Gold and Combined Fluorescent/Gold Labels for Solid-Phase DNA Synthesis

提出了一种新的亚磷酰胺试剂，用于在自动固相合成过程中将金簇合物和结合的荧光/金标记引入到寡核苷酸中。标签将被合成修饰，以经受合成和脱保护条件。通过可控孔玻璃载体的衍生化，在寡核苷酸的5‘端、3’端和任何中间位置制备金和荧光/金标记的亚磷酰胺；还提出了合成标记在2‘位置的腺苷亚磷酰胺。这些标记将被合并到寡核苷酸探针中，这些探针将在斑点杂交研究和原位电子显微镜杂交研究中进行测试。标记的核苷酸的灵敏度、穿透性和分辨率将相对于带有相同标记的二级探针检测到的类似生物素化探针进行评估。预计包含新标记的合成探针将在电子显微镜中获得前所未有的分辨率，并且杂交灵敏度将等于或高于当前的放射性同位素或化学发光技术。

Novel phosphoramidite reagents are proposed which will be used to incorporate gold cluster complexes and combined fluorescent/gold labels into oligonucleotides during automated solid state synthesis. The labels will be synthetically modified to withstand the synthesis and deprotection conditions. Gold and fluorescent/gold conjugated phosphoramidites will be prepared in forms specific for labeling at the 5' end of the oligonucleotide, at the 3' end via derivatization of a controlled pore glass support, and at any intermediate position; the synthesis of an adenosine phosphoramidite labeled in the 2' position is also proposed. The labels will be incorporated into oligonucleotide probes which will be tested in hybridization studies on blots, and in situ electron microscopy hybridization studies. The sensitivity, penetration and resolution of the labeled nucleotides will be evaluated relative to analogous biotinylated probes detected by a secondary probe bearing the same label. It is anticipated that synthetic probes which incorporate the new labels will achieve unprecedented resolution in the electron microscope, and blot sensitivities equal to or better than current radioisotopic or chemiluminescent techniques.