Biotinylated nucleic acid

While enzymes may be covalently attached directly to primary probe molecules, as noted above for reasons of reagent versatility, steric factors, and potential signal amplification, indirect detection systems appear to be the more popular. Consequently, enzyme-probe conjugates are typically complexes of a desired enzyme marker and a secondary level probe that is, a probe molecule that can specifically identify a primary level probe molecule, such as an alkaline phosphatase-streptavidin conjugate can identify a biotinylated nucleic acid probe by virtue of the binding affinity between streptavidin and biotin. Other examples of enzyme-probe systems are given in the preceding section on direct and indirect detection systems. 

Nucleic acids have been linked to a variety of surfaces including polystyrene beads, glass, silicon, gold, and even cells. Immobilization of nucleic acids may occur through a number of covalent linkages that are the subject of other chapters. Strept(avidin) may then be bound to a surface through a biotinylated nucleic acid linker. Alternatively, strept(avidin) may be linked to a surface directly with methods used for other proteins. This chapter will describe the biotin-strept(avidin) system and focus on the use of the biotin-strept(avidin) to link nucleic acids to surfaces. 

Elegant and efficient subtractive hybridization procedures have been developed from the original procedure described by Sive and St John (1988) and Duguid et al. (1988) in which driver cDNA is photobi-otinylated, allowed to react with the (-I-) DNA and the hybrids then eliminated with streptavidin agarose, Schweinfest et al. (1990) used the lambda ZAP II phage vector system to produce by in vivo excision a large amount of DNA for biotinylation. Recent methods take advantage of the property of biotinylated nucleic acids... 

Visualize the bands by placing the gel on an intensifying screen and shadowing with a UV light source. The fluoresceinated bands can be observed by shadowing at 320 nm. The biotinylated nucleic acid bands can be visualized by shadowing at 260 nm see Note 12). 

Streptavidin-single-stranded DNA covalent conjugates were described as the building blocks for assembling nanostructured scaffolds [31], The amount and type of biotinylated ligands were used to modulate the affinity of duplex formation between solid-phase-bound nucleic acid templates and DNA-streptavidin conjugates. This system has been proposed for the design of fine-tuned sequence detection systems. 

Psoralen, or derivatives of 9-methoxy-7H-furo[3,2-g]chromen-7-one tricyclic ring structures, are used as photoreactive groups in crosslinkers, biotinylation compounds, and nucleic acid probes. Psoralens have been used for many years as photochemotherapy agents for treatment of psoriasis and vitiligo (Smith and Barker, 2006). Psoralens react when exposed to UV light... 

Another popular tag for use with immunoglobulins is biotin. Modification reagents that can add a functional biotin group to proteins, nucleic acids, and other molecules now come in many shapes and reactivities (Chapter 11). Depending on the reactive group present on the biotinylation... 

Didenko, V.V. (1993) Biotinylation of DNA on membrane supports A procedure for preparation and easy control of labeling of nonradioactive single-stranded nucleic acid probes. Anal. Biochem. 213, 75-78. 

If cellular localization of the antigen-antibody complex is not required, enzyme immunolabeling can be performed on cells adherent to a microtiter plate, and the color change resulting from the enzymatic reaction can be detected as a change in absorbance with an automatic plate reader (see Chapter 28). Biotinylation of antibodies and the use of the avidin-biotin complex has further extended the versatility and sensitivity of the enzymatic techniques (see Chapters 7 and 25-27). Most recently, the principles behind these techniques have been applied in combination with in situ hybridization techniques. Using nucleic acid-antibody complexes as probes, specific DNA or RNA sequences can be localized (see Chapters 46 9). 

Not only biotinylated proteins, but also nucleic acids or peptides synthesized in the presence of biotin derivatives or carbohydrate-biotin conjugates, are separated by immobilized avidin and streptavidin, respectively, or are identified by avidin-enzyme or avidin-gold conjugates. 

The bottom-up synthesis of metallic nanowires was further applied to construct a nanotransistor device.93 The sequence-specific winding of the homologous nucleic acid carried by the RecA-protein into the duplex DNA was used to address the nucleic acid/protein complex on the DNA scaffold (Fig. 12.27). The subsequent association of the anti-RecA antibody to the protein DNA complex, followed by the association of the biotinylated antiantibody, and the linkage of streptavidin-modified carbon nanotube deposited the tubes in the specific domain of the DNA scaffold. The further... 

Chan, W. T.-W., Fleming, K. A., and McGee, J. O D. (1985) Detection of subpicogram quantities of specific DNA sequences on blot hybridization with biotinylated probes Nucleic Acids Res. 13,8083-8091... 

Fig. 2. Diagrammatic representation of three-step digoxigemn detection. The target nucleic acid (1) is labeled with digoxigemn (Dig.). Monoclonal antidigoxigenm (2) is linked via biotinylated (Bio) rabbit antimouse (3) to avidin alkaline phosphatase (4)...

Detection of the sites of hybridization-dependent binding of biotinylated probe to the filters is most readily conducted with commercially available kits. Favorable results have been obtained with the BluGene Nonradioactive Nucleic Acid Detection System from BRL. Follow the manufacturer s instructions when carrying out the following steps. After washing, sequentially expose the filters to streptavidin and biotinylated alkaline phosphatase (or to a conjugate of these two proteins) This causes the immobilization of alkaline phosphatase at sites of positive hybridization... 

Nucleic acids can also be biotinylated by nonenzymatic methods with photobiotin, a photoactivatable biotin analog (6), which can be commercially obtained from BRL, Sigma, and other commercial sources I have not compared the suitability of this method of biotin incorporation with that reported here, but expect that the method would be fully acceptable FMC (Rockland, ME) markets an alternate nonradioactive sequence detection kit known as Chemiprobe. The basis of this system is a chemical modification of cytosine residues m the probe DNA. After hybridization, the probe is detected by means of a monoclonal antibody that specifically recognizes the sulfonated DNA. Detection of the bound monoclonal antibody is achieved by means of an alkaline phosphatase-conjugated second antibody. 

Another important application of this interaction is in the formation of self-assembled bilayers on silver or gold surfaces. Ebersole et al. [457] showed that avidin and streptavidin molecules will adsorb onto clean layers of Au or Ag from an aqueous solution. Such layers can then be employed to capture biotinylated compounds. These authors used this technique to attach fragments of nucleic acid derived from the herpes virus to a solid support. A more popular approach to this general problem has, however, been to start by adsorption of a suitable biotin... 

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