Carpenter W R, Schutzbank T E, Tevere V J, Tocyloski K R, Dattagupta N, Yeung K K
Miles Inc., Diagnostics Division, Tarrytown, NY 10591.
Clin Chem. 1993 Sep;39(9):1934-8.
Transcriptionally amplified DNA probes are valuable tools in the development of sensitive nucleic acid-based diagnostic assays. Here we describe a model assay using a novel oligonucleotide hairpin probe that encodes a T7 RNA polymerase promoter. The hairpin probe and an adjacently hybridizing biotinylated capture probe were hybridized to target DNA and the duplex was captured onto streptavidin-coated magnetic particles. After ligation of the immobilized probes, which served to maintain specificity, the hairpin probe was transcribed by T7 RNA polymerase. The amplified RNA product was hybridized to the capture probe and bound to the streptavidin-coated magnetic particles. The immobilized heteroduplex was detected with an antibody-alkaline phosphatase conjugate specific for DNA:RNA hybrids, and the chemiluminescent substrate adamantyl-1,2-dioxetane phenyl phosphate. Ten attomoles of target DNA could be detected in a background of 5 micrograms of unrelated DNA. The chemiluminescent immunoassay was as sensitive as radioactive detection of specific product after gel electrophoresis.
转录扩增DNA探针是基于核酸的灵敏诊断检测方法开发中的重要工具。在此，我们描述了一种使用新型寡核苷酸发夹探针的模型检测方法，该探针编码T7 RNA聚合酶启动子。将发夹探针和相邻杂交的生物素化捕获探针与靶DNA杂交，然后将双链体捕获到链霉亲和素包被的磁性颗粒上。在连接固定化探针（用于保持特异性）后，发夹探针被T7 RNA聚合酶转录。扩增的RNA产物与捕获探针杂交，并结合到链霉亲和素包被的磁性颗粒上。使用对DNA:RNA杂交体特异的抗体-碱性磷酸酶偶联物和化学发光底物金刚烷基-1,2-二氧杂环丁烷苯基磷酸检测固定化的异源双链体。在5微克无关DNA的背景下可检测到10个阿托摩尔的靶DNA。化学发光免疫测定与凝胶电泳后特异性产物的放射性检测一样灵敏。
