Assays for single molecule detection and use thereof
First Claim
1. A method of detecting nucleic acid copy numbers in a genetic sample obtained from a subject, comprisingcontacting first and second probe sets to the genetic sample,wherein the first probe set comprises a first labeling probe and a first tagging probe, andthe second probe set comprises a second labeling probe and a second tagging probe,hybridizing at least parts of the first and second probe sets to first and second nucleic acid regions of interest in nucleotide molecules present in the genetic sample, respectively,optionally amplifying the first and second probe sets to form first and second amplified probe sets, respectively,labeling at least parts of the first and second labeling probes and/or first and second amplified probe sets with first and second labels, respectively,immobilizing at least parts of the first and second probe sets and/or first and second amplified probe sets to a substrate at a density in which the first and second labels of the first and second probe sets and/or first and second amplified probe sets are optically resolvable after immobilization,detecting (i) a first number of the first label corresponding to a number of the first probe set and/or the first amplified probe set immobilized to the substrate, and (ii) a second number of the second label corresponding to a number of the second probe set and/or the second amplified probe set immobilized to the substrate to detect the nucleic acid copy numbers.
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Abstract
The invention relates to methods of detecting a genetic variation in a genetic sample from a subject using labeled probes and counting the number of labels in the probes.
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Citations
35 Claims
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1. A method of detecting nucleic acid copy numbers in a genetic sample obtained from a subject, comprising
contacting first and second probe sets to the genetic sample, wherein the first probe set comprises a first labeling probe and a first tagging probe, and the second probe set comprises a second labeling probe and a second tagging probe, hybridizing at least parts of the first and second probe sets to first and second nucleic acid regions of interest in nucleotide molecules present in the genetic sample, respectively, optionally amplifying the first and second probe sets to form first and second amplified probe sets, respectively, labeling at least parts of the first and second labeling probes and/or first and second amplified probe sets with first and second labels, respectively, immobilizing at least parts of the first and second probe sets and/or first and second amplified probe sets to a substrate at a density in which the first and second labels of the first and second probe sets and/or first and second amplified probe sets are optically resolvable after immobilization, detecting (i) a first number of the first label corresponding to a number of the first probe set and/or the first amplified probe set immobilized to the substrate, and (ii) a second number of the second label corresponding to a number of the second probe set and/or the second amplified probe set immobilized to the substrate to detect the nucleic acid copy numbers.
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29. A method of detecting nucleic acid copy numbers in a genetic sample obtained from a subject, comprising
forming a first probe product comprising a plurality of first oligonucleotides by hybridizing one or more first oligonucleotide probe to a first nucleic acid region of interest in nucleotide molecules present in the genetic sample, forming a second probe product comprising a plurality of second oligonucleotides by hybridizing one or more second oligonucleotide probe to a second nucleic acid region of interest in nucleotide molecules present in the genetic sample, ligating at least two oligonucleotides of the plurality of first oligonucleotides to form a first ligated probe product, ligating at least two oligonucleotides of the plurality of second oligonucleotides to form a second ligated probe product, optionally amplifying at least portions of the first and second ligated probe products to form first and second amplified probe products, respectively, labeling at least parts of the first and second ligated probe products and/or first and second amplified probe products with first and second labels, respectively, immobilizing at least parts of the first and second ligated probe products and/or first and second amplified probe products to a substrate at a density in which the first and second labels of the first and second ligated probe products and/or first and second amplified probe products are optically resolvable after immobilization, detecting (i) a first number of the first label corresponding to a number of the first ligated probe products and/or the first amplified probe product immobilized to the substrate, and (ii) a second number of the second label corresponding to a number of the second ligated probe products and/or the second amplified probe product immobilized to the substrate.
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35. A method of detecting nucleic acid copy numbers in a genetic sample obtained from a subject, comprising
contacting first and second probe sets to the genetic sample, wherein the first probe set comprises a first labeling probe and a first tagging probe, and the second probe set comprises a second labeling probe and a second tagging probe, hybridizing at least parts of the first and second probe sets to first and second nucleic acid regions of interest in nucleotide molecules present in the genetic sample, respectively, joining the first probe set at least by joining the first labeling probe and the first tagging probe, joining the second probe set at least by joining the second labeling probe and the second tagging probe, optionally amplifying the first and second probe sets to form first and second amplified probe sets, respectively, each of which comprising an amplified labeling probe and an amplified tagging probe, labeling at least parts of the first and second labeling probes and/or the amplified labeling probes in the first and second amplified probe sets with first and second labels, respectively, immobilizing at least parts of the first and second tagging probes and/or the amplified tagging probes in the first and second amplified probe sets to a substrate at a density in which the first and second labels of the first and second probe sets and/or first and second amplified probe sets are optically resolvable after immobilization, and detecting (i) a first number of the first label corresponding to a number of the first probe set and/or the first amplified probe set immobilized to the substrate, and (ii) a second number of the second label corresponding to a number of the second probe set and/or the second amplified probe set immobilized to the substrate to detect the nucleic acid copy numbers.
Specification