Nonisotopic DNA Probe Techniques by Larry J. Kricka

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By Larry J. Kricka

Lately many nonisotopic tools of probing particular DNA sequences were built as replacements for radioactive labels, resembling 32phosphorous and 125iodine. This ebook brings all of those new equipment jointly in a single handy, simply available resource. It allows researchers to choose the nonisotopic approach most suitable to their software and to take advantage of it to greatest virtue by way of following the straightforward Read more...

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DNA/RNA hybrids (Van Prooijen-Knegt et al, 1982; Stollar and Rashtchian, 1987; Rashtchian et al, 1987;Coutlee et al, 1989a; 1989b; 1989c)]. Among the indirect systems, only the biotin and digoxigenin systems are characterized by subpicogram sensitivities; all other systems are of less sensitivity. Even though the biotin system is characterized by high sensitivity and a wide range of applications, the disadvantage of this system is that an endogenous ubiquitous vitamin, vitamin H, is used as modification group (Lardy and Peanasky, 1953; Chaiet and Wolf, 1964; Greene, 1975).

The digoxigenin system, characterized by analogous sensitivity, circumvents these background side-reactions by using the cardenolide digoxigenin occurring only in Digitalis plants (Pataki et al, 1953; Reichstein and Weiss, 1962; Hegnauer, 1971). Besides various methods for linking fluorescent dyes of marker enzymes directly to nucleic acid probes, a broad repertoire of enzymatic, photochemical, and chemical labeling methods can be applied for introduction of modification groups into nucleic acid probes (Table III).

Noncovalent Labeling with Ethidium Bromide This labeling system is based on the observation that the lanthanide cation terbium quenches the fluorescence of ethidium bromide by 40-fold when bound to double-stranded RNA (Al-Hakeem and Sommer, 1987). 5-fold, and quenching of single-stranded RNA is less than 5-fold. This selective quenching permits specific detection of double-stranded RNA in yeast or some viruses. Terbium ions exhibit a low level of intrinsic fluorescence in aqueous solution. This is greatly enhanced when it is chelated to organic ligands, such as the aromatic ring systems of the nucleic acid bases, to form charge transfer complexes (Gross and Simpkins, 1981).

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