Probes labelling

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. 

Yano Y, Matsuzaki K (2009) Tag-probe labeling methods for live-cell imaging of membrane proteins. Biochim Biophys Acta-Biomembranes 1788 2124—2131... 

This section covers early indirect fiber optic chemical sensors (FOCS) for species that cannot be sensed directly but require the use of indicators, probes, labeled biomolecules, or color-forming reactions. 

Similar techniques can be used to devise (strept)avidin-biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind... 

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

Oser, A., Roth, W.K., and Valet, G. (1988) Sensitive non-radioactive dot-blot hybridization using DNA probes labeled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence. Nucleic Acids Res. 16, 1181-1196. 

Miller reported several monomeric, photolabile CCK agonists and antagonists. The photoreactive residue (L-Bpa) was placed at the N-terminal and a fluorescent reporter group was also linked to it. The CCK receptor in the study was expressed on Chinese hamster ovary-CCKR cells. To identify the labeled domains on the receptor capillary electrophoresis was used with laser induced fluorescence detection. Separate regions were labeled with the two photoprobes, one labeled the first extracellular loop (96-121), while the other probe labeled a fragment in the second extracellular loop (174-195). 

Armed with the common techniques of molecular biology and immu-nocytochemistry, an investigator is in a good position to apply in situ hybridization to EM for localization of nucleic acids at the ultrastructural level. McFadden (9) has a review on such use of in situ hybridization techniques. In the review, McFadden has included details of some of his laboratory protocols needed in in situ hybridization from fixation and labeling to probe labeling to the hybridization steps for localization of specific RNAs at the EM level. His protocol for hybridization is outlined below ... 

Traditionally, Northern blot analysis of isolated total RNA or mRNA has been used to determine differences in levels of mRNA between and among different animal groups, tissues or time-points. Standard protocols for performing Northern blot hybridization are available (Sambrook and Russell, 2001). Radioisotopic and colorimetric methods are available for probe labeling. The advantage of Northern blot analysis, especially... 

Probes can be differently labeled with hapten labels, for example carboxyfluorescein (6-FAM), digoxigenin (DIG) and biotin can be bound to LNA oligos. The choice of probe label depends on experimental design and the techniques available in the laboratory. The hapten label provides a template for crucial signal amplification since the FITC label on the oligo itself is not sufficient to allow detection in standard epifluorescence. In this study, the fluorescence signal was obtained with the TSA-FITC substrate, which allowed detection of miR-21 and miR-205. 

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.

The sensitivity of probes labeled by this method is not as high as for those labeled by enzymatic methods. Furthermore, photobiotinylated probes usually give higher nonspecific background owing to the cationic spacer arm of photobiotm, which nonspecifically binds to anionic molecules (e g., DNA and RNA). 

In situ hybridization may be defined as the detection of nucleic acids in situ in cells, tissues, chromosomes, and isolated cell organelles. The technique was described in 1969 by two separate groups who demonstrated repetitive riboso-mal sequences in nuclei of Xenopus oocytes using radiolabeled probes (1,2). Refinements in recombinant DNA technology and the development of nonisotopic probe labeling and detection (3) obviate the need for radiation protection and disposal facilities, and have converted nonisotopic in situ hybridization (NISH) from a purely research technique to one that can be used in routine laboratory testing. 

Multiple in situ nucleic acid detection can be achieved in two ways- by sequential hybridization with probes labeled with the same reporter and by simultaneous hybridization using probes labeled with different reporters. Any of the reporters listed above can be combined to allow dual nucleic acid detection, but we have found biotm and digoxigenin to be the most generally useful, since they are safe and sensitive Probes labeled with these reporters can be combined m NISH and detected differentially according to the scheme presented m Fig. 1 (6) 2. Pretreatment of cells/tissues Having chosen the reporter and incorporated it into a probe, the cell/tissue to be analyzed is prepared for hybridization The following requirements must be met to allow a successful hybridization reaction to take place. 

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).

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