Ellwood M S, Collins M, Fritsch E F, Williams J I, Diamond S E, Brewen J G
Clin Chem. 1986 Sep;32(9):1631-6.
This novel method for the detection of specific nucleic acid sequences has potential applications to clinical diagnosis. During hybridization, a signal-bearing nucleic acid strand is displaced by the target nucleic acid from a partially single-stranded complementary probe strand of nucleic acid. The probe:signal strand complex is prepared by hybridizing single-stranded probe that is entirely complementary to the target nucleic acid with a shorter signal sequence that is complementary to a portion of the probe strand. The sample nucleic acid is added to this hybrid complex under hybridization conditions. The target sequence, if present in the sample, will hybridize first to the unoccupied probe sequences, and then will displace the labeled strand by branch migration. By this "strand displacement" the signal strands are freed in solution, where they may be separated from those still hybridized; the quantity of label measured is directly proportional to the amount of analyte sequences in the sample. This method, demonstrated here for model and synthetic DNAs, can easily be adapted for the detection of any RNA or DNA sequence and obviates the need for immobilization of sample. A wide variety of labeling techniques can be used, and the displacement can be performed in solution or with the hybrid complex attached to a solid support. This assay circumvents nonspecific binding of label to the filter matrix and the laborious washing steps inherent in other assays involving nucleic acid probes.
这种检测特定核酸序列的新方法在临床诊断方面具有潜在应用价值。在杂交过程中，携带信号的核酸链被靶核酸从核酸的部分单链互补探针链上置换下来。探针：信号链复合物是通过将与靶核酸完全互补的单链探针与与探针链一部分互补的较短信号序列杂交而制备的。在杂交条件下，将样品核酸加入到这种杂交复合物中。如果样品中存在靶序列，它将首先与未占据的探针序列杂交，然后通过分支迁移置换标记链。通过这种“链置换”，信号链在溶液中被释放出来，在那里它们可以与仍杂交的链分离；测量的标记量与样品中分析物序列的量直接成正比。这里针对模型DNA和合成DNA展示的这种方法，可以很容易地适用于检测任何RNA或DNA序列，并且无需固定样品。可以使用多种标记技术，并且置换可以在溶液中进行，或者使杂交复合物附着在固体支持物上。这种检测方法避免了标记物与滤膜基质的非特异性结合以及其他涉及核酸探针的检测中固有的繁琐洗涤步骤。
