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[69] Preparation and uses of photobiotin

1990; Academic Press; Linguagem: Inglês

10.1016/0076-6879(90)84323-9

1557-7988

James McInnes, Anthony Forster, Derek C. Skingle, Robert H. Symons,

bioluminescence and chemiluminescence research

This chapter describes the synthesis of photobiotin, its use as a labeling reagent for the preparation of both DNA and RNA probes, and examples of their use. Photobiotin acetate consists of biotin attached by a charged linker arm to a photoreactive arylazide group. When a mixture of nucleic acid and photobiotin acetate is exposed to strong visible light for 15–20 minutes under defined conditions, the arylazide group is converted to an extremely reactive arylnitrene, which allows the formation of linkages to the nucleic acid. Photobiotin acetate, in addition to labeling RNA, labels single stranded DNA and linear or supercoiled double-stranded DNA. Because photobiotin acetate reacts with any organic material, it is essential that nucleic acids to be used as probes be highly purified and free from any contaminating DNA, RNA, proteins, agarose, or buffers, such as Tris. Inorganic salts may also inhibit the biotinylation of nucleic acids. A strong source of white light is required for the photoactivation reaction. A 250- to 500-W mercury vapor discharge lamp containing a tungsten filament with a built-in reflector to direct the output of light is the most suitable.