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

提出了一种新型的磷酰胺试剂，用于在自动固体合成过程中将金簇配合物和组合荧光/金标记物结合到寡核苷酸中。该标签将被合成修饰以承受合成和脱保护条件。金和荧光/金共轭磷酰胺将以特定的形式制备，用于标记寡核苷酸的5‘端，在3’端通过受控孔玻璃载体衍生化，以及在任何中间位置；还提出了在2'位置标记的腺苷磷酸酯的合成。这些标签将被纳入寡核苷酸探针，将在杂交研究中进行测试，并在原位电子显微镜杂交研究。将评估标记的核苷酸的灵敏度、穿透性和分辨率，相对于用带有相同标记的二次探针检测的类似生物素化探针。预计包含新标签的合成探针将在电子显微镜下达到前所未有的分辨率，并且印迹灵敏度等于或优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.