Viscidi R P, Connelly C J, Yolken R H
J Clin Microbiol. 1986 Feb;23(2):311-7. doi: 10.1128/jcm.23.2.311-317.1986.
A novel chemical method was used to prepare biotin-labeled nucleic acids for nonisotopic hybridization. The method involves the transamination of unpaired cytosine residues in polynucleotides with sodium bisulfite and ethylenediamine. Primary amino groups on the cytosine derivatives are then reacted with biotinyl-e-aminocaproic acid N-hydroxysuccinimide ester. Biotinylated probes hybridized with 1 to 2 pg of nitrocellulose filter-bound DNA and were visualized with a colorimetric detection technique. This method is simpler and less expensive than other methods for the preparation of nonisotopic probes. In addition, it is more versatile since the chemically modified bases can potentially react with other "indicator" molecules or proteins such as an enzyme. The specificity for unpaired cytosine residues is another advantage which could allow for the selective labeling of a specific region of a double-stranded nucleic acid. This improved labeling method should lead to the wider application of hybridization techniques in diagnostic microbiology and basic research in infectious diseases.
一种新的化学方法被用于制备用于非同位素杂交的生物素标记核酸。该方法涉及用亚硫酸氢钠和乙二胺对多核苷酸中未配对的胞嘧啶残基进行转氨作用。然后使胞嘧啶衍生物上的伯氨基与生物素化的ε-氨基己酸N-羟基琥珀酰亚胺酯反应。生物素化探针与1至2皮克硝酸纤维素滤膜结合的DNA杂交，并用比色检测技术进行可视化。该方法比其他制备非同位素探针的方法更简单、成本更低。此外，它更具通用性，因为化学修饰的碱基可能与其他“指示剂”分子或蛋白质（如酶）发生反应。对未配对胞嘧啶残基的特异性是另一个优点，这可以允许对双链核酸的特定区域进行选择性标记。这种改进的标记方法应会导致杂交技术在诊断微生物学和传染病基础研究中得到更广泛的应用。
