Riley L K, Marshall M E, Coleman M S
DNA. 1986 Aug;5(4):333-7. doi: 10.1089/dna.1986.5.333.
A new method is described for biotinylation of oligonucleotide probes for use in molecular hybridization reactions. Biotin-11-dUTP residues were added enzymatically, using terminal deoxynucleotidyl transferase, to the 3' terminus of a synthetic oligonucleotide prepared from the known nucleotide sequence for adenosine deaminase. The biotinylated probe was hybridized to DNA and mRNA selectively immobilized on nitrocellulose and detected by sequential incubation of the nitrocellulose membrane with avidin and biotinylated polyalkaline phosphatase, followed by colorimetric development. The biotinylated oligonucleotide probe proved useful for the qualitative detection of complementary DNA and mRNA sequences but was unsatisfactory for quantitative determinations using reflective densitometry.
描述了一种用于寡核苷酸探针生物素化的新方法，该探针用于分子杂交反应。使用末端脱氧核苷酸转移酶，将生物素-11-dUTP残基酶促添加到由腺苷脱氨酶的已知核苷酸序列制备的合成寡核苷酸的3'末端。将生物素化探针与选择性固定在硝酸纤维素上的DNA和mRNA杂交，并通过硝酸纤维素膜与抗生物素蛋白和生物素化多碱性磷酸酶的顺序孵育，然后进行比色显影来检测。生物素化寡核苷酸探针被证明可用于互补DNA和mRNA序列的定性检测，但对于使用反射光密度测定法进行定量测定并不令人满意。
