METHODS, KITS, AND SYSTEMS FOR MULTIPLEXED DETECTION OF TARGET MOLECULES AND USES THEREOF
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Abstract
Described herein are methods, compositions, kits and systems for multiplexed detection of target molecules from a sample. In some embodiments, the methods, compositions, kits and systems can be used to perform multiplexed protein analysis of a sample (e.g., a sample comprising a small number of cells or a single-cell sample). In some embodiments, the same sample subjected to a multiplexed protein analysis using the methods, compositions, kits and systems described herein can also be subjected to a nucleic acid (e.g., RNAs, microRNAs, and/or DNA) analysis, thereby creating an integrated expression profiling from a limited amount of sample.
32 Citations
80 Claims
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1-66. -66. (canceled)
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67. A method for detecting a plurality of target molecules in a sample comprising:
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a. contacting a sample with a composition comprising a plurality of target probes, wherein each target probe in the plurality comprises; i. a target-binding molecule that specifically binds to a distinct target molecule in the sample; ii. an identification nucleotide sequence that identifies the target-binding molecule; and iii. a cleavable linker between the target-binding molecule and the identification nucleotide sequence; b. separating unbound target probes from a plurality of complexes in the sample, each complex having a target molecule and a single target probe bound thereto, wherein the complex does not have a second target probe binding to a different region of the target molecule; c. releasing the identification nucleotide sequences from the plurality of complexes; d. coupling the released identification nucleotide sequences from the releasing step (c) to a detection composition comprising a plurality of reporter probes, wherein each reporter probe in the plurality comprises a detectable label that identifies the reporter probe, wherein the label creates a unique distinguishable signal for each reporter probe; and e. detecting signals from the released identification nucleotide sequences based on a non-gel electrophoresis method, wherein the signals are distinguishable for the identification nucleotide sequences, thereby identifying the corresponding target-binding molecules and detecting a plurality of different target molecules in the sample. - View Dependent Claims (68, 69, 70, 71, 72, 73, 74, 75, 76, 77)
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78. A kit for multiplexed detection of a plurality of different target molecules from a sample comprising:
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a. a plurality of target probes, wherein each target probe in the plurality comprises; i. a target-binding molecule that specifically binds to a distinct target molecule in the sample; ii. an identification nucleotide sequence that identifies the target-binding molecule; and iii. a cleavable, non-hybridizable linker between the target-binding molecule and the identification nucleotide sequence; b. a plurality of reporter probes, wherein each reporter probe comprises; i. a first target probe-specific region that is capable of binding a first portion of the identification nucleotide sequence; and ii. a detectable label that identifies the reporter probe; and c. a plurality of capture probes, wherein each capture comprises; i. a second target probe-specific region that is capable of binding a second portion of the identification nucleotide sequence; and ii. an affinity tag for immobilization of the identification nucleotide sequence to a solid substrate surface. - View Dependent Claims (79, 80)
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Specification