Multiplexed analysis by chromatographic separation of molecular tags
First Claim
1. A method for determining the presence or absence of one or more target compounds in a sample, the method comprising the steps of:
- providing a plurality of binding compounds, such that there is at least one binding compound specific for each of the one or more target compounds, each binding compound having one or more molecular tags, each molecular tag being attached by a cleavable linkage, and the molecular tags of each binding compound being distinguishable from those of every other binding compound by one or more physical and/or optical characteristics;
combining with the sample the plurality of binding compounds specific for the one or more target compounds such that in the presence of a target compound a complex is formed between such target compound and a binding compound specific therefor;
cleaving the cleavable linkage of each binding compound forming such complex so that molecular tags are released; and
chromatographically separating and identifying the released molecular tags by the one or more physical characteristics to determine the one or more target compounds in the sample.
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Abstract
Methods and kits are disclosed for determining, either in a homogeneous or heterogeneous assay format, one or more target analytes in a sample using binding compositions coupled to molecular tags by cleavable linkages. Generally, an assay mixture is formed comprising a sample and a reagent comprising multiple such binding compositions under conditions that permit stable complexes to form between the binding compositions and analytes. In one aspect of the invention, the interaction between the binding compositions and their respective binding sites brings a cleavage-inducing moiety into close proximity to cleavable linkages or provides a recognizable substrate for a cleavage-inducing moiety. In this way, one or more molecular tags for each of the analytes are released from the complexes. Released molecular tags are chromatographically separated and the presence and/or amount of the target analytes are determined based on the analysis of the released and separated molecular tags.
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Citations
20 Claims
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1. A method for determining the presence or absence of one or more target compounds in a sample, the method comprising the steps of:
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providing a plurality of binding compounds, such that there is at least one binding compound specific for each of the one or more target compounds, each binding compound having one or more molecular tags, each molecular tag being attached by a cleavable linkage, and the molecular tags of each binding compound being distinguishable from those of every other binding compound by one or more physical and/or optical characteristics;
combining with the sample the plurality of binding compounds specific for the one or more target compounds such that in the presence of a target compound a complex is formed between such target compound and a binding compound specific therefor;
cleaving the cleavable linkage of each binding compound forming such complex so that molecular tags are released; and
chromatographically separating and identifying the released molecular tags by the one or more physical characteristics to determine the one or more target compounds in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method of detecting the presence or absence of a plurality of polynucleotides in a sample, the method comprising the steps of:
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providing for each polynucleotide, a helper probe complementary to a region of the polynucleotide, and a detection probe complementary to the polynucleotide adjacent to said region, each detection probe having a molecular tag attached by a cleavable linkage, and the molecular tag of each detection probe having one or more physical and/or optical characteristics distinct from those of molecular tags attached to other detection probes so that each molecular tag forms a distinguishable peak in a separation profile;
mixing under hybridization conditions a nuclease, the sample, the detection probes, and the helper probes to form an assay mixture, such that the detection probes and the helper probes hybridized to the target polynucleotides to form complexes recognized by the nuclease so that a detection probe in a complex is cleaved at a cleavage site to produce in the assay mixture released molecular tags, uncleaved detection probes, and nonspecific degradation products;
treating the assay mixture to exclude from the separation profile uncleaved detection probes and nonspecific degradation products; and
chromatographically separating and identifying the released molecular tags to determine each of the plurality of polynucleotides. - View Dependent Claims (12, 13, 14, 15)
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16. A method for detecting a plurality of target analyte in a sample, the method comprising the steps of:
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providing a binding compound for each of a plurality of target analytes, each binding compound having one or more molecular tags attached thereto by a cleavable linkage, the one or more molecular tags of each binding compound having one or more physical and/or optical characteristics distinct from those of molecular tags attached to other binding compounds so that each molecular tag forms a distinguishable peak in a chromatogram;
providing a second binding compound for each of the plurality of target analytes, each second binding compound having a sensitizer for generating an active species;
combining with the sample a binding compound and a second binding compound for each of the plurality of target analytes such that in the presence of a target analyte a complex is formed between the target analyte and the binding compound and the second binding compound specific therefor, and such that the sensitizer of the second binding compound causes the generation of an active species and the cleavage of one or more cleavable linkages to release one or more molecular tags; and
chromatographically separating and identifying the released molecular tags by the one or more physical characteristics to determine the target analytes in the sample. - View Dependent Claims (17, 18, 19, 20)
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Specification