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

提出了新型亚磷酰胺试剂，其将用于在自动化固态合成期间将金簇复合物和组合的荧光/金标记物并入寡核苷酸中。将对标记进行合成修饰以耐受合成和脱保护条件。金和荧光/金偶联的亚磷酰胺将以特异性标记寡核苷酸5'端、通过可控孔径玻璃载体衍生化标记3'端和任何中间位置的形式制备;还拟定了2'位标记的腺苷亚磷酰胺的合成。将标记掺入寡核苷酸探针中，在印迹杂交研究和原位电子显微镜杂交研究中进行检测。将相对于通过带有相同标记的二级探针检测的类似生物素化探针，评价标记核苷酸的灵敏度、穿透性和分离度。据预计，合成探针，纳入新的标签将达到前所未有的分辨率，在电子显微镜和印迹灵敏度等于或优于目前的放射性同位素或荧光技术。

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.