"Another technique for amplifying a signal from a hybridization event is the branched DNA (bDNA) approach, in which a pre-amplifier strand hybridizes to a portion of the probe, which in turn serves as a nucleation site for the hybridization of a fixed number of multiply-labeled amplifier strands. (Schweitzer et al. Curr Opin Biotechnol, 12:21-27, 2001; Qian et al. Diagnostic Molecular Pathology, 12(1):1-13, 2003; Collinset al. Nucleic Acids Res, 25(15):2979-2984, 1997; Bushnell, et al.
In addition to obtaining background information on this patent, NewsRx editors also obtained the inventors' summary information for this patent: "Methods and compositions for detecting one or more analytes within a biological sample (in situ) using HCR are provided. The advantages of HCR for in situ imaging include, without limitation, the ability to rapidly amplify a signal based on a small amount of analyte present and the ability to image a diversity of analytes in the same sample.
"In one aspect of the invention, methods are provided for detecting an analyte in a biological sample. Preferably, the sample is contacted with a probe comprising a target region and an initiation region. The target region is able to specifically bind to the analyte of interest, while the initiation region is able to initiate the polymerization of labeled nucleic acid monomers. Thus, the sample is contacted with a first metastable monomer comprising an initiator region that is complementary to the initiation region of the probe and a second metastable monomer comprising a region complementary to a portion of the first monomer. One or both of the monomers is preferably labeled with a fluorescent dye. They may also be labeled with a fluorescence quencher such that prior to polymerization the fluorescence is quenched. A fluorescent signal is thus generated upon formation of a polymer and background is reduced.
"The analyte to be detected is not limited in any way and may be, for example, a nucleic acid such as mRNA or a gene of interest, or a polypeptide. In some preferred embodiments the analyte is a nucleic acid and the target region of the probe is complementary to at least a portion of the analyte.
"In some embodiments, a triggered probe is utilized, such that the initiation region is only made available to interact with the monomers when the probe is bound to the analyte of interest. For example, in some embodiments the probe undergoes a conformational change upon binding of the target region to the analyte such that the initiation region is available to stimulate polymerization. In this way, non-specific polymerization resulting from non-specific probe binding is reduced.
"The in situ HCR reactions can be multiplexed to identify the presence of multiple analytes of interest simultaneously.
"In another aspect, methods of in situ imaging are provided in which a biological sample is contacted with a probe comprising a target region capable of specifically binding to an analyte of interest and an initiator region, such that the probe binds to the analyte of interest. The sample is then contacted with at least two fluorescently labeled monomers, whereby the initiator region of the bound probe hybridizes to at least one of the monomers. As a result, the monomers form a fluorescently labeled polymer tethered to the analyte via the probe. The fluorescently labeled polymer can then be visualized.
"In a further aspect, kits are provided for the in situ detection of an analyte of interest, preferably a nucleic acid. The kits preferably comprise a first metastable nucleic acid monomer comprising an initiator complement region and a fluorescent label and a second metastable nucleic acid monomer comprising a propagation region that is substantially complementary to a portion of the first nucleic acid. A probe is included comprising a target region that is complementary to at least a portion of the nucleic acid to be detected and an initiator strand that is complementary to the initiator complement region of the first monomer. The first and second monomers are preferably hairpin monomers. In some embodiments the kits comprise one or more additional monomers and may comprise one or more additional probes, for identifying multiple analytes."
For more information, see this patent: Pierce, Niles A.; Dirks, Robert;
Keywords for this news article include: Antibodies, Immunology, Blood Proteins, Immunoglobulins, Molecular Probes, Nucleic Acid Probes,
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