Reisfeld A, Rothenberg J M, Bayer E A, Wilchek M
Biochem Biophys Res Commun. 1987 Jan 30;142(2):519-26. doi: 10.1016/0006-291x(87)90305-6.
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.
已开发出一种新颖的一步化学方法，用于将生物素引入核酸以进行非同位素杂交。该方法基于生物素酰肼与未配对胞嘧啶残基的相互作用。这种相互作用由亚硫酸氢钠催化，在缓冲pH约为4.5时达到最佳效果。在生物素酰肼浓度为10 mg/ml的情况下孵育24小时后，反应达到最大值。使用链霉亲和素-碱性磷酸酶偶联物，检测直接吸附在硝酸纤维素上或与滤膜结合的靶DNA杂交的生物素化探针的极限分别为0.3和0.9 pg。此处所述方法相对于先前使用的生物素衍生物的显著之处在于它快速（一步法）、简单，并且在引入报告基团时不涉及任何酶促或仪器介导的步骤。此外，其他低分子量和高分子量的酰肼（例如荧光或酶酰肼）可作为报告基团。相同的程序可用于游离胞苷的单步生物素化。
