CA-2921628-C - ASSAYS FOR SINGLE MOLECULE DETECTION AND USE THEREOF

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.

Inventors

• Adrian Nielsen Fehr
• Patrick James Collins
• Jill Lyndon Herschleb
• Hywel Bowden Jones

Assignees

• SINGULAR BIO, INC.

Dates

Publication Date

20260505

Application Date

20140819

Priority Date

20130819

Claims (1)

1. 101 Claims 1. A method of detecting a genetic variation in a genetic sample from a subject, comprising (a) 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 comprising an affinity tag, wherein the affinity tag comprises a nucleic acid sequence, and wherein the first labeling probe hybridizes adjacent to the first tagging probe on a first nucleic acid region of interest in the genetic sample, and the second probe set comprises a second labeling probe and a second tagging probe comprising the affinity tag, wherein the second labeling probe hybridizes adjacent to the second tagging probe on a second nucleic acid region of interest in the genetic sample; (b) ligating the first labeling probe to the first tagging probe, thereby providing a first ligated probe set, and ligating the second labeling probe to the second tagging probe, thereby providing a second ligated probe set; (c) amplifying the first ligated probe set with a first primer comprising a first label and a second primer, wherein the first primer hybridizes to a portion of the first labeling probe or complement thereof, and the second primer hybridizes to a portion of the first tagging probe or complement thereof, thereby providing a first amplified ligated probe set comprising the first label, and the affinity tag or a complement thereof, and amplifying the second ligated probe set with a third primer comprising a second label and the second primer, wherein the third primer hybridizes to a portion of the second labeling probe, or complement thereof, and the second primer hybridizes to a portion of the second tagging probe or complement thereof, thereby providing a second amplified ligated probe set comprising the second label, and the affinity tag or a complement thereof, and wherein the first and second labels are different; (d) hybridizing the affinity tag or complement thereof, of the first and second amplified ligated probe sets to a pre-determined location on a substrate of an array, thereby providing immobilized first and second ligated probe sets comprising the first and second labels, respectively, wherein the first and second labels are optically resolvable at the pre-determined location on the substrate of the array; (e) counting (i) a first number of the first label immobilized to the pre-determined location on the array, and (ii) a second number of the second label immobilized to the pre-determined location on the array; and (f) comparing the first and second numbers to determine the genetic variation in the genetic sample. 102 2. The method according to claim 1, wherein the first labeling probe comprises a first priming sequence, the first and second tagging probes comprise a second priming sequence and the second labeling probe comprises a third priming sequence, different from the first priming sequence. 3. The method according to claim 1 or 2, wherein the first and second labeling probes comprise first and second reverse priming sequences, respectively, and the first and second tagging probes comprise first and second forward priming sequences, respectively. 4. 5. 6. The method according to any one of claims 1-3, wherein the first labeling probe is at a 3’-end of the first ligated probe set, or the second labeling probe is at a 3’-end of the second ligated probe set. The method according to any one of claims 1-3, wherein the first labeling probe is at a 5’-end of the first ligated probe set, or the second labeling probe is at a 5’-end of the second ligated probe set. The method according to any one of claims 1-5, wherein after (c) and prior to (d), the method further comprises contacting an exonuclease to the first and second amplified ligated probe sets, and digesting single-stranded molecules that do not have a label at the 5’-end. 7. The method according to claim 6, wherein single-stranded molecules of the amplified ligated probe sets comprising a label at the 5’-end are protected from exonuclease digestion. 8. The method according to any one of claims 1-7, wherein the detecting of the genetic variation comprises detecting a presence or absence of cancer. 9. The method according to any one of claims 1 to 7, wherein the genetic variation indicates pharmacokinetic variability, drug toxicity, or transplant rejection in the subject. 10. The method according to any one of claims 1-9, wherein the genetic variation is a single nucleotide polymorphism. 11. 103 The method according to any one of claims 1-9, wherein the genetic variation is a copy number variation. 12. The method according to any one of claims 1-7, wherein the subject is a pregnant female, and the genetic sample comprises a mixture of maternal DNA and fetal DNA. 13. The method according to claim 12, wherein the detecting of the genetic variation comprises detecting a presence or absence of the genetic variation in a fetus of the pregnant female. 14. 15. The method according to claim 13, wherein the genetic variation is an aneuploidy. The method according to claim 14, wherein the genetic variation is selected from the group consisting of trisomy 13, trisomy 18, trisomy 21, aneuploidy of X, and aneuploidy of Y in the fetus of the pregnant female. 16. The method according to claim 14, wherein the genetic variation is trisomy 21 in the fetus of the pregnant female. 17. The method according to claim 13, wherein the genetic variation is a variation in the fetus of the pregnant female in a region selected from the group consisting of 22q11.2, 1q21.1, 9q34, 1p36, and 22q13. 18. The method according to any one of claims 1-17, wherein the first and second labels have different optical properties. 19. 20. The method according to claim 14, wherein the genetic variation is trisomy 21 in the fetus of the pregnant subject, the first nucleic acid region of interest is located in chromosome 21, and the second nucleic acid region of interest is not located in chromosome 21. The method according to claim 14, wherein the genetic variation is trisomy 21 in the fetus of the pregnant subject, the first nucleic acid region of interest is located in chromosome 21, and the second nucleic acid region of interest is located in chromosome 18. 104 21. 22. The method according to any one of claims 1-20, wherein the subject is human. The method according to any one of claims 1-21, wherein the genetic sample is selected from the group consisting of whole blood, serum, plasma, urine, saliva, sweat, fecal matter, and tears. 23. The method according to claim 22, wherein the genetic sample is plasma, and the method further comprises isolating the plasma from a blood sample of the subject. 24. The method according to claim 22, wherein the genetic sample is serum, and the method further comprises isolating the serum from a blood sample of the subject. 25. sample. 26. The method according to any one of claims 1-24, wherein the genetic sample is a cell free DNA The method according to any one of claims 1-25, further comprising amplifying nucleic acids of the genetic sample prior to the contacting. 27. The method according to any one of claims 1-26, wherein the counting comprises an optical analysis. 28. The method according to any one of claims 1-27, wherein the counting comprises reading the substrate in first and second imaging channels that correspond to the first and second labels, respectively, and producing one or more images of the substrate, wherein the first and second labeling probes are resolvable in the one or more images. 29. 30. The method according to any one of claims 1-28, wherein the counting comprises spatial filtering. The method according to any one of claims 1-29, wherein the counting comprises watershedding analysis. 31. The method according to any one of claims 1-30, wherein the immobilized first or second labels are separated by a distance of at least 250 nm. 32. 105 The method according to any one of claims 1-31, wherein the first and second labels are fluorescent dyes. 33. The method according to any one of claims 1-32, wherein the substrate comprises a binding partner that hybridizes to and immobilizes the affinity tag. 34. The method according to any one of claims 1-33, wherein the first labeling probe and first tagging probe are separated by no nucleotides when hybridized to the first nucleic acid region of interest; and the second labeling probe and second tagging probe are separated by no nucleotides when hybridized to the second nucleic acid region of interest. 35. The method according to any one of claims 1-12, wherein the first nucleic acid region of interest and the second nucleic acid region of interest are at the same genetic locus. 36. The method according to claim 35, wherein the first nucleic acid region of interest comprises a first allele of a single nucleotide polymorphism and the second nucleic acid region of interest comprises a second allele of the single nucleotide polymorphism. 37. A method of detecting a cancer in a genetic sample from a subject, comprising (a) 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 comprising an affinity tag, wherein the affinity tag comprises a nucleic acid sequence, and wherein the first labeling probe hybridizes adjacent to the first tagging probe on a first nucleic acid region of interest in the genetic sample, and the second probe set comprises a second labeling probe and a second tagging probe comprising the affinity tag, wherein the second labeling probe hybridizes adjacent to the second tagging probe on a second nucleic acid region of interest in the genetic sample; wherein one of the first nucleic acid region of interest and the second nucleic acid region of interest comprises a cancer-associated genetic variation, (b) ligating the first labeling probe to the first tagging probe, thereby providing a first ligated probe set, and ligating the second labeling probe to the second tagging probe, thereby providing a second ligated probe set; 106 (c) amplifying the first ligated probe set with a first primer comprising a first label and a second primer, wherein the first primer hybridizes to a portion of the first labeling probe or complement thereof, and the second primer hybridizes to a portion of the first tagging probe or complement thereof, thereby providing a first amplified ligated probe set comprising the first label, and the affinity tag or a complement thereof, and amplifying the second ligated probe set with a third primer comprising a second label and the second primer, wherein the third primer hybridizes to a portion of the second labeling probe, or complement thereof, and the second primer hybridizes to a portion of the second tagging probe or complement thereof, thereby providing a second amplified ligated probe set comprising the second label, and the affinity tag or a complement thereof, and wherein the first and second labels are different; (d) hybridizing the affinity tag or complement thereof, of the first and second amplified ligated probe sets to a pre-determined location on a substrate on an array, thereby providing immobilized first and second ligated probe sets comprising the first and second labels, respectively, wherein the first and second labels are optically resolvable at the pre-determined location on the substrate on the array; (e) counting (i) a first number of the first label immobilized to the pre-determined location on the array, and (ii) a second number of the second label immobilized to the pre-determined location on the array; and (f) comparing the first and second numbers to determine the genetic variation in the genetic sample. 38. The method of claim 37, wherein the genetic sample is selected from the group consisting of whole blood, serum, plasma, urine, saliva, sweat, fecal matter, and tears. 39. 40. The method according to claim 37 or 38, wherein the genetic sample is a cell free DNA sample. The method according to claim 39, wherein the method further comprises isolating the cell free DNA sample from a blood sample of the subject. 41. The method according to any one of claims 37-40, further comprising amplifying the nucleotide molecules of the genetic sample prior to the contacting. 42. 107 The method of any one of claims 37-41, wherein the cancer-associated genetic variation is selected from the group consisting of single base mutations, SNPs, copy number variants, copy neutral variants, and combinations thereof. 43. 44. The method of claim 42, wherein the copy number variants comprise amplification variants. The method of claim 43, wherein the amplification variants comprise amplification of a cancer associated gene selected from the group consisting of Cyclin D1 (CCND1), Epidermal Growth Factor Receptor (EGFR), MYC Proto-Oncogene, BHLH Transcription Factor (MYC), Telomerase RNA Component (TERC), Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), Cyclin E1 (CCNE1), MCL1 Apoptosis Regulator, BCL2 Family Member (MCL1), MDM2 Proto-Oncogene (MDM2), Integrator Complex Subunit 4 (INTS4), Wolf-Hirschhorn Syndrome Candidate 1-Like Protein 1 (WHSC1L1), Cyclin Dependent Kinase 4 (CDK4), Lysine Acetyltransferase 6A (KAT6A), SRY-Box Transcription Factor 2 (SOX2), Platelet Derived Growth Factor Receptor Alpha (PDGFRA), 3-Hydroxybutyrate Dehydrogenase 1 (BDH1), MDM4 Regulator Of P53 (MDM4), Telomerase Reverse Transcriptase (TERT), Lysine Demethylase 5A (KDM5A), MYCL Proto-Oncogene, BHLH Transcription Factor (MYCL1), Insulin Like Growth Factor 1 Receptor (IGF1R), Poly(ADP-Ribose) Polymerase Family Member 10 (PARP10), Glucose-6-Phosphate Dehydrogenase (G6PD), PHD Finger Protein 12 (PHF12), PAF1 Homolog, Paf1/RNA Polymerase II Complex Component (PAF1), BCL2 Like 1 (BCL2L1), Tubulin Delta 1 (TUBD1), Zinc Finger Protein 703 (ZNF703), Bromodomain Containing 4 (BRD4), KRAS Proto-Oncogene, GTPase (KRAS), NK2 Homeobox 1 (NKX2-1), NFE2 Like BZIP Transcription Factor 2 (NFE2L2), Lysine Acetyltransferase 6B (KAT6B), Nuclear Receptor Binding SET Domain Protein 1 (NSD1), Fibroblast Growth Factor Receptor 3 (FGFR3), Cytochrome C Oxidase Assembly Factor COX18 (COX18), SRY-Box Transcription Factor 17 (SOX17), Chromobox 8 (CBX8), AKT Serine/Threonine Kinase 1 (AKT1), Cyclin Dependent Kinase 6 (CDK6), ETS Homologous Factor (EHF), Bromodomain PHD Finger Transcription Factor (BPTF), E2F Transcription Factor 3 (E2F3), Lysine Demethylase 2A (KDM2A), Neural Precursor Cell Expressed, Developmentally Down-Regulated 9 (NEDD9), Long Intergenic Non-Protein Coding RNA 536 (LINC00536), PHD Finger Protein 3 (PHF3), and Paired Box 8 (PAX8). 45. The method of claim 43, wherein the amplification variants comprise amplification of a cancer associated genetic locus selected from the group consisting of 1q44, 20q13.33, 1q23.3, 8q22.2, 13q34, 9p13.3, 7q36.3, 18q11.2, 11q22.2, 6p21.1, 6q21, 19q13.42, 17q21.33, 19p13.2, 17q25.1, 8q21.13, 2p15, 14q11.2, 5p13.1, 10p15.1, 22q11.21, and 9p24.2. 108 46. 47. The method of claim 42, wherein the copy number variants comprise deletion variants. The method of claim 46, wherein the deletion variants comprise deletion of a cancer-associated gene selected from the group consisting of Cyclin Dependent Kinase Inhibitor 2A (CDKN2A), Serine/Threonine Kinase 11 (STK11), Phosphodiesterase 4D (PDE4D), Parkin RBR E3 Ubiquitin Protein Ligase (PARK2), LDL Receptor Related Protein 1B (LRP1B), CUB And Sushi Multiple Domains 1 (CSMD1), AT-Rich Interaction Domain 1A (ARID1A), Phosphatase And Tensin Homolog (PTEN), WW Domain Containing Oxidoreductase (WWOX), RB Transcriptional Corepressor 1 (RB1), Family With Sequence Similarity 190, Member A (FAM190A), Long Intergenic Non-Protein Coding RNA 290 (LINC00290), Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), RNA Binding Fox-1 Homolog 1 (RBFOX1), Protein Tyrosine Phosphatase Receptor Type D (PTPRD), FAT Atypical Cadherin 1 (FAT1), M-Phase Phosphoprotein 8 (MPHOSPH8), Neurofibromin 1 (NF1), Mono-ADP Ribosylhydrolase 2 (MACROD2), Zinc Finger Protein 132 (ZNF132), Myeloid/Lymphoid Or Mixed Lineage Leukemia 3 (MLL3), Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A), IKAROS Family Zinc Finger 2 (IKZF2), Contactin-4 (CNTN4), RAD51 Paralog B (RAD51B), Inner Mitochondrial Membrane Peptidase Subunit 2 (IMMP2L), Neuronal Growth Regulator 1 (NEGR1), BRCA1 DNA Repair Associated (BRCA1), Ankyrin Repeat And Sterile Alpha Motif Domain Containing 1B (ANKS1B), Dystrophin (DMD), Zinc Finger MYND-Type Containing 11 (ZMYND11), Protein Kinase CGMP-Dependent 1 (PRKG1), Forkhead Box K2 (FOXK2), AGBL Carboxypeptidase 4 (AGBL4), Cyclin Dependent Kinase Inhibitor 1B (CDKN1B), PTTG1 Interacting Protein (PTTG1IP), SMAD Family Member 4 (SMAD4), and SET And MYND Domain Containing 3 (SMYD3). 48. The method of claim 46, wherein the deletion variants comprise deletion of a cancer-associated genetic locus selected from the group consisting of 1p36.23, 2q37.3, 22q13.32, 11p15.5, 18q23, 15q15.1, 11q25, 1p13.2, 7p22.3, 6p25.3, 21q11.2, 9p13.1, 5q15, 19q13.32, 15q12, 12q24.33, 10q26.3, 6q21, 3p12.2, 11q23.1, 9q34.3, 14q32.33, 14q11.2, 2p25.3, 5q35.3, 22q11.1, 17p13.3, 4p16.3, 9p21.2, 10q25.1, 8p11.21, and Xp22.33. 49. 50. The method of claim 42, wherein the copy neutral variants comprise inversions or translocations. The method according to any one of claims 37-49, wherein the first labeling probe comprises a first priming sequence, the first and second tagging probes comprise a second priming sequence and the second labeling probe comprises a third priming sequence, different from the first priming sequence. 109 51. The method according to any one of claims 37-50, wherein the first and second labeling probes comprise first and second reverse priming sequences, respectively, and the first and second tagging probes comprise first and second forward priming sequences, respectively. 52. The method according to any one of claims 37-51, wherein the first labeling probe is at a 3’-end of the first ligated probe set, or the second labeling probe is at a 3’-end of the second ligated probe set. 53. The method according to any one of claims 37-51, wherein the first labeling probe is at a 5’-end of the first ligated probe set, or the second labeling probe is at a 5’-end of the second ligated probe set. 54. The method according to any one of claims 37-53, wherein after (c) and prior to (d), the method further comprises contacting an exonuclease to the first and second amplified ligated probe sets, and digesting single-stranded molecules that do not have a label at the 5’-end. 55. The method according to claim 54, wherein single-stranded molecules of the amplified ligated probe sets comprising a label at the 5’-end are protected from exonuclease digestion. 56. The method according to any one of claims 37-55, wherein the counting comprises an optical analysis. 57. The method according to any one of claims 37-56, wherein the counting comprises reading the substrate in first and second imaging channels that correspond to the first and second labels, respectively, and producing one or more images of the substrate, wherein the first and second labeling probes are resolvable in the one or more images. 58. The method according to any one of claims 37-56, wherein the counting comprises spatial filtering. 59. The method according to any one of claims 37-56, wherein the counting comprises watershedding analysis. 60. 110 The method according to any one of claims 37-59, wherein the immobilized first or second labels are separated by a distance of at least 250 nm. 61. The method according to any one of claims 37-60, wherein the first and second labels are fluorescent dyes. 62. The method according to any one of claims 37-61, wherein the substrate comprises a binding partner that hybridizes to and immobilizes the affinity tag. 63. The method according to any one of claims 37-62, wherein the first labeling probe and first tagging probe are separated by no nucleotides when hybridized to the first nucleic acid region of interest; and the second labeling probe and second tagging probe are separated by no nucleotides when hybridized to the second nucleic acid region of interest. 64. The method according to any one of claims 37-63, wherein the first nucleic acid region of interest and the second nucleic acid region of interest are at the same genetic locus. 65. The method according to claim 64, wherein the first nucleic acid region of interest comprises a first allele of a single nucleotide polymorphism and the second nucleic acid region of interest comprises a second allele of the single nucleotide polymorphism. 66. The method according to any one of claims 37-65, wherein the method comprises detecting low level recurrence of a cancer. 67. The method according to any one of claims 37-66, wherein the method comprises monitoring genetic variation of a cancer. 68. The method according to any one of claims 37-67, wherein the method comprises detecting heterogeneity of a cancer. 69. The method according to any one of claims 37-68, wherein the subject is human.

Description

1 ASSAYS FOR SINGLE MOLECULE DETECTION AND USE THEREOF Background of the Invention [0001] The invention relates to methods of detecting a genetic variation in a genetic sample from a subject. Detecting a genetic variation is important in many aspects of human biology. Summary [0002] The invention relates to methods of detecting a genetic variation in a genetic sample from a subject. The invention further 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. Brief Description of the Drawings [0003] Figure 1 depicts exemplary array members comprising binding partners, tags, affinity tags, tagging probes, probe sets, and/or litigated probe sets described herein on a substrate. [0004] Figure 2 depicts a normalized histogram of signal intensity measured from both single label samples and multi-label antibodies. [0005] Figure 3 depicts average bleaching profiles from various labels. [0006] Figures 4-13 show the integrated label intensity graphs over time for various Alexa 488 labels. [0007] Figure 14 depicts excitation spectrum and emission spectrum through a standard operation when excitation of a fluorophore is achieved by illuminating with a narrow spectral band aligned with the absorption maxima of that species. [0008] Figure 15 depicts excitation spectrum and emission spectrum through interrogation with various excitation colors and collected emission bands different from ( or in addition to) the case for the standard operation. [0009] Figure 16 shows results when the light from these various imaging configurations, e.g., various emission filters, is collected and compared to calibration values for the fluorophores of interest. The circles (dots) are the experimental measurement; the triangles are the expected calibration/reference data for that fluorophore; the squares are an alternate hypothesis. [0010] Figure 17 shows results collected with various references, including contaminant 1 with a flat emission profile or contaminant 2 with a blue-weighted profile. The circles (dots) are the experimental measurement; the triangles are contaminant 1; the stars are contaminant 2 and the squares are an alternate hypothesis. [0011] Figure 18 depicts significantly-different excitation bands of two fluorophores. [0012] Figure 19 depicts an exemplary system flow chart. Date Re9ue/Date Received 2020-11-10 lA [0013] Figure 20 depicts an exemplary system flow chart including various methods for analyzing data. [0014] Figures 21-46 depict exemplary probe sets described herein. Date Re9ue/Date Received 2020-11-10 2 [0015] Figures 47 and 48 show the resulting fluorescence patterns when products contain unique affinity tag sequences and the underlying substrate contains complements to each of the unique affinity tags within the same location ( e.g., as the same member) on a substrate. [0016] Figures 49 and 51 show the resulting fluorescence patterns when different products contain identical affinity tag sequences and the underlying substrate contains the complement to the affinity tag. [0017] Figures 50 and 52 show zoomed-in locations of Figures 49 and 51, respectively. [0018] Figures 53 and 54 show the resulting fluorescence patterns when products contain unique affinity tag sequences and the underlying substrate has one location (e.g., as one member) containing the complement to one affinity tag complement, and another separate location (e.g., as another member) containing the complement to the other affinity tag. [0019] Figure 55 depicts two probe sets; one probe set for Locus 1 and one probe set for Locus 2 - although as aforementioned, multiple probes sets may be designed for each genomic locus. [0020] Figure 56 depicts the procedural workflow that would be applied to the collection of probe sets. [0021] Figure 57 depicts a modified version of the procedural workflow illustrated in Figure 56. [0022] Figure 58 provides an example of how probe products for Locus 1 and Locus 2 may be labeled with different label molecules. [0023] Figure 59 provides evidence that probe products representing a multitude of genomic locations for one locus may be generated in a ligase enzyme specific manner using the hybridizationligation process. [0024] Figure 60 provides data indicating that probe sets may be used to detect relative changes in copy number state. [0025] Figure 61 provides evidence that mixtures of probe products may be used to generate quantitative microarray data. [0026] Figures 62-64 illustrate modifications of the general procedure described in Figures 55 to 58. [0027] Figure 65 depicts a further embodiment of the modified procedure described in Figure 62. [0028] Figure 66 depicts yet another embodiment of the procedure depicted in Figure 65. [0029] Figure 67 depicts exemplary probe sets used in methods described herein. [0030] Figure 68 depicts exemplary probe sets used in methods described herein when translocations that