Gulilat Gebeyehu

[66] Biotinylated nucleotides for labeling and detecting DNA

Publisher Summary The sensitivity, selectivity, and ease of use of nucleic acid probes and the availability of efficient protocols for the isolation and cloning of specific DNA sequences have led to the development of a wide selection of probes for biomedical and clinical applications. In the basic hybridization protocol, a labeled nucleic acid probe is annealed to a complementary DNA or RNA target sequence, which either is in solution or is immobilized on an inert support. The labeled nucleic acid probe is used to determine the presence or absence of the target sequence in the reaction mixture. An effective nonradioactive detection system should recognize the annealed nucleic acid probe with a degree of precision comparable to that obtained in the primary hybridization reaction. The presence of either a free amine or biotin may also be ascertained by chemical analysis. Compounds containing primary amines give positive color development when spotted on thin layer chromatography (TLC) plates and sprayed with ninhydrin. The first approach uses iminobiotin, an analog of biotin in which the ureido group has been replaced with a guanido group. Iminobiotin binds to avidin with a binding constant that increases with increasing pH and nonprotonated iminobiotin binds efficiently to avidin.

DNA isolation using methidium-spermine-sepharose.

Publisher Summary This chapter describes protocols for isolating DNA from a variety of complex samples using a DNA capture reagent consisting of the intercalator, methidium, attached to a Sepharose bead by a spermine linker. In designing novel reagents for rapid isolation of DNA from complex samples, one reasoned that a very efficient “capture reagent” would consist of an intercalating dye attached to a solid support by a molecular linker. The intercalator binds the DNA with high affinity and the solid support allows rapid separation of the bound material from unwanted contaminants. The second important feature of the capture reagent is its versatility. It can be used to isolate DNA from many different biological samples. Capture is independent of the salt concentration of the sample and occurs in the presence of detergents, proteinase K, and EDTA, which are often used in protocols for the isolation of viral and cellular DNAs from complex samples. Very small amounts of DNA can be isolated from large volumes of sample and analyzed using a sensitive assay, such as hybridization or the polymerase chain reaction. These features should facilitate analysis of DNA from pathogens or other DNAs of interest in complex biological samples.