| 摘要 |
This invention describes methods for the generation of nucleic acid probes that improve the sensitivity of hybridization assays. The sensitivity increase results from structural modifications of nucleic acids that promote network formation during hybridization with the result that a single target molecule becomes attached to a complex of many probe molecules. The structural modification involves fragmentation of the probe nucleic acid followed by joining the fragments together such that their order and orientation and number is altered from the original probe molecule. The result is the generation of permuted probe libraries. Various fragmentation and joining methods are described. Labeling can be done by standard methods before during or after formation of permuted probe libraries. Individual members of permuted probe libraries can be isolated and amplified and perpetuated. Fixed mixtures of such isolated library members can be used as permuted probe libraries of limited variability to control network complexity. Libraries can be prepared with additional sequences not present in the target and the fraction of the library made up by such sequences can be controlled. Such extra-target additions can be used as targets for detection by secondary probes. Since their number can be far greater than equimolar with the probe sequences represented in the target they can be detected with higher sensitivity in the networks. Probes for different targets can incorporate different non-target sequences in hyper-molar quantities permitting sensitive detection of multiple hybridization targets in the same sample. Probes made according to this invention can be used in many kinds of hybridization assay including Southern. blots, Northern blots, Dot blots, Nucleic acid Array hybridization, ‘in situ’ hybridization with fluorescent or other labels (FISH) and various kinds of sandwich hybridization assays.
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