Nonradioactive hybridization probes prepared by the reaction of biotin hydrazide with DNA

A novel one-step chemical method has been developed for the introduction of biotin into nucleic acids for non-isotopic hybridization. The method is based on the interaction of biotin hydrazide with unpaired cytosine residues. The interaction is catalyzed by sodium bisulfite with an optimum at a buffered pH of about 4.5. The reaction reached its maximum after 24 h incubation at a biotin hydrazide concentration of 10 mg/ml. Using streptavidin-alkaline phosphatase conjugates, the limits for detecting the biotinylated probe, either adsorbed directly to nitrocellulose or hybridized to filter-bound target DNA, were 0.3 and 0.9 pg, respectively. The salience of the approach described here over previously used biotin derivatives is that it is quick (one-step), simple and does not involve any enzymatic or instrument-mediated step to introduce the reporter moiety. In addition, other low- and high-molecular-weight hydrazides (e.g. fluorescent or enzyme hydrazides) can serve as the reporter group. The same procedure may be employed for the single-step biotinylation of free cytidine.