Assays for single molecule detection and use thereof
View Patent ↗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.
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.
2. The method according to claim 1 , wherein the labeling is performed prior to the contacting, and the labeling comprises labeling the first and second labeling probes with the first and second labels.
3. The method according to claim 1 , wherein the method comprises simultaneously performing the amplifying and the labeling.
4. The method according to claim 1 , further comprising contacting an exonuclease to the first and second amplified probe sets, and
digesting 5′-end of the first and second amplified probe sets without a label at the 5′-end,
wherein the 5′-end of the first and second amplified probe sets comprising a label at the 5′-end is protected from exonuclease digestion.
5. The method according to claim 1 , wherein the difference between the first and second copy numbers indicates presence or absence of cancer, presence or absence of metastatic cancer, recurrence of cancer, tumor load, tumor heterogeneity, phamacokinetic variability, drug toxicity, transplant rejection, efficacy of treatment, or aneuploidy in the subject.
6. The method according to claim 1 , wherein the subject is a pregnant subject, and the nucleic acid copy numbers indicate a nucleic acid copy number variation selected from the group consisting of trisomy 13, trisomy 18, trisomy 21, aneuploidy of X, aneuploidy of Y, 22q11.2, 1q21.1, 9q34, 1p36, and 22q13 in the fetus of the pregnant subject.
7. The method according to claim 1 , wherein the first and second labels have different optical properties.
8. The method according to claim 1 , wherein the genetic sample is selected from the group consisting of a cell-free DNA sample, whole blood, serum, plasma, urine, saliva, sweat, fecal matter, and tears from the subject.
9. The method according to claim 1 , wherein
the first labeling probe and the first tagging probe are hybridized to the first nucleic acid region of interest, and
the second labeling probe and the second tagging probes are hybridized to the second nucleic acid region of interest.
10. The method according to claim 1 , wherein the detecting comprises spatial filtering and/or watershedding analysis.
11. The method according to claim 1 , further comprising obtaining an estimate of a relative number of the nucleotide molecules having the first and second nucleic acid regions of interest.
12. The method according to claim 1 , wherein the immobilized labels of the same type are separated by a distance of at least 250 nm in both dimensions.
13. The method according to claim 1 , wherein the first and second labels are fluorescent dyes.
14. The method according to claim 1 , wherein
the at least parts of the first and second probe sets and/or first and second amplified probe sets labeled with the first and second labels comprises first and second tags, respectively; and
the substrate comprises a binding partner that contacts and immobilizes the first and second tags.
15. The method according to claim 1 , wherein
the at least parts of the first and second probe sets and/or first and second amplified probe sets labeled with the first and second labels comprise first and second tagging nucleotide sequences, respectively; and
the immobilizing comprises hybridizing at least a part of the first and second tagging nucleotide sequences to a corresponding nucleotide molecule immobilized on the substrate.
16. The method according to claim 15 , wherein
the first and second tagging nucleotide sequences share a common tagging nucleotide sequence, and the common tagging nucleotide sequence are immobilized to the same member of an array on the substrate.
17. The method according to claim 15 , wherein each of the first and second tagging nucleotide sequences independently has a non-genomic sequence.
18. The method according to claim 1 , wherein the first and second probe sets and/or first and second amplified probe sets are immobilized to an array of multiple pre-determined locations on a substrate.
19. The method according to claim 1 , wherein the subject is a pregnant subject, the nucleic acid copy numbers are nucleic acid copy numbers in the fetus of the pregnant subject, and the method further comprises
contacting maternal and paternal probe sets to the genetic sample, wherein
the maternal probe set comprises a maternal labeling probe and a maternal tagging probe, and
the paternal probe set comprises a paternal labeling probe and a paternal tagging probe,
hybridizing at least parts of the maternal and paternal probe sets to a third nucleic acid region of interest in the nucleotide molecules of the genetic sample, the third nucleic acid region of interest comprising a predetermined Single Nucleotide Polymorphism (SNP) site, wherein
the at least a part of the maternal probe set hybridizes to a first allele at the predetermined SNP site, the at least a part of the paternal probe set hybridizes to a second allele at the predetermined SNP site, and the first and second alleles are different from each other,
optionally amplifying the maternal and paternal probe sets to form maternal and paternal amplified probe sets,
labeling the maternal and paternal labeling probes and/or maternal and paternal amplified probe sets with maternal and paternal labels, respectively,
immobilizing at least a part of the maternal and paternal probe sets and/or maternal and paternal amplified probe sets to the substrate,
detecting the numbers of the maternal and paternal labels.
20. The method according to claim 1 , wherein the subject is a pregnant subject, the nucleic acid copy numbers are nucleic acid copy numbers in the fetus of the pregnant subject, and the method further comprises
contacting allele A and B probe sets that are allele-specific to the genetic sample, wherein
the allele A probe set comprises an allele A labeling probe and an allele A tagging probe, and
the allele B probe set comprises an allele B labeling probe and an allele B tagging probe,
hybridizing at least parts of the allele A and allele B probe sets to a fourth nucleic acid region of interest in the nucleotide molecules of the genetic sample, the fourth nucleic acid region of interest comprising a predetermined single nucleotide polymorphism (SNP) site for which a maternal allelic profile differs from a fetal allelic profile at the SNP site, wherein
the at least a part of the allele A probe set hybridizes to a first allele at the predetermined SNP site, the at least a part of the allele B probe set hybridizes to a second allele at the predetermined SNP site, and the first and second alleles are different from each other,
ligating at least parts of the allele A and B probe sets by ligating (i) the allele A labeling and tagging probes, and (ii) the allele B labeling and tagging probes to form allele A and B probe sets,
optionally amplifying the probe sets to form allele A and B amplified probe sets,
labeling the allele A and B labeling probes and/or allele A and B amplified probe sets with allele A and B labels, respectively,
immobilizing at least a part of the allele A and B probe sets and/or allele A and B amplified probe sets to the substrate,
detecting the numbers of the allele A and allele B labels.
21. The method according to claim 1 , wherein the detecting comprises
measuring optical signals from the immobilized labels, and
calibrating the first and second numbers by distinguishing an optical signal from a single label from the rest of the optical signals from background and/or multiple labels.
22. The method according to claim 1 , further comprising
contacting first and second control probe sets to the genetic sample, wherein
the first probe set comprises a first control labeling probe and a first control tagging probe, and
the second probe set comprises a second control labeling probe and a second control tagging probe,
hybridizing at least parts of the first and second control probe sets to the first and second nucleic acid regions of interest, respectively,
ligating at least parts of the first control probe set by ligating the first control labeling probe and the first control tagging probe to form a first control ligated probe set,
ligating at least parts of the second control probe set by ligating the second control labeling probe and the second control tagging probe to form a second control ligated probe set,
optionally amplifying the first and second control ligated probe sets to form first and second control amplified ligated probe sets, respectively,
labeling the first and second control labeling probes and/or first and second control amplified ligated control probe sets with first and second control labels, respectively,
immobilizing at least parts of the first and second control ligated probe sets and/or first and second control amplified control ligated probe sets to a substrate at a density in which the first and second control labels of the first and second control probe sets and/or first and second control amplified first and second control ligated probe sets are optically resolvable after immobilization, and
detecting (i) a first control number of the first control label corresponding to a sum of the first control ligated probe set and/or the first control amplified ligated probe set immobilized to the substrate, and (ii) a second control number of the second control label corresponding to a sum of the second control ligated probe set and/or the second control amplified ligated probe set immobilized to the substrate.
23. The method according to claim 22 , wherein
the first and second labels are different from the first and second control labels, respectively.
24. The method according to claim 1 , wherein
the method comprises the amplifying;
the first probe set is amplified with a first forward primer and a first reverse primer;
the second probe set is amplified with a second forward primer and a second reverse primer; and
(i) the first and second forward primers do not include a label and have the same nucleotide sequence, and/or (ii) the first and second reverse primers do not include a label and have the same nucleotide sequence.
25. The method according to claim 1 , further comprising
calibrating the first and second numbers by distinguishing a first optical signal from a single first label from the rest of the optical signals from background, and distinguishing a second optical signal from a single second label from the rest of the optical signals from background.
26. The method according to claim 1 , wherein
the method excludes sequencing of the first and second probe sets or the first and second amplified probe sets, and/or
the detecting excludes bulk array readout of the first and/or second labels.
27. The method according to claim 1 , wherein the nucleic acid copy numbers indicate a nucleic acid copy number variation selected from the group consisting of substitutions, inversions, insertions, deletions, mutations, single nucleotide polymorphism (SNP) and translocations in nucleotide sequences.
28. The method according to claim 1 , further comprising ligating at least a portion of the first probe set, and ligating at least a portion of the second probe set.
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.
30. The method according to claim 29 , wherein
the first probe product comprises at least the first oligonucleotide probe, a portion of the nucleotide molecules comprising the first nucleic acid region, and a third oligonucleotide probe, and
the second probe product comprises at least the first oligonucleotide probe, a portion of the nucleotide molecules comprising the first nucleic acid region, and a fourth oligonucleotide probe.
31. The method according to claim 1 , wherein a difference between the first number of the first label and the second number of the second label indicates the presence of a nucleic acid copy number variation in the genetic sample.
32. The method according to claim 1 , wherein the method excludes sequencing the first and second probe sets or the first and second amplified probe sets.
33. The method according to claim 1 , wherein the method excludes quenching the first and second labels.
34. The method according to claim 1 , further comprising detecting a difference between the first number of the first label and the second number of the second label to detect a difference between said nucleic acid copy numbers.
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.