Assays for Single Molecule Detection and Use Thereof

A method of detecting a genetic variation in a genetic sample from a subject, comprising

• contacting probe sets to a genetic sample under a condition effective to hybridize the probe sets to a nucleic acid region of interest in nucleotide molecules of the genetic sample, wherein
• each of the probe sets comprises a first oligonucleotide probe at the 5′ end of the probe set and a second oligonucleotide probe at the 3′ end of the probe set,
• each of the oligonucleotide probes is configured to hybridize to a part of the nucleic acid region of interest,
• the first oligonucleotide probe comprises phosphorothioate bonds at four or more nucleotide bonds from the 5′ end, and
• the second oligonucleotide probe comprises phosphorothioate bonds at four or more nucleotide bonds from the 3′ end,
• ligating the first and second oligonucleotide probes that are hybridized to the nucleotide molecules,
• digesting terminal phosphodiester bonds in non-hybridized oligonucleotide probes and/or partially hybridized nucleic acid molecules with one or more exonuclease,
• detecting non-digested and ligated probe sets to determine the presence or absence of the genetic variation in a genetic sample from a subject.

A method of detecting a genetic variation in a genetic sample from a subject, comprising

• contacting first and second probe sets to the genetic sample under a condition effective to hybridize the probe sets to a nucleic acid region of interest in nucleotide molecules of 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, and
• each of the probes is configured to hybridize to a part of the nucleic acid region of interest in the nucleic acid molecules and comprises phosphorothioate bonds at four or more nucleotide bonds from the 5′ or 3′ end,
• hybridizing at least parts of the first and second probe sets to first and second nucleic acid regions of interest in nucleotide molecules of the genetic sample, respectively,
• ligating the first probe set by ligating the first labeling probe and the first tagging probe to produce a first ligated probe set comprising the phosphorothioate bonds at the 5′ and 3′ ends,
• ligating the second probe set by ligating the second labeling probe and the second tagging probe to produce a second ligated probe set comprising the phosphorothioate bonds at the 5′ and 3′ ends,
• digesting terminal phosphodiester bonds in non-hybridized probe sets and/or partially hybridized nucleic acid molecules with one or more exonuclease,
• immobilizing the tagging probes to a pre-determined location on a substrate, wherein
• the first and second labeling probes and/or the amplified labeling probes thereof ligated to the immobilized tagging probes comprise first and second labels, respectively,
• the first and second labels are different,
• the immobilized labels are optically resolvable, and
• the immobilized first and second tagging probes and/or the amplified tagging probes thereof comprise first and second tags, respectively,
• counting (i) a first number of the first label immobilized to the substrate, and (ii) a second number of the second label immobilized to the substrate, and
• comparing the first and second numbers to determine the genetic variation in the genetic sample.

A method of detecting a genetic variation in a genetic sample from a subject, comprising

• contacting probes or probe sets to the genetic sample,
• hybridizing the probes or probe sets to a nucleic acid region of interest in nucleotide molecules of the genetic sample,
• amplifying the probes or probe sets with forward and reverse primers, wherein the forward or reverse primers comprise one or more label,
• digesting terminal phosphodiester bonds in single-stranded oligonucleotides of the probes or probe sets with a first exonuclease after the amplifying,
• after the digesting with the first exonuclease, inactivating the first exonuclease,
• after the inactivating, digesting an end of the amplified double-stranded probes or probe sets with a second exonuclease to produce an amplified single-stranded probe or probe sets, and
• after the digesting with the second exonuclease, detecting single-stranded oligonucleotides of the probes or probe sets to determine the presence or absence of the genetic variation in a genetic sample from a subject.

A method of isolating amplified products of a ligated probe set comprising

• immobilizing a composition comprising single-stranded ligated probe sets and second probes on a substrate, wherein
• each of the single-stranded ligated probe set comprises a labeling probe and a tagging probe ligated to each other,
• each of the second probes comprises the labeling probe or the tagging probe, and
• the labeling probe or the tagging probe comprises an isolating tag configured to bind to the substrate,
• amplifying one or more of the single-stranded ligated probe sets with forward and reverse primers after the immobilizing to form one or more double-stranded ligated probe set, wherein
• the forward or reverse primer hybridizing to the labeling probe of the one or more of the single-stranded ligated probe sets comprises a label,
• digesting terminal phosphodiester bonds in the second probes and/or the single-stranded ligated probe sets with one or more exonuclease after the amplifying, and
• isolating the non-digested and ligated probe sets after the digesting.

A method of detecting a genetic variation in a genetic sample from a subject, comprising

• contacting single-stranded probe sets to the genetic sample, wherein each of the single-stranded probe sets comprises a labeling probe and a tagging probe, and the labeling probe or the tagging probe comprises an isolating tag configured to bind to the substrate,
• hybridizing the single-stranded probe sets to a nucleic acid region of interest in nucleotide molecules of the genetic sample,
• ligating the single-stranded probe sets at least by ligating the labeling probe and the tagging probe to produce first single-stranded ligated probe sets,
• immobilizing the first single-stranded ligated probe sets on a substrate,
• amplifying the first single-stranded ligated probe sets with forward and reverse primers after the immobilizing to form double-stranded ligated probe set, wherein
• the forward or reverse primer hybridizing to the labeling probe of the first single-stranded ligated probe set comprises one or more label,
• digesting terminal phosphodiester bonds in the single-stranded probe sets with a first exonuclease after the amplifying,
• after the digesting with the first exonuclease, inactivating the first exonuclease,
• after the inactivating, digesting an end of the amplified double-stranded ligated probe sets with a second exonuclease to produce second single-stranded ligated probe set, and
• after the digesting with the second exonuclease, detecting the second single-stranded ligated probe sets to determine the presence or absence of the genetic variation in a genetic sample from a subject.

A method of detecting a genetic variation in a genetic sample from a subject, comprising

• contacting non-ligated first and second probe sets to the genetic sample, wherein each of the first probe sets comprises a first labeling probe and a first tagging probe, and each of the second probe sets comprises a second labeling probe and a second tagging probe,
• hybridizing one or more of the first probe set and one or more of the second probe set to first and second nucleic acid regions of interest in nucleotide molecules of the genetic sample, respectively, to produce hybridized first and second probe set,
• ligating the hybridized first probe set at least by ligating the first labeling probe and the first tagging probe to produce a ligated first probe set,
• ligating the hybridized second probe set at least by ligating the second labeling probe and the second tagging probe to produce a ligated second probe set,
• immobilizing one or more of the tagging probes or the labeling probes of each of the non-ligated and ligated probe sets on one or more bead,
• amplifying the ligated probe set with forward and reverse primers after the immobilizing, wherein
• the forward or reverse primer hybridizing to the labeling probe of the ligated probe set comprises a label,
• digesting terminal phosphodiester bonds in non-ligated probes with one or more exonuclease after the amplifying,
• immobilizing the tagging probes to a pre-determined location on a substrate, wherein
• the first and second labeling probes and/or the amplified labeling probes thereof ligated to the immobilized tagging probes comprise first and second labels, respectively,
• the first and second labels are different,
• the immobilized labels are optically resolvable, and
• the immobilized first and second tagging probes and/or the amplified tagging probes thereof comprise first and second tags, respectively,
• counting (i) a first number of the first label immobilized to the substrate, and (ii) a second number of the second label immobilized to the substrate, and
• comparing the first and second numbers to determine the genetic variation in the genetic sample.

A method of isolating a ligated probe set hybridized to a genetic sample, comprising

• contacting a probe set to a genetic sample under a condition effective to hybridize the probe set to a nucleic acid region of interest in nucleotide molecules of the genetic sample, wherein the probe set comprises first and second oligonucleotide probes,
• ligating the first and second oligonucleotide probes to form a ligated probe set,
• denaturing the ligated probe set from the genetic sample,
• hybridizing at least a part of a junction capture probe to the ligated probe set, wherein the junction capture probe is hybridized to (i) at least a part of the first oligonucleotide probe and (ii) at least a part of the second oligonucleotide probe, and
• isolating the ligated probe set hybridized to the junction capture probe.

A method of detecting a genetic variation in a genetic sample from a subject, comprising

• contacting a probe set to a genetic sample under a condition effective to hybridize the probe set to a nucleic acid region of interest in nucleotide molecules of the genetic sample, wherein
• the probe set comprises first and second oligonucleotide probes, and
• each of the oligonucleotide probes is configured to hybridize to a part of the nucleic acid region of interest,
• ligating the first and second oligonucleotide probes that are hybridized to the nucleotide molecules to form a ligated probe set,
• denaturing the ligated probe set from the genetic sample,
• hybridizing at least a part of a junction capture probe to the ligated probe set, wherein the junction capture probe is hybridized to (i) at least a part of the first oligonucleotide and (ii) at least a part of the second oligonucleotide,
• isolating the ligated probe set hybridized to the junction capture probe,
• amplifying the ligated probe set to form amplified ligated probe sets, and
• detecting the amplified ligated probe sets to determine the presence or absence of the genetic variation in a genetic sample from a subject.

A method of detecting a genetic variation in a genetic sample from a subject, comprising

• contacting first and second probe sets to the genetic sample under a condition effective to hybridize the probe sets to a nucleic acid region of interest in nucleotide molecules of 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,
• denaturing the ligated probe set from the genetic sample,
• ligating at least parts of the first probe set at least by ligating the first labeling probe and the first tagging probe to form a first ligated probe set,
• ligating at least parts of the second probe set at least by ligating the second labeling probe and the second tagging probe to form a second ligated probe set,
• hybridizing at least a part of each of first and second junction capture probes to the first and second ligated probe sets, respectively, wherein the first junction capture probe is hybridized to at least a part of each of the first labeling probe and the first tagging probe, and the second junction capture probe is hybridized to at least a part of each of the second labeling probe and the second tagging probe,
• isolating at least a part of the first and second ligated probe sets that are hybridized to the first and second junction capture probes, respectively, to form first and second isolated ligated probe sets,
• amplifying (i) the first isolated ligated probe set with first forward and reverse primers, wherein at least one of the first forward and reverse primers comprises a first label, and (ii) the second isolated ligated probe set with second forward and reverse primers, wherein at least one of the second forward and reverse primers comprises a second label, to form amplified first and second ligated probe sets comprising the first and second labels, respectively, wherein the first and second labels are different,
• immobilizing at least parts of the amplified first and second ligated probe sets on a substrate, wherein the first and second labels of the amplified first and second ligated probe sets are optically resolvable after immobilization,
• counting (i) a first number of the first label in the amplified first probe set immobilized to the substrate, and (ii) a second number of the second label in the amplified second probe set immobilized to the substrate, and
• comparing the first and second numbers to determine the presence or absence of the genetic variation in a genetic sample from a subject.

A method of isolating a ligated probe set hybridized to a genetic sample, comprising

• contacting a probe set to a genetic sample under a condition effective to hybridize the probe set to a nucleic acid region of interest in nucleotide molecules of the genetic sample, wherein the probe set comprises first and second oligonucleotide probes,
• ligating the first and second oligonucleotide probes to form a ligated probe set,
• hybridizing at least a part of a junction capture probe to the ligated probe set, wherein the junction capture probe is hybridized to (i) at least a part of the first oligonucleotide probe and (ii) at least a part of the second oligonucleotide probe, and
• isolating the ligated probe set hybridized to the junction capture probe.

A method of detecting a genetic variation in a genetic sample from a subject, comprising

• contacting a probe set to a genetic sample under a condition effective to hybridize the probe set to a nucleic acid region of interest in nucleotide molecules of the genetic sample, wherein
• the probe set comprises first and second oligonucleotide probes, and
• each of the oligonucleotide probes is configured to hybridize to a part of the nucleic acid region of interest,
• ligating the first and second oligonucleotide probes that are hybridized to the nucleotide molecules to form a ligated probe set,
• hybridizing at least a part of a junction capture probe to the ligated probe set, wherein the junction capture probe is hybridized to (i) at least a part of the first oligonucleotide and (ii) at least a part of the second oligonucleotide,
• isolating the ligated probe set hybridized to the junction capture probe,
• amplifying the ligated probe set to form amplified ligated probe sets, and
• detecting the amplified ligated probe sets to determine the presence or absence of the genetic variation in a genetic sample from a subject.

A method of detecting a genetic variation in a genetic sample from a subject, comprising

• contacting first and second probe sets to the genetic sample under a condition effective to hybridize the probe sets to a nucleic acid region of interest in nucleotide molecules of 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,
• ligating at least parts of the first probe set at least by ligating the first labeling probe and the first tagging probe to form a first ligated probe set,
• ligating at least parts of the second probe set at least by ligating the second labeling probe and the second tagging probe to form a second ligated probe set,
• hybridizing at least a part of each of first and second junction capture probes to the first and second ligated probe sets, respectively, wherein the first junction capture probe is hybridized to at least a part of each of the first labeling probe and the first tagging probe, and the second junction capture probe is hybridized to at least a part of each of the second labeling probe and the second tagging probe,
• isolating at least a part of the first and second ligated probe sets that are hybridized to the first and second junction capture probes, respectively, to form first and second isolated ligated probe sets,
• amplifying (i) the first isolated ligated probe set with first forward and reverse primers, wherein at least one of the first forward and reverse primers comprises a first label, and (ii) the second isolated ligated probe set with second forward and reverse primers, wherein at least one of the second forward and reverse primers comprises a second label, to form amplified first and second ligated probe sets comprising the first and second labels, respectively, wherein the first and second labels are different,
• immobilizing at least parts of the amplified first and second ligated probe sets on a substrate, wherein the first and second labels of the amplified first and second ligated probe sets are optically resolvable after immobilization,
• counting (i) a first number of the first label in the amplified first probe set immobilized to the substrate, and (ii) a second number of the second label in the amplified second probe set immobilized to the substrate, and
• comparing the first and second numbers to determine the presence or absence of the genetic variation in a genetic sample from a subject.