Oligonucleotide Probe, biotin

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. 

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. 

Perhaps the most common method of DNA biotinylation is through enzymatic incorporation with the use of a biotin-labeled deoxynucleoside triphosphate. First reported by Langer et al. and Leary et al. in 1981, the procedure is probably the most popular nonradioactive labeling technique reported for oligonucleotide probes. Although biotinylated derivatives of dCTP and dATP are reported in the literature, by far the most frequently employed derivative is biotin—dUTP prepared from the reaction of an amine-modified dUTP with an amine-reactive biotinylation reagent, such as NHS-LC—biotin (Chapter 8, Section 3.1). 

Presently, nonradioactive probes, especially biotin or digoxigenin, are favored because they are less hazardous to work with, can be more rapidly developed, and provide better spatial resolution. Thus, introduction of nonradioactive detection systems has made ISH, using formalin-fixed and paraffin-embedded tissues, more accessible for application to molecular cell biology and diagnostic pathology. However, radioactive detection systems are more sensitive than nonradioactive probes, especially oligonucleotide probes used instead of cRNA probes (Sperry et al., 1996). 

Free-solution (gel-free) CE separation has been used in the detection of a target gene sequence amplified using the CPT reaction (see Chapter 9, section 9.2.1.7 for details). The intact chimeric oligonucleotide probe (fluorescein-labeled at the 5 end and biotin-labeled at the 3 end) was separated from a cleaved probe (only fluorescein-labeled at the 5 end). Owing to the presence of biotin in the intact probe, it migrated later than the cleaved probe, even in the absence of a gel sieving matrix [606]. 

Fig. 4. Schematic diagram of the steps in the automated PCR/OLA procedure performed with a robotic workstation. The assay contains three steps (1) DNA target amplification (2) analysis of target nucleotide sequences with biotin (B)-labeled and digoxigenin (D)-labeled oligonucleotide probes and T4 DNA ligase (L) and (3) capture of the biotin-labeled probes on streptavidin (SA)-coated microtiter wells and analysis for covalently linked digoxigenin by using an ELISA procedure with alkaline phosphatase (AP)-conjugated antidigoxigenin (aD) antibodies and a substrate (S). Reprinted with the permission of Nickerson el al. (N2) and the Proc. Natl. Acad. Sci. (U.SA.).

Wang JY, Montone KT. A rapid simple in situ hybridization method for herpes simplex virus employing a synthetic biotin-labeled oligonucleotide probe a comparison with immunohistochemical methods for HSV detection. J Clin Lab Anal. 1994 8 105-115. 

Fig. 43. General scheme for sandwich-type gene probe assays utilizing dioxetanes of the type shown in Fig. 41. Both alkaline phosphatase (Aik. Phos.) and biotin (B) are covalently attached to oligonucleotide probes. In the scheme shown on the right-hand side, the enzyme is covalently attached to streptavidin (SA), which is in turn captured by hybridizing to a biotinylated probe.

A hybridization-based approach has also been developed to quantitate levels of ribozymes in serum using paired complementary oligonucleotide probes, one labeled with biotin and the other with digoxigenin [90]. The annealed triplex is first collected on streptavidin-coated 96-well plates, then an anti-digoxigenin antibody conjugated with alkaline phosphatase is added, followed by addition of the enzyme substrate p-nitrophenyl phosphate, which is cleaved into a soluble colored product that is quantitated by absorbance at 405 nm. 

Moreover, the significance of the attachment of biotinylated oligonucleotide probes through the streptavidin/biotin interaction has been tested in a previous work [15]. When a double-labeled (biotin and fluorescein) poly-T was attached to the electrode surface through the streptavidin/biotin interaction, the peak currents were much higher than those obtained when it was accumulated on the electrode surface by physical adsorption. This fact means that streptavidin/biotin interaction allows to attach and orient the oligonucleotide strands on electrode surface, whereas the direct adsorption of the oligonucleotide on the electrode surface results... 

---