for reassociation has elapsed, the unbound probe is washed away and the presence and location of the hybridized probe can be determined from its radioactivity or other tag, indicating the presence of the complementary sequence on the original blot (Fig. 2.15). 

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