Oligonucleotide probe

Homology screening. Using oligonucleotide probes based on known receptor sequences, search cDNA libraries for homologous sequences which may code for related receptors. The clones are then isolated and sequenced and used in expression studies to confirm the identity of the receptor. 

With the use of oligonucleotide probes based on the amino acid sequences of these protease V8-obtained peptides and of cyanogen bromide fragments of the porcine H,K-ATPase P subunit, cDNA clones for the rat [12,25] and rabbit [74] H,K-ATPase P subunit were then isolated. 

Saiki RK, Walsh PS, Levbnson CH., Erlich HA (1989) Genetic analysis of amplified DNA with immobilized sequence specific oligonucleotide probes. Proc. Natl Acad Sd USA 86 6230-6234. 

R. E. Hicks, R. I. Amann, and D. A. Stahl, Dual staining of natural bacterioplankton with 4, 6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences, Appl. Environ. Microbiol. 571 2158 (1992). 

R. 1. Amann, L. Krumholz, and D. A. Stahl, Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology, J. Bacteriol. 772 762 (1990). 

Lebaron, P. Catala, P. Fajon, C. Joux, F. Baudart, J. Bernard, L. A new sensitive, whole-cell hybridization technique for detection of bacteria involving a biotinylated oligonucleotide probe targeting rRNA and tyramide signal amplification. Appl. Environ. Microbiol. 1997, 63, 3274-3278. 

Manz, W. In situ analysis of microbial biofilms by rRNA-targeted oligonucleotide probing. Methods Enzymol. 1999,310,79-91. 

Manz, W. Szewzyk, U. Ericsson, P. Amann, R. Schleifer, K. H. Stenstrom, T. A. In situ identification of bacteria in drinking water and adjoining biofilms by hybridization with 16S and 23S rRNA-directed fluorescent oligonucleotide probes. Appl. Environ. Microbiol. 1993,59, 2293-2298. 

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. 

Regnault, B. Martin-Delautre, S. Lejay-Collin, M. Lefevre, M. Grimont, P. A. D. Oligonucleotide probe for the visualization of Escherichia coli Escherichia fergu-sonii cells by in situ hybridization Specificity and potential applications. Res. Microbiol. 2000,151,521-533. 

Lewis FD, Zhang Y, Letsinger RL (1997) Bispyrenyl excimer fluorescence a sensitive oligonucleotide probe. J Am Chem Soc 119 5451-5452... 

Hessner MJ, Budish MA, Friedman KD. Genotyping of factor V G1691A (Leiden) without the use of PCR by invasive cleavage of oligonucleotide probes [In Process Citation]. Clin Chem 2000 46(8 Pt 1) 1051—1056. 

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... 

The power and advantages of assessing cellular processes at their most fundamental level is propelling the science of oligonucleotide probe detection into one of the most prominent positions in bioconjugate chemistry. Oligonucleotide arrays containing hundreds or thousands of... 

Figure 1.57 Base-pairing can occur between complementary bases in opposing oligonucleotide strands. These predictable interactions form the basis for using synthetic oligonucleotide probes to target particular DNA sequences.

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. 

---