Oligonucleotide probe labeling

Fuchs, B. M. Wallner, G. Beisker, W. Schwippl, I. Ludwig, W. Amann, R. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 1998, 64, 4973 1982. 

Oligonucleotide probes were labeled at their 3 end using terminal transferase and digoxigenin-11 -UTP, according to the manufacturer s recommendations (Roche Pharmaceuticals). Ten ml of cells are pre-fixed in 3.7% formaldehyde for 15 min at room temperature. Five ml of cells are harvested and resuspended in 6 ml of 4% paraformaldehyde, 0.1 MKPO4 (pH 6.5), and 5 mM MgCb. After 3 h, the cells are washed twice in solution B (1.2 M sorbitol, 0.1 M KPO4 (pH 6.5)). The cells are then resuspended in 2.8 ml... 

Cyanine dyes also are used as labels for oligonucleotide probes. Unlike the hydrophilic cyanine dyes valuable for protein labeling, the use of dye-phosphoramidite compounds to synthesize DNA or RNA probes typically requires the use of more hydrophobic dye structures to make them compatible with the solvents and reactions of oligonucleotide synthesis. Thus, indol cyanines containing few or no sulfonates are used in these applications to label oligos for applications such as array detection, hybridization assays, and RT-PCR. 

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays better than HRP, AP often is the enzyme of choice for labeling oligonucleotide probes. AP also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody-enzyme conjugates (behind HRP), being used in almost 20 percent of all commercial enzyme-linked assays. 

To modify the unique chemical groups on nucleic acids, novel methods have been developed that allow derivatization through discrete sites on the available bases, sugars, or phosphate groups (see Chapter 1, Section 3 for a discussion of RNA and DNA structure). These chemical methods can be used to add a functional group or a label to an individual nucleotide or to one or more sites in oligonucleotide probes or full-sized DNA or RNA polymers. 

When photobiotin is irradiated in the presence of DNA the reaction process nonselectively couples a biotin label to every 100-200 base residues. The result is an oligonucleotide probe detectable by the use of (strept)avidin conjugates. The uses of photobiotin for DNA or RNA modification are summarized in Chapter 11, Section 4. 

Oligonucleotide probes may be labeled with small fluorescent molecules for detection of hybridization by luminescence. Fluorescent probes are widely used in assay systems involving bio-specific interactions (Chapter 9). Receptors for ligands may be localized in tissues or cells by modification of the ligand with the appropriate fluorophore. Targeted molecules may be... 

Kumar, A., Tchen, P., Roullet, F., and Cohen, J. (1988) Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase. Anal. Biochem. 169, 376-382. 

FIGURE 14.1 Scanometric DNA array detection with enlarged nanoparticle probes. Use of oligonucleotide targets, labeled with gold nanoparticles, for recognizing DNA segments on a chip (reproduced from [13] with permission). 

Assay for human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood monuclear cells can be performed by PCR followed by detection of PCR products by electrochemiluminescence-labeled oligonucleotide probe [Tris-bipyridine ruthenium (II) complex]. Since one of the PCR primers is biotin-labeled at the 5 end, facile capture of the PCR product-probe complex can be accomplished on streptavidin-conjugated magnetic particles, prior to analysis in an electrochemiluminescence analyzer (S3). 

Tata, AM. 2001. An in situ hybridization protocol to detect rare mRNA expressed in neural tissue using biotin-labelled oligonucleotide probes. Brain Res Prot 6 178-184. 

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