Methods, systems and compositions for counting nucleic acid molecules

Abstract

The present invention relates to methods, systems, and compositions for counting nucleic acid molecules. The present invention relates to a method for detecting changes in the number of molecules without digital sequencing, in particular for example due to gene duplication or normal euploid complements of chromosomes, compositions and methods, systems, and kits for detecting and quantifying changes in gene dose, such as changes in trisomes of one or more chromosomes typically found in diploid pairs.

Description

[0001] This application is a divisional application of application no. 201980035125.9, filed on April 02, 2019, entitled "Methods, systems, and compositions for counting nucleic acid molecules".

[0002] This application claims priority to U.S. provisional application serial no. 62 / 651,676, filed on April 2, 2018, and U.S. provisional application serial no. 62 / 660,699, filed on April 20, 2018, each of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present invention relates to compositions and methods for determining the copy number of individual molecules, such as nucleic acid molecules, without digital sequencing. The present technology can be used, for example, to analyze changes in the copy number of particular nucleic acid sequences that can be caused by, for example, changes in chromosome number, gene copy number, expression level, etc. The present technology can be particularly useful in genetic screening, for example, prenatal testing, particularly for non-invasive prenatal testing (NIPT). NIPT involves analyzing cell-free DNA (cfDNA) from a fetus circulating in the blood of a woman carrying the fetus in utero. Analysis of cell-free DNA in maternal blood can be used to assess the health of the fetus. The present technology herein relates to methods, systems, and kits for detecting and quantifying changes in the number of molecules, particularly changes in gene dosage caused, for example, by changes in gene duplication or normal euploid complements of chromosomes, for example, trisomy of one or more chromosomes typically found in diploid pairs. BACKGROUND

[0004] Detection of the presence or change in number of molecules in a sample is a useful way to characterize a sample and the source of the sample. For example, changes in gene dosage are clinically significant indicators of disease states, e.g., of the subject from which the sample was taken. Changes in gene dosage arise from errors in DNA replication and can occur in germline cells leading to congenital defects and even embryonic death or in somatic cells leading to cancer generally. These replication abnormalities can result in deletions or duplications of portions of genes, full-length genes and the regulatory regions surrounding them, megabase-long portions of chromosomes, or entire chromosomes. Analysis of other biological molecules is also clinically important. For example, changes in the amount of RNA or protein can indicate changes in the expression of genes associated with disease states. While embodiments of the technology presented herein are discussed in relation to specific applications, e.g., measuring DNA, it should be understood that the technology is not limited to these applications and readily applies to the analysis of many different types of molecules or moieties that are capable of binding to a partner molecule in a specific manner, e.g., antigens with antibodies, nucleic acids with complementary nucleic acids, nucleic acid structures with proteins that bind such structures (e.g., stem loops, bulging nucleotides, flaps, promoter sequences), lectins with carbohydrates, proteins with protein binding partners, proteins with lipids (e.g., SH2 domains with lipids), etc.

[0005] Chromosomal abnormalities can affect the number or structure of chromosomes. The absence or presence of one or more complete chromosomes or fragments of chromosomes in addition to the normal euploid complement of chromosomes in a cell, tissue, or individual can be referred to as an aneuploidy. Germline replication errors due to failure of chromosomes to segregate result in monosomy (one copy of a chromosome instead of the usual two or only one sex chromosome) or trisomy (three copies). When such events do not result in complete embryonic death, they often result in a wide variety of conditions that are generally considered syndromes, e.g., 21 trisomy and Down's syndrome, 18 trisomy and Edward's syndrome, and 13 trisomy and Patau's syndrome. Structural chromosomal abnormalities affecting portions of chromosomes result from chromosome breaks and result in deletions, inversions, translocations, or duplications of large blocks of genetic material. These events are as disruptive as gaining or losing entire chromosomes and can result in conditions such as Prader-Willi syndrome (del 15ql l-13), retinoblastoma (del 13ql4), Cri du Chat syndrome (del 5p), and others listed in U.S. Patent No. 5,888,740, incorporated herein by reference in its entirety.

[0006] Major chromosomal abnormalities were detected in nearly 1 in 140 live births and in a higher proportion of preterm or non-live births. Hsu (1998) Prenatal diagnosis of chromosomal abnormalities through amniocentesis in Milunsky A, ed. Genetic Disorders and the Fetus. 4th ed. Baltimore: The Johns Hopkins University Press. 179-180; Staebler et al. (2005) "Should determination of the karyotype be systematic for all malformations detected by obstetrical ultrasound?" Prenat Diagn 25:567-573. The most common aneuploidy is trisomy 21 (Down syndrome), which currently occurs in 1 in 730 births. Hsu; Staebler et al. Although less common than trisomy 21, trisomy 18 (Edward syndrome) and trisomy 13 (Patau syndrome) occur in 1 in 5,500 and 1 in 17,200 live births, respectively. Hsu. Various congenital defects, growth deficiencies, and intellectual disabilities are found in children with chromosomal aneuploidies, and these pose lifelong challenges to families and society. Jones (2006) Smith's recognizable patterns of human malformation. Philadelphia: Elsevier Saunders. Various prenatal tests can indicate an increased risk of fetal aneuploidy, including invasive diagnostic tests such as amniocentesis or chorionic villus sampling, which are the current gold standard but are associated with a non-negligible risk of fetal loss. American College of Obstetrics and Gynecology (2007) ACOG Practice Bulletin No. 88, December 2007.Invasive prenatal testing for aneuploidy Obstet Gynecol 110: 1459-1467. Thus, there has been a long search for more reliable non-invasive testing for fetal aneuploidy. The most promising of these is based on detection of fetal DNA in maternal plasma. It has been demonstrated that massively parallel sequencing of libraries generated from maternal plasma can reliably detect chromosome 21 abnormalities. See, e.g., Chiu et al., Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma Proc Natl Acad Sci U S A 105:20458-20463 (2008); Fan et al., Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood Proc Natl Acad Sci U S A 105:16266-16271 (2008). See also U.S. Patent No. 7,888,017.

[0007] Current methods for quantifying changes in the number of molecules, e.g., for aneuploidy screening, that rely on next generation sequencing (NGS) are typically time consuming, expensive, and require extensive bioinformatics analysis. SUMMARY

[0008] The present invention provides compositions, methods, and systems for detecting and characterizing samples by counting specific molecules (e.g., small molecules, haptens, proteins, antibodies, lipids, carbohydrates, and nucleic acids, such as genes or other DNA molecules or fragments and / or RNA (e.g., messenger RNA, microRNA, and other non-coding RNA)) that can be represented in a sample. The present technology can be applied, for example, to monitor gene expression, measure non-coding RNA abundance, and analyze genetic variations, including but not limited to changes in gene dosage, such as aneuploidy. In preferred embodiments, the present technology provides methods for detecting and thereby counting single copies of target molecules without using "next generation" sequencing (NGS) technologies, such as those described by Chiu et al. and Fan et al. (supra), or single molecule amplification techniques that rely on separate amplification reactions for individual target molecules (including nucleic acids) in different physically discrete elements (e.g., microcontainers or emulsion droplets).

[0009] In general, these compositions, methods, and systems provide improved ways to detect genomic deletions and duplications of various sizes, including whole chromosomes, chromosome arms, microdeletions and duplications, submicroscopic deletions and duplications, and single nucleotide features, including single nucleotide polymorphisms, deletions, and insertions. In certain embodiments, the methods of the present disclosure can be used to detect subchromosomal genetic lesions, such as microdeletions. Exemplary applications of the methods include pediatric and prenatal diagnosis of aneuploidy, testing for risk of a pregnancy product or a miscarriage, non-invasive prenatal testing (both qualitative and quantitative genetic testing, such as detecting Mendelian disorders, insertions / deletions, and chromosomal imbalances), testing preimplantation genetics, tumor characterization, postnatal testing (including cytogenetics), and mutagenic effect monitoring.

[0010] In some embodiments, the present technology herein provides methods to characterize nucleic acids, preferably DNA, more preferably circulating cell-free DNA from blood or plasma, in a sequence-specific and quantitative manner. In preferred embodiments, single copies of DNA are detected and counted without polymerase chain reaction or DNA sequencing. Embodiments of the present technology provide methods, compositions, and systems for detecting target DNA using methods for amplifying signals indicative of the presence of target DNA in a sample. In preferred embodiments, the detectable signal from a single target molecule is amplified in a degree and manner such that the signal originating from the single target molecule is detectable and identifiable from signals from other targets and from other copies of the target molecule.

[0011] In some embodiments, the present technology provides a method for counting target molecules on a solid support, the method comprising: forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a solid support; and detecting formation of the at least one complex in a process comprising: i) extending the primer in the complex in a rolling circle amplification (RCA) reaction to form a RCA product; ii) hybridizing a plurality of labeled probes to the RCA product; and iii) detecting hybridized labeled probes, wherein hybridized labeled probes are indicative of the presence of the target molecule on the solid support. In some embodiments, the solid support comprises a silanized surface, preferably a surface comprising glass.

[0012] In some embodiments, the present technology provides a method for counting target molecules on a solid support, the method comprising: a) providing a silanized surface comprising at least one of: an acrylic group and a reactive amine group; b) forming a plurality of complexes on a glass surface, the plurality of complexes comprising at least one of: a RCA product comprising a plurality of hybridized labeled probes; and a double stranded scaffold product comprising a plurality of catenated labeled scaffold oligonucleotides, wherein formation of a complex is indicative of the presence of a target molecule on the glass surface, and wherein forming the plurality of complexes comprises exposing the glass surface to a solution comprising graphene oxide; and c) counting the plurality of complexes. In some embodiments, the silanized surface is glass. In certain preferred embodiments, the silanized surface comprises a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate.

[0013] The surface is not limited to any particular format. For example, in any of the embodiments described above, the solid support can comprise a surface in an assay plate, preferably a glass bottom assay plate. In some embodiments, the assay plate is a multi-well assay plate, preferably a microtiter plate.

[0014] In some embodiments of the present technology, the primer of any of the embodiments described above is directly bound to the solid support, preferably covalently linked to the solid support. For example, in some embodiments, the primer comprises a biotin moiety and the solid support comprises avidin, preferably streptavidin. In certain embodiments, one or more complexes comprise an antibody bound to an antigen or hapten, and in some embodiments, the complex comprises an antigen or hapten directly bound to the solid support. In certain embodiments, the antigen or hapten is covalently linked to the solid support.

[0015] In any of the embodiments described herein, forming a complex or complexes can comprise exposing the solid support to a solution comprising a crowding agent. In some embodiments, the crowding agent comprises polyethylene glycol (PEG), preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG. In certain preferred embodiments, the PEG has an average molecular weight of between 200 and 8000, preferably between 200 and 1000, preferably between 400 and 800, preferably 600.

[0016] In any of the embodiments described above, forming a complex or complexes can comprise the step of exposing the solid support to a solution comprising graphene oxide. In preferred embodiments, the solid support is exposed to graphene oxide prior to the step of detecting hybridized labeled probes. In particularly preferred embodiments, the solid support is exposed to a solution comprising a mixture of labeled probes and graphene oxide. In some embodiments, the solid support or the glass surface exposed to a solution comprising graphene oxide is washed with a solution comprising a detergent prior to the detecting or counting. In certain preferred embodiments, the detergent comprises Tween 20.

[0017] The present technology can be used to detect many different kinds of molecules, including, for example Figure 38 molecules depicted schematically in FIG. 1. In some embodiments, the target molecules comprise nucleic acids, preferably DNA from a sample of a subject, preferably a blood or blood product sample. In certain preferred embodiments, the DNA is cell-free DNA from a blood or blood product sample. In some embodiments, the cell-free DNA comprises maternal and / or fetal DNA from a maternal blood sample.

[0018] Any of the embodiments described above herein can comprise forming a RCA product in a process comprising extending a primer on a circularized nucleic acid probe in a reaction mixture comprising at least 0.2 units per μΐ of Phi29 DNA polymerase, preferably at least 0.8 units per μΐ, and at least 400 μΜ, preferably at least 600 μΜ, more preferably at least 800 μΜ total dNTPs. In some embodiments, forming a RCA product comprising a plurality of hybridized labeled probes comprises forming the RCA product in a reaction mixture further comprising more than 100 nM molecular beacon probe, preferably at least 1000 nM molecular beacon probe in the reaction mixture.

[0019] In certain embodiments of the technology provided herein, the plurality of RCA products hybridized to the labeled probes is immobilized on the solid support in a dispersed manner, wherein at least a portion of the plurality of RCA products is individually detectable by detection of a label. In some embodiments, the dispersion of RCA products is irregular, while in some embodiments, the dispersion of RCA products is in an addressable array.

[0020] In any of the embodiments described herein, the complex immobilized on the surface can include at least one polypeptide (e.g., an antibody) and / or the complex can include at least one specific binding molecule selected from a hapten, a lectin, and a lipid.

[0021] In some embodiments, at least one labeled probe of the technology described herein includes a fluorescent label, while in some embodiments, at least one labeled probe includes a quencher moiety. In certain preferred embodiments, at least one labeled probe includes a fluorophore and a quencher moiety. In preferred embodiments, the at least one labeled probe is a molecular beacon probe.

[0022] In some embodiments of the technology, the plurality of RCA products is hybridized to labeled probes that all include the same label, while in some embodiments, the plurality of RCA products is hybridized to labeled probes that include two or more different labels, preferably two or more different fluorescent dyes.

[0023] Embodiments of the technology are not limited to any particular means of detecting or counting complexes bound to a surface. In some embodiments, the detecting or counting includes detecting fluorescence. In certain preferred embodiments, the detecting or counting includes fluorescence microscopy, while in some embodiments, the detecting or counting includes flow cytometry.

[0024] In some embodiments of the technology, forming the RCA products includes incubating the reaction mixture at at least 37°C, preferably at least 42°C, preferably at least 45°C. In certain embodiments, the reaction mixture includes PEG, preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG.

[0025] The technology also provides compositions related to practicing the methods. In some embodiments, the technology provides a composition comprising a silanized surface bound to a plurality of complexes each comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a solid support, and a reaction mixture comprising: at least 0.2 units of Phi29 DNA polymerase per μΐ, preferably at least 0.8 units per μΐ; a buffer; at least 400 μΜ, preferably at least 600 μΜ, more preferably at least 800 μΜ total dNTPs; and PEG, preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG. In some embodiments, the PEG has an average molecular weight of between 200 and 8000, preferably between 200 and 1000, preferably between 400 and 800, preferably 600. In some embodiments, the reaction mixture further comprises at least 100 nM molecular beacon probe, preferably at least 1000 nM molecular beacon probe.

[0026] In some embodiments of the composition, the primers are bound to the solid support in a random dispersion, while in some embodiments the primers are bound to the solid support in an addressable array. In certain embodiments, the primers are covalently linked to the solid support, while in some embodiments, wherein the primers comprise a biotin moiety and the solid support comprises avidin, preferably streptavidin. In some embodiments, the complexes comprise an antibody bound to an antigen or hapten, and in some embodiments, the complexes comprise an antigen or hapten bound directly to the solid support. In some embodiments, the antigen or hapten is covalently linked to the solid support.

[0027] In some embodiments of the composition herein, a complex comprises at least one polypeptide. In some preferred embodiments, the at least one polypeptide comprises an antibody. In some embodiments, the complex comprises at least one specific binding molecule selected from the group consisting of a hapten, a lectin, and a lipid.

[0028] Embodiments of the compositions described above can comprise a silanized surface bound to a plurality of complexes each comprising an RCA product comprising a plurality of hybridized labeled probes, and a solution comprising graphene oxide. In some embodiments, the silanized surface is glass. In some preferred embodiments, the silanized surface comprises a surface, preferably a glass surface, treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate.

[0029] In some embodiments, the solution comprising graphene oxide further comprises molecular beacon probes, preferably over 100 nM molecular beacon probes, preferably at least 1000 nM molecular beacon probes.

[0030] In some embodiments of the composition, the solution comprising graphene oxide comprises a buffer solution, the buffer solution comprising MgCl2. In certain embodiments, the buffer comprising MgCl2is Phi29 DNA polymerase buffer.

[0031] The present technology provided herein is not limited to any particular use or application. In some embodiments, the present technology can be used to analyze chromosomal aberrations (e.g., aneuploidy), preferably in the context of non-invasive prenatal testing. For example, some embodiments of the application of the present technology include obtaining a maternal sample comprising both maternal genetic material and fetal genetic material and measuring a plurality of target nucleic acids, wherein the target nucleic acids comprise specific sequences associated with a first chromosome, wherein the first chromosome is suspected to be a variant (e.g., in terms of gene dosage or chromosome count) of the fetal material, and wherein the target nucleic acids further comprise specific sequences associated with a second chromosome, which is not suspected to be a variant of the fetal material. The method includes analyzing the amount of the target nucleic acids associated with the first chromosome and the amount of the target nucleic acids associated with the second chromosome in the sample to determine whether the amount of the target nucleic acids associated with the first chromosome is significantly different from the amount of the target nucleic acids associated with the second chromosome to indicate a chromosomal or gene dosage variant in the fetus. In preferred embodiments, the target nucleic acids associated with the first and second chromosomes are present in both the maternal genetic material and the fetal genetic material and are maternal and fetal nucleic acids, the determination is not specific to. In preferred embodiments, the maternal sample is cell-free DNA from maternal blood. Statistical methods for analyzing chromosomal aberrations based on the amount of DNA in a sample, including determining aberrations in fetal DNA when the fetal DNA is a small fraction of the total DNA in the maternal sample, are known in the art. See, e.g., U.S. Patent No. 6,100,029, incorporated herein by reference.

[0032] The present technology includes the following embodiments:

[0033] 1. A method for counting target molecules on a solid support, the method comprising:

[0034] a) forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a solid support;

[0035] b) detecting the formation of the at least one complex in a process comprising:

[0036] i) extending the primers in the complex in a rolling circle amplification (RCA) reaction to form an RCA product;

[0037] ii) hybridizing a plurality of labeled probes to the RCA product; and

[0038] iii) detecting hybridized labeled probes;

[0039] wherein hybridized labeled probes are indicative of the presence of the target molecule on the solid support.

[0040] 2. The method of clause 1, wherein the solid support comprises a silanized surface.

[0041] 3. The method of clause 2, wherein the silanized surface comprises glass.

[0042] 4. A method for counting target molecules on a solid support, the method comprising:

[0043] a) providing a silanized surface comprising at least one of:

[0044] - an acrylic group;

[0045] - a reactive amine group;

[0046] b) forming a plurality of complexes on the glass surface, the plurality of complexes comprising at least one of:

[0047] - an RCA product comprising a plurality of hybridized labeled probes;

[0048] - a double stranded scaffold product comprising a plurality of catenated labeled scaffold oligonucleotides;

[0049] wherein the formation of complexes is indicative of the presence of target molecules on the glass surface, and wherein forming the plurality of complexes comprises exposing the glass surface to a solution comprising graphene oxide; and

[0050] c) counting the plurality of complexes;

[0051] 5. The method of clause 4, the silanized surface is glass.

[0052] 6. The method of clause 1 or clause 4, wherein the silanized surface comprises a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate.

[0053] 7. The method according to clause 1 or clause 4, wherein the solid support comprises a surface in an assay plate, preferably a glass-bottomed assay plate.

[0054] 8. The method according to clause 7, wherein the assay plate is a multi-well assay plate, preferably a microtiter plate.

[0055] 9. The method according to clause 1, wherein the primer is directly bound to the solid support, preferably covalently linked to the solid support.

[0056] 10. The method according to clause 1, wherein the primer comprises a biotin moiety and the solid support comprises avidin, preferably streptavidin.

[0057] 11. The method according to clause 1 or clause 4, wherein one or more complexes comprise an antibody bound to an antigen or hapten.

[0058] 12. The method according to clause 11, wherein the complex comprises an antigen or hapten directly bound to the solid support.

[0059] 13. The method according to clause 12, wherein the antigen or hapten is covalently linked to the solid support.

[0060] 14. The method according to clause 1 or clause 4, wherein forming the complex or the plurality of complexes comprises exposing the solid support to a solution comprising a crowding agent.

[0061] 15. The method according to clause 14, wherein the crowding agent comprises polyethylene glycol (PEG), preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG.

[0062] 16. The method according to clause 15, wherein the PEG has an average molecular weight of between 200 and 8000, preferably between 200 and 1000, preferably between 400 and 800, preferably 600.

[0063] 17. The method according to clause 1, comprising the step of exposing the solid support to a solution comprising graphene oxide.

[0064] 18. The method according to clause 17, wherein the solid support is exposed to graphene oxide prior to step b) iii).

[0065] 19. The method according to clause 17, wherein the solid support is exposed to graphene oxide prior to step b) ii).

[0066] 20. The method of clause 1, wherein the target molecule comprises a nucleic acid.

[0067] 21. The method of clause 4, wherein the target molecule comprises a nucleic acid.

[0068] 22. The method of clause 20 or 21, wherein the nucleic acid comprises DNA from a sample of a subject, preferably a blood or blood product sample.

[0069] 23. The method of clause 22, wherein the DNA is cell-free DNA from a blood or blood product sample.

[0070] 24. The method of clause 23, wherein the cell-free DNA comprises fetal DNA from a maternal blood sample.

[0071] 25. The method of clause 1 or clause 4, wherein forming a RCA product comprises extending a primer on a circularized nucleic acid probe in a reaction mixture comprising:

[0072] - at least 0.2 units of Phi29 DNA polymerase per μΐ, preferably at least 0.8 units per μΐ;

[0073] - at least 400 μΜ, preferably at least 600 μΜ, more preferably at least 800 μΜ total dNTPs.

[0074] 26. The method of clause 25, wherein hybridizing a labeled probe to the RCA product comprises forming the RCA product in a reaction mixture further comprising more than 100 nM molecular beacon probe, preferably at least 1000 nM molecular beacon probe in the reaction mixture.

[0075] 27. The method of clause 25, wherein forming a RCA product comprising a plurality of hybridized labeled probes comprises forming the RCA product in a reaction mixture further comprising more than 100 nM molecular beacon probe, preferably at least 1000 nM molecular beacon probe in the reaction mixture.

[0076] 28. The method of clause 25, wherein the reaction mixture further comprises PEG, preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG.

[0077] 29. The method of clause 28, wherein the PEG has an average molecular weight of between 200 and 8000, preferably between 200 and 1000, preferably between 400 and 800, preferably 600.

[0078] 30. The method of any one of clauses 1, 4, and 25, wherein a plurality of RCA products hybridized to labeled probes are immobilized in a dispersed manner on the solid support, wherein at least a portion of the plurality of RCA products are individually detectable by detecting a label.

[0079] 31. The method of clause 30, wherein the dispersion of RCA products is irregular.

[0080] 32. The method of clause 30, wherein the dispersion of RCA products is in an addressable array.

[0081] 33. The method of clause 30, wherein the complex comprises at least one polypeptide.

[0082] 34. The method of clause 33, wherein the at least one polypeptide comprises an antibody.

[0083] 35. The method of clause 30, wherein the complex comprises at least one specific bindable molecule selected from the group consisting of a hapten, a lectin, and a lipid.

[0084] 36. The method of any one of clauses 1, 4, and 25, wherein at least one labeled probe comprises a fluorescent label.

[0085] 37. The method of any one of clauses 1, 4, and 25, wherein at least one labeled probe comprises a quencher moiety.

[0086] 38. The method of any one of clauses 1, 4, and 25, wherein at least one labeled probe comprises a fluorophore and a quencher moiety.

[0087] 39. The method of clause 38, wherein the at least one labeled probe is a molecular beacon probe.

[0088] 40. The method of any one of clauses 1, 4, and 25, wherein a plurality of RCA products are hybridized to labeled probes that all comprise the same label.

[0089] 41. The method of any one of clauses 1, 4, and 25, wherein a plurality of RCA products are hybridized to labeled probes that comprise two or more different labels.

[0090] 42. The method of clause 41, wherein the two or more different labels comprise two or more different fluorescent dyes.

[0091] 43. The method of any one of clauses 1, 4, and 25, wherein the detecting or counting comprises detecting fluorescence.

[0092] 44. The method according to item 43, wherein detecting fluorescence comprises fluorescence microscopy.

[0093] 45. The method according to any one of items 1, 4 and 25, wherein forming RCA products comprises incubating the reaction mixture at at least 37°C, preferably at least 42°C, preferably at least 45°C.

[0094] 46. The method according to item 45, wherein the reaction mixture comprises PEG, preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG.

[0095] 47. The method according to any one of items 4, 17 to 19 and 25, wherein the solid support or the glass surface exposed to a solution comprising graphene oxide is washed with a solution comprising a detergent prior to the detecting or counting.

[0096] 48. The method according to item 47, wherein the detergent comprises Tween 20.

[0097] 49. A composition comprising a silanized surface bound to a plurality of complexes each comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a solid support, and a reaction mixture comprising

[0098] - at least 0.2 units of Phi29 DNA polymerase per μΐ, preferably at least 0.8 units per μΐ;

[0099] - a buffer;

[0100] - at least 400 μΜ, preferably at least 600 μΜ, more preferably at least 800 μΜ total dNTPs;

[0101] - PEG, preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG.

[0102] 50. The composition according to item 49, wherein the PEG has an average molecular weight of between 200 and 8000, preferably between 200 and 1000, preferably between 400 and 800, preferably 600.

[0103] 51. The composition according to item 49, wherein the reaction mixture further comprises at least 100 nM molecular beacon probe, preferably at least 1000 nM molecular beacon probe.

[0104] 52. The composition according to any one of clauses 49 to 51, wherein the primers are bound to the solid support in a random distribution.

[0105] 53. The composition according to any one of clauses 49 to 51, wherein the primers are bound to the solid support in an addressable array.

[0106] 54. The composition according to clause 49, wherein the primers are covalently linked to the solid support.

[0107] 55. The composition according to clause 49, wherein the primers comprise a biotin moiety and the solid support comprises avidin, preferably streptavidin.

[0108] 56. The composition according to clause 49, wherein the complex comprises an antibody bound to an antigen or hapten.

[0109] 57. The composition according to clause 49, wherein the complex comprises an antigen or hapten bound directly to the solid support.

[0110] 58. The composition according to clause 57, wherein the antigen or hapten is covalently linked to the solid support.

[0111] 59. The composition according to clause 49, wherein the complex comprises at least one polypeptide.

[0112] 60. The composition according to clause 59, wherein the at least one polypeptide comprises an antibody.

[0113] 61. The composition according to clause 49, wherein the complex comprises at least one specific binding molecule selected from the group consisting of a hapten, a lectin and a lipid.

[0114] 62. A composition comprising a silanized surface bound to a plurality of complexes each comprising an RCA product comprising a plurality of hybridized labeled probes, and a solution comprising graphene oxide.

[0115] 63. The composition according to clause 62, wherein the silanized surface is glass.

[0116] 64. The composition according to clause 62, wherein the silanized surface comprises a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate.

[0117] 65. The composition of clause 62, wherein the solution comprising graphene oxide further comprises molecular beacon probes, preferably more than 100 nM molecular beacon probes, preferably at least 1000 nM molecular beacon probes.

[0118] 66. The composition of clause 62, wherein the solution comprising graphene oxide comprises a buffer solution, the buffer solution comprising MgCl2.

[0119] 67. The composition of clause 66, wherein the buffer comprising MgCl2is Phi29 DNA polymerase buffer.

[0120] 68. The method of any one of clauses 1 to 4, wherein the solid support comprises a surface in an assay plate, preferably a glass-bottomed assay plate.

[0121] 69. The method of clause 68, wherein the assay plate is a multi-well assay plate, preferably a microtiter plate.

[0122] 70. The method of any one of clauses 1 to 3 and 68 to 69, wherein the primer is directly bound to the solid support, preferably covalently linked to the solid support.

[0123] 71. The method of any one of clauses 13 and 68 to 70, wherein the primer comprises a biotin moiety and the solid support comprises avidin, preferably streptavidin.

[0124] 72. Any one of clauses 1 to 4 and 68 to 71, wherein one or more complexes comprise an antibody bound to an antigen or hapten.

[0125] 73. The method of clause 72, wherein the complex comprises an antigen or hapten directly bound to the solid support.

[0126] 74. The method of clause 73, wherein the antigen or hapten is covalently linked to the solid support.

[0127] 75. The method of any one of clauses 1 to 4 and 68 to 74, wherein forming the complex or the plurality of complexes comprises exposing the solid support to a solution comprising a crowding agent.

[0128] 76. The method of clause 75, wherein the crowding agent comprises polyethylene glycol (PEG), preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG.

[0129] 77. The method according to Clause 76, wherein the average molecular weight of the PEG is between 200 and 8000, preferably between 200 and 1000, preferably between 400 and 800, preferably 600.

[0130] 78. The method according to any one of Clauses 1 to 3 and 68 to 77, comprising the step of exposing the solid support to a solution comprising graphene oxide.

[0131] 79. The method according to Clause 78, wherein the solid support is exposed to graphene oxide prior to step b) iii).

[0132] 80. The method according to Clause 78, wherein the solid support is exposed to graphene oxide prior to step b) ii).

[0133] 81. The method according to any one of Clauses 1 to 4 and 68 to 80, wherein the one or more target molecules comprise a nucleic acid.

[0134] 82. The method according to Clause 81, wherein the nucleic acid comprises DNA from a sample of a subject, preferably a blood or blood product sample.

[0135] 83. The method according to Clause 82, wherein the DNA is cell-free DNA from a blood or blood product sample.

[0136] 84. The method according to Clause 83, wherein the cell-free DNA comprises fetal DNA from a maternal blood sample.

[0137] 85. The method according to any one of Clauses 1 to 4 and 68 to 84, wherein forming the RCA product comprises extending a primer on a circularised nucleic acid probe in a reaction mixture comprising:

[0138] - at least 0.2 units of Phi29 DNA polymerase per μΐ, preferably at least 0.8 units per μΐ;

[0139] - at least 400 μΜ, preferably at least 600 μΜ, more preferably at least 800 μΜ total dNTPs.

[0140] 86. The method according to Clause 85, wherein hybridising a labelled probe to the RCA product comprises forming the RCA product in a reaction mixture further comprising more than 100 nM molecular beacon probe, preferably at least 1000 nM molecular beacon probe in the reaction mixture.

[0141] 87. The method according to any one of clauses 85 or 86, wherein forming RCA products comprising a plurality of hybridized labeled probes comprises forming the RCA products in a reaction mixture further comprising more than 100 nM molecular beacon probes, preferably at least 1000 nM molecular beacon probes in the reaction mixture.

[0142] 88. The method according to any one of clauses 85 to 87, wherein the reaction mixture further comprises PEG, preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG.

[0143] 89. The method according to clause 88, wherein the PEG has an average molecular weight of between 200 and 8000, preferably between 200 and 1000, preferably between 400 and 800, preferably 600.

[0144] 90. The method according to any one of clauses 1 to 4 and 68 to 89, wherein a plurality of RCA products hybridized to labeled probes are immobilized in a dispersed manner on the solid support, wherein at least a portion of the plurality of RCA products are individually detectable by detecting the label.

[0145] 91. The method according to clause 90, wherein the dispersion of RCA products is irregular.

[0146] 92. The method according to clause 90, wherein the dispersion of RCA products is in an addressable array.

[0147] 93. The method according to any one of clauses 90 to 92, wherein the complex comprises at least one polypeptide.

[0148] 94. The method according to clause 93, wherein the at least one polypeptide comprises an antibody.

[0149] 95. The method according to any one of clauses 90 to 94, wherein the complex comprises at least one specific binding molecule selected from a hapten, a lectin, and a lipid.

[0150] 96. The method according to any one of clauses 1 to 4 and 68 to 95, wherein at least one labeled probe comprises a fluorescent label.

[0151] 97. The method according to any one of clauses 1 to 4 and 68 to 96, wherein at least one labeled probe comprises a quencher moiety.

[0152] 98. The method according to any one of clauses 1 to 4 and 68 to 97, wherein at least one labeled probe comprises a fluorophore and a quencher moiety.

[0153] 99. The method of item 98, wherein the at least one labeled probe comprises a molecular beacon probe.

[0154] 100. The method of any one of items 1-4 and 68-99, wherein the plurality of RCA products hybridize to labeled probes that all comprise the same label.

[0155] 101. The method of any one of items 1-4 and 68-100, wherein the plurality of RCA products hybridize to labeled probes that comprise two or more different labels.

[0156] 102. The method of item 101, wherein the two or more different labels comprise two or more different fluorescent dyes.

[0157] 103. The method of any one of items 1-4 and 68-102, wherein the detecting or counting comprises detecting fluorescence.

[0158] 104. The method of item 103, wherein detecting fluorescence comprises fluorescence microscopy.

[0159] 105. The method of any one of items 1-4 and 68-104, wherein forming RCA products comprises incubating the reaction mixture at at least 37°C, preferably at least 42°C, preferably at least 45°C.

[0160] 106. The method of item 105, wherein the reaction mixture comprises PEG, preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG.

[0161] 107. The method of any one of items 78-106, wherein the solid support or the glass surface exposed to a solution comprising graphene oxide is washed with a solution comprising a detergent prior to the detecting or counting.

[0162] 108. The method of item 107, wherein the detergent comprises Tween 20.

[0163] 109. The composition of item 49 or item 50, wherein the reaction mixture further comprises at least 100 nM molecular beacon probe, preferably at least 1000 nM molecular beacon probe.

[0164] 110. The composition of any one of items 49 and 109, wherein the primers are covalently attached to the solid support.

[0165] 111. The composition of any one of clauses 49 and 109-110, wherein the primer comprises a biotin moiety and the solid support comprises avidin, preferably streptavidin.

[0166] 112. The composition of any one of clauses 49 and 109-111, wherein the complex comprises an antibody bound to an antigen or hapten.

[0167] 113. The composition of any one of clauses 49 and 109-112, wherein the complex comprises an antigen or hapten bound directly to the solid support.

[0168] 114. The composition of any one of clauses 49 and 109-113, wherein the antigen or hapten is covalently linked to the solid support.

[0169] 115. The composition of any one of clauses 49 and 109-114, wherein the complex comprises at least one polypeptide.

[0170] 116. The composition of any one of clauses 49 and 109-115, wherein the at least one polypeptide comprises an antibody.

[0171] 117. The composition of any one of clauses 49 and 109-116, wherein the complex comprises at least one specific binding molecule selected from the group consisting of a hapten, a lectin, and a lipid.

[0172] 118. The composition of clause 62 or 63, wherein the silanized surface comprises a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate.

[0173] 119. The composition of any one of clauses 62, 63, and 118, wherein the solution comprising graphene oxide further comprises molecular beacon probes, preferably more than 100 nM molecular beacon probes, preferably at least 1000 nM molecular beacon probes.

[0174] 120. The composition of clauses 62-63 and 118-119, wherein the solution comprising graphene oxide comprises a buffer solution, the buffer solution comprising MgCl2.

[0175] 121. The composition of clause 120, wherein the buffer comprising MgCl2is Phi29 DNA polymerase buffer.

[0176] 122. The method of any one of clauses 1-48 and 68-108, comprising detecting or counting for diagnostic purposes.

[0177] 123. The method of clause 123, wherein the diagnostic purpose comprises detecting an aneuploidy.

[0178] 124. The method of clause 123, wherein the aneuploidy in is a fetal aneuploidy detected in a maternal blood sample.

[0179] 125. The method of clause 123 or 124, wherein detecting an aneuploidy comprises detecting or counting cfDNA molecules from a maternal blood sample.

[0180] 126. The method of clause 125, wherein the cfDNA from a maternal blood sample comprises maternal DNA and fetal DNA.

[0181] 127. The method of clause 126, wherein maternal DNA and fetal DNA are detected by the same MIP probe in a single reaction mixture.

[0182] Definitions

[0183] To facilitate an understanding of the present application, a number of terms and phrases are defined below:

[0184] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Also, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, though it may. Thus, individual embodiments can be readily combined, so long as the

[0185] Additionally, as used in this document, unless otherwise clear from context, the term “or” is the inclusive “or” operator and is equivalent to the term “and / or”. Unless otherwise clear from context, the term “based on” is not exclusive and allows for being based on other factors not described. Also, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.”

[0186] The transitional phrase "consisting essentially of" is used in the claims of this application to limit the scope of the claims to the specified materials or steps "plus an insubstantial number of material or steps," as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition "consisting essentially of recited elements can contain a certain level of unrecited contaminants such that, despite the contaminants, the recited composition does not change in function as compared to a pure composition (i.e., a composition "consisting of recited components").

[0187] As used herein, the terms "subject" and "patient" refer to any organism, including plants, microorganisms, and animals (e.g., mammals such as dogs, cats, livestock, and humans).

[0188] The term "sample" in the specification and claims is used in its broadest sense. In one aspect it means to include a specimen or culture (e.g., a microbial culture). In another aspect it means to include both biological samples and environmental samples. A sample can include a specimen of synthetic origin. A biological sample can be an animal, including a human, a fluid, a solid (e.g., fecal matter) or tissue, as well as liquid and solid food and feed products and ingredients, such as dairy products, vegetables, meats and meat byproducts, and waste. Biological samples can be obtained from all different families of domesticated animals as well as undomesticated or wild animals, including but not limited to, ungulates, bear species, fish species, rabbit species, rodents, and the like.

[0189] Environmental samples include environmental materials, such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing instruments, instruments, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the types of samples suitable for use in the present application.

[0190] As used herein, the term "target" refers to a molecule sought to be sorted from other molecules for evaluation, measurement, or other characterization. For example, a target nucleic acid can be sorted from other nucleic acids in a sample, e.g., by probe binding, amplification, isolation, capture, etc. When used in reference to a hybridization-based detection (e.g., polymerase chain reaction), "target" refers to the region of nucleic acid defined by the primers used in the polymerase chain reaction, while when used in assays that do not amplify the target DNA (e.g., when captured by a molecular inversion probe (MIP)), the target includes the site defined by the hybridization of the target-specific arm of the MIP, such that the MIP can be ligated and the presence of the target nucleic acid can be detected.

[0191] The term "source of target nucleic acid" refers to any sample containing nucleic acid (RNA or DNA). A particularly preferred source of target nucleic acid is a biological sample, including but not limited to blood, plasma, serum, saliva, urine, stool, gastrointestinal fluid, cerebrospinal fluid, pleural fluid, milk, lymphatic fluid, saliva, and semen.

[0192] As used herein, the term "gene dosage" refers to the copy number of a gene, a gene region, a chromosome, or a fragment or portion thereof. Normal individuals carry two copies of most genes or gene regions, one on each of two chromosomes. However, there are certain exceptions, for example, when a gene or gene region is located on the X or Y chromosome, or when a gene sequence exists in a pseudogene.

[0193] As used herein, the term "aneuploidy" refers to a condition in which a cell, tissue, or individual has one or more complete chromosomes or chromosome fragments absent or in addition to the normal euploid complement of chromosomes.

[0194] As used herein, the "sensitivity" of a given assay (or a set of assays used together) refers to the percentage of samples reporting a particular form or variant, e.g., a mutation, gene duplication, chromosomal duplication, above a threshold value that differentiates between samples exhibiting a variant phenotype (e.g., cancerous cells, aneuploidy) and samples exhibiting a normal or wild-type phenotype (e.g., non-cancerous cells, euploidy). In some embodiments, a "positive" is defined as reporting a clinically confirmed variant of an assay result associated with the presence of a disease or condition being detected, and a false negative is defined as reporting a clinically confirmed variant of an assay result associated with the absence of a disease or condition. Thus, the value of sensitivity reflects the probability that a given diagnostic assay will produce a result indicating the presence of a variation or disease when performed on a known variant or diseased sample. As defined here, the clinical relevance of a calculated sensitivity value represents an estimate of the probability that a given assay will detect the presence of a clinical condition when applied to a subject having said condition. Using the present technology described herein, a level of accuracy can be achieved without the need to generate sequence reads. The accuracy can refer to sensitivity, the accuracy can refer to specificity, or the accuracy can refer to some combination thereof. The desired level of accuracy can be between 90% and 95%; the desired level of accuracy can be between 95% and 98%; the desired level of accuracy can be between 98% and 99%; the desired level of accuracy can be between 99% and 99.5%; the desired level of accuracy can be between 99.5% and 99.9%; the desired level of accuracy can be between 99.9% and 99.99%; the desired level of accuracy can be between 99.99% and 99.999%; the desired level of accuracy can be between 99.999% and 100%. An accuracy level higher than 95% can be referred to as high accuracy.

[0195] As used herein, the "specificity" of a given assay (or set of assays used together) refers to the percentage of normal samples reporting an assay result associated with the presence of the disease or condition being detected, and a false positive is defined as a clinically confirmed normal sample reporting an assay result associated with the presence of the disease or condition. Thus, the value of specificity reflects the probability that a given diagnostic assay performed on a known normal sample will yield a result indicating the presence of a variant or disease. The clinical relevance of a calculated specificity value, as defined here, represents an estimate of the probability that a given marker, when applied to a subject not suffering from a clinical condition, will detect the absence of that condition.

[0196] The term "gene" refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA (e.g., ribosomal RNA, or transfer RNA), a polypeptide, or a precursor. The RNA or polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.

[0197] As used herein, the term "genomic region" refers to a gene, its exons, its introns, and regions flanking it upstream and downstream, e.g., 5 to 10 kilobases 5' and 3' of the transcription start site and the transcription termination site, respectively.

[0198] As used herein, the term "genomic sequence" refers to a sequence of a gene, its introns, and regions flanking it upstream and downstream, e.g., 5 to 10 kilobases 5' and 3' of the transcription start site and the transcription termination site, respectively.

[0199] As used herein, the term "chromosome-specific" refers to a sequence found only in the particular type of chromosome.

[0200] As used herein, the term "hybridization" is used to refer to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of association between nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, the m The process of "hybridization" involves annealing one nucleic acid to another complementary nucleic acid, i.e., a nucleic acid having a complementary sequence of nucleotides. The ability of two nucleic acid polymers containing complementary sequences to find and anneal to each other and interact through base pairing is a well-recognized phenomenon. Following the initial observations of the process of "hybridization" by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960), the process was refined into an important tool of modern biology.

[0201] As used herein, the term "oligonucleotide" is defined as a molecule that includes two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides, and more preferably at least about 15 to 30 nucleotides. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or combinations thereof.

[0202] Because mononucleotides are reacted to form oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is linked by a phosphodiester bond to the 3' oxygen of its adjacent mononucleotide pentose ring, the end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring, and the end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to the 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence can be referred to as having 5' and 3' ends even if it is internal to a larger oligonucleotide. A first region along a nucleic acid chain is referred to as being upstream of another region if the 3' end of the first region precedes the 5' end of the other region when moving in the 5' to 3' direction along the nucleic acid chain.

[0203] When two different non-overlapping oligonucleotides anneal to different regions of the same single-stranded complementary nucleic acid sequence and the 3' end of one oligonucleotide is directed toward the 5' end of the other oligonucleotide, the former can be referred to as the "upstream" oligonucleotide and the latter as the "downstream" oligonucleotide. Similarly, when two overlapping oligonucleotides hybridize to the same single-stranded complementary nucleic acid sequence and the first oligonucleotide is positioned such that its 5' end is upstream of the 5' end of the second oligonucleotide and the 3' end of the first oligonucleotide is upstream of the 3' end of the second oligonucleotide, the first oligonucleotide can be referred to as the "upstream" oligonucleotide and the second oligonucleotide can be referred to as the "downstream" oligonucleotide.

[0204] The term "primer" refers to an oligonucleotide that is capable of acting as a point of initiation for synthesis when placed under conditions in which primer extension is initiated, e.g., in the presence of nucleotides and a suitable nucleic acid polymerase. Oligonucleotide "primers" can occur naturally, can be made using molecular biology methods (e.g., purification of restriction digests), or can be produced synthetically. In preferred embodiments, primers are composed of or include DNA.

[0205] Primers are selected to be "substantially" complementary to a specific sequence of the template. The primer must be sufficiently complementary to the template strand to hybridize with the template strand such that extension of the primer can occur. The primer sequence need not reflect the exact sequence of the template. For example, a fragment of non-complementary nucleotides can be attached to the 5' end of the primer, and the remainder of the primer sequence can be substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer sequence, provided the primer sequence is sufficiently complementary to the sequence of the template to hybridize and thereby form a template-primer complex for synthesis of the extension product of the primer.

[0206] As used herein, the term "sequence variant" refers to a difference in nucleic acid sequence between two nucleic acids. For example, a wild-type structural gene and a mutant form of this wild-type structural gene can differ in sequence by the presence of a single base substitution and / or a deletion or insertion of one or more nucleotides. These two forms of the structural gene are said to differ from each other in sequence. A second mutant form of the structural gene can exist. This second mutant form can be said to differ in sequence from both the wild-type gene and the first mutant form of the gene.

[0207] As used herein, the term "nucleotide analog" refers to a modified or non-naturally occurring nucleotide, including but not limited to, analogs with altered stacking interactions, such as 7-deazapurines (i.e., 7-deaza-dATP and 7-deaza-dGTP); base analogs with alternative hydrogen bonding configurations (e.g., Iso-C and Iso-G and other non-standard base pairs as described in U.S. Patent No. 6,001,983 to S. Benner); non-hydrogen bonding analogs (e.g., non-polar aromatic nucleoside analogs such as 2,4-difluorotoluene, as described by B. A. Schweitzer and E. T. Kool, J. Org. Chem., 1994, 59, 7238-7242, B. A. Schweitzer and E. T. Kool, J. Am. Chem. Soc, 1995, 117, 1863-1872); "universal" bases such as 5-nitroindole and 3-nitropyrrole; and universal purines and pyrimidines (as "K" nucleotides and "P" nucleotides, respectively; P. Kong et al., Nucleic Acids Res., 1989, 17, 10373-10383, P. Kong et al., Nucleic Acids Res., 1992, 20, 5149-5152). Nucleotide analogs include base analogs and encompass modified forms of deoxyribonucleotides as well as ribonucleotides, and include but are not limited to modified bases and nucleotides described in U.S. Patent Nos. 5,432,272; 6,001,983; 6,037,120; 6,140,496; 5,912,340; 6,127,121; and 6,143,877, each of which is incorporated herein by reference in its entirety; heterocyclic base analogs and other heterocyclic bases based on purine or pyrimidine ring systems.

[0208] As used herein, the term "continuous nucleic acid strand" means a nucleic acid strand having a continuous, covalently linked backbone structure without nicks or other disruptions. The disposition of the base portion of each nucleotide, whether base-paired, single-stranded, or mismatched, is not an element of the definition of a continuous strand. The backbone of a continuous strand is not limited to the ribo-phosphate composition or the deoxyribo-phosphate composition found in naturally occurring, unmodified nucleic acids. The nucleic acids of the present application can include modifications in the structure of the backbone, including but not limited to, phosphorothioate residues, phosphonate residues, 2' substituted ribose residues (e.g., 2'-O-methyl ribose), and alternative sugars containing residues (e.g., arabinose).

[0209] As used herein, the term "continuous duplex" refers to a region of a double-stranded nucleic acid in which there is no disruption to the progression of base pairs within the duplex (i.e., the base pairs along the duplex do not twist to accommodate gaps, bulges, or mismatches with the extent of the region of continuous duplex). As used herein, the term refers only to the arrangement of base pairs within the duplex, without implying continuity in the backbone portion of the nucleic acid strands. Double-stranded nucleic acids with uninterrupted base pairing but with nicks in one or both strands are within the definition of continuous duplex.

[0210] The term "duplex" refers to the state of a nucleic acid in which the base portions of the nucleotides on one strand are bound by hydrogen bonding to their complementary bases arranged on the second strand. The condition of being in duplex form is reflected in the state of the bases of the nucleic acid. By virtue of base pairing, the nucleic acid strands also typically adopt a triple helical structure with a major groove and a minor groove in the duplex form. Adoption of the helical form is implicit in the act of becoming duplexed.

[0211] The term "template" refers to a nucleic acid strand on which a complementary copy is constructed from nucleoside triphosphates by the activity of a template-dependent nucleic acid polymerase. By convention, the template strand is depicted and described as the "bottom" strand within a duplex. Similarly, the non-template strand is typically depicted and described as the "top" strand.

[0212] The term "substantial identity" when applied to a polynucleotide indicates a property of the polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85% sequence identity, preferably at least 90% to 95% sequence identity, more usually at least 99% sequence identity, over a comparison window of at least 20 nucleotide positions, usually 25-50 nucleotide positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which can include up to 20% or less deletions or additions in the comparison window of reference sequence. The reference sequence can be a subset of a larger sequence, for example a splice variant of a full-length sequence.

[0213] The term "substantial identity" when applied to polypeptides, indicates that two polypeptide sequences, when optimally aligned, share at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or more (e.g., 99% sequence identity) as measured by the program GAP or BESTFIT using default parameters. Preferably, differing residues are conservative amino acid substitutions. Conservative amino acid substitutions are those in which the replaced residue and the replacing residue have similar side chains. For example, one group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a second group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a third group of amino acids having amide-containing side chains is asparagine and glutamine; a fourth group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; and a fifth group of amino acids having basic side chains is lysine, arginine, and histidine. Preferred conservative amino acids substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0214] As used herein, the term "label" refers to anything that can be used to provide a detectable, preferably quantifiable, effect and can be attached to any atom or molecule of a nucleic acid or protein. Labels include, but are not limited to, dyes: radioactive labels, such as 32P; a binding moiety, such as biotin; a hapten, such as digoxgenin; a luminescent, phosphorescent, or fluorescent moiety; a mass tag; and a fluorescent dye alone or in combination with a moiety that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). FRET is a distance-dependent interaction between the excited states of two molecules (e.g., two dye molecules or one dye molecule and one non-fluorescent quencher molecule), in which excitation is transferred from the donor molecule to the acceptor molecule without emission of a photon. (Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol. 246:300, each incorporated herein by reference). As used herein, the term "donor" refers to a fluorophore that absorbs at a first wavelength and emits at a second, longer wavelength. The term "acceptor" refers to a moiety, such as a fluorophore, chromophore, or quencher, whose absorption spectrum overlaps with the emission spectrum of the donor and is capable of absorbing some or most of the emitted energy from the donor when in close proximity (typically between 1-100 nm) to the donor group. If the acceptor is a fluorophore, it typically re-emits at a third, still longer wavelength; if it is a chromophore or quencher, it releases the energy absorbed from the donor without emission of a photon. In some embodiments, a change in detectable emission from the donor dye (e.g., when the acceptor moiety is brought into or removed from proximity) is detected. In some embodiments, a change in detectable emission from the acceptor dye is detected. In preferred embodiments, the emission spectrum of the acceptor dye is different from the emission spectrum of the donor dye, such that emission from the dyes can be distinguished from one another (e.g., spectrally resolved).

[0215] In some embodiments, a donor dye is used in combination with multiple acceptor moieties. In preferred embodiments, the donor dye is used in combination with a non-fluorescent quencher and with an acceptor dye, such that when the donor dye is in proximity to the quencher, its excitation is transferred to the quencher rather than the acceptor dye, and when the quencher is removed (e.g., by cleavage of the probe), excitation of the donor dye is transferred to the acceptor dye. In particularly preferred embodiments, emission from the acceptor dye is detected. See, e.g., Tyagi et al., Nature Biotechnology 18:1191 (2000), incorporated herein by reference.

[0216] The label can provide a signal that can be detected by fluorescence (e.g., simple fluorescence, FRET, time-resolved fluorescence, fluorescence polarization, etc.), radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, mass or mass-affected behavior (e.g., MALDI time-of-flight mass spectrometer), etc. The label can be a charged moiety (positive or negative charge) or can alternatively be charge neutral. The label can comprise or consist of a nucleic acid or protein sequence, so long as the sequence including the label is detectable.

[0217] In some embodiments, the label comprises a particle for detection. In preferred embodiments, the particle is a phosphor particle. In particularly preferred embodiments, the phosphor particle is an up-converting phosphor particle (see, e.g., Ostermayer, F. W. Preparation and properties of infrared-to-visible conversion phosphors. Metall. Trans. 752, 747-755

[1971] ). In some embodiments, rare earth doped ceramic particles are used as the phosphor particle. The phosphor particle can be detected by any suitable method, including but not limited to up-converting phosphor technology (UPT), in which up-converting phosphors convert low-energy infrared (IR) radiation into high-energy visible light. While the present invention is not limited to any particular mechanism, in some embodiments, UPT up-converts infrared light to visible light through multi-photon absorption and subsequent emission of dopant-dependent phosphorescence. See, e.g., Zarling et al. U.S. Patent No. 6,399,397, issued June 4, 2002; van De Rijke et al., Nature Biotechnol. 19(3):273-6

[2001] ; Corstjens et al., IEE Proc. Nanobiotechnol. 152(2):64

[2005] , each of which is incorporated herein by reference in its entirety.

[0218] As used herein, the term "solid phase support" or "support" refers to any material that provides a solid or semi-solid structure to which another material can be attached. Such materials include smooth supports (e.g., smooth metal, glass, quartz, plastic, silicon, wafer, carbon (e.g., diamond), and ceramic surfaces, etc.) as well as textured and porous materials. Such materials also include, but are not limited to, gels, rubbers, polymers, and other non-rigid materials. The solid phase support need not be flat. Supports include any type of shape, including spherical shapes (e.g., beads).

[0219] As used herein, the term "bead" refers to a small solid phase support that is capable of moving about when in solution (e.g., its size is less than the size of the enclosure or vessel in which the solution is located). In some embodiments, the beads can settle out of solution when the solution is not mixed (e.g., by shaking, thermal mixing, vortexing), while in other embodiments, the beads can be suspended in the solution in a colloidal form. In some embodiments, the beads are completely or partially spherical or cylindrical. However, the beads are not limited to any particular three-dimensional shape.

[0220] The material attached to the solid phase support can be attached to any portion of the solid phase support (e.g., can be attached to an interior portion of a porous solid phase support material or to a flat portion on a support that is otherwise not flat, or vice versa). In preferred embodiments of the present technology, a biological molecule, such as a nucleic acid or protein molecule, is attached to the solid phase support. The biological material is "attached" to the solid phase support when it is affixed to the solid phase support through a chemical or physical interaction. In some embodiments, the attachment is through a covalent bond. However, the attachment need not be covalent and need not be permanent. In some embodiments, the attachment can be reversed or dissociated by a change in conditions (e.g., by a change in temperature, a change in ionic strength, the addition or removal of a chelating agent, or other changes in the conditions of the solution to which the surface and bound molecule are exposed).

[0221] In some embodiments, the target molecule (e.g., biological material) is attached to the solid phase support through a "spacer molecule" or "linker group." Such spacer molecules are molecules that have a first portion attached to the biological material and a second portion attached to the solid phase support. The spacer molecule typically includes a chain of atoms (e.g., carbon atoms) that provides additional distance between the first and second portions. Thus, the spacer molecule allows for separation between the solid phase support and the biological material when attached to the solid phase support, but is attached to both.

[0222] As used herein, the terms "array" and "microarray" refer to a surface or vessel comprising a plurality of predefined loci that are addressable for analysis at the loci (e.g., to determine the results of an assay). Analysis at loci in an array is not limited to any particular type of analysis and includes, for example, analysis for detection of atoms, molecules, chemical reactions, light or fluorescent emission, inhibition, or changes in a property of the locus (e.g., intensity or wavelength) indicative of a result at the locus. Examples of predefined loci include a grid or any other pattern in which loci to be analyzed are determined by their known position in the array pattern. Microarrays are generally described, for example, in Schena, Microarray Biochip Technology, Eaton Publishing, Natick, MA, 2000. Examples of arrays include, but are not limited to, a support having a plurality of molecules non-randomly bound to the surface (e.g., in a grid or other regular pattern) and a vessel comprising a plurality of defined reaction loci (e.g., wells) in which molecules or signal generating reactions can be detected. In some embodiments, an array comprises a patterned distribution of wells that receive beads, e.g., as described above for SIMOA technology. See also U.S. Patent Nos. 9,057,730; 9,556,429; 9,481,883; and 9,376,677, each of which is incorporated herein by reference in its entirety for all purposes.

[0223] As used herein, the term "irregular distribution" used with respect to loci on a solid support or surface refers to a distribution of loci on or in a surface in a non-arrayed manner. For example, molecules can be irregularly distributed on a surface by applying a solution of a particular concentration that provides a desired approximate average distance between molecules on the surface, but not at loci that are predefined or addressable by any pattern on the surface or by virtue of the application of the solution (inkjet printing). In such embodiments, analysis of the surface can include finding loci of molecules by detecting signals wherever they can occur (e.g., scanning the entire surface to detect fluorescence anywhere on the surface). This is in contrast to locating signals by analyzing the surface or vessel only at predetermined loci (e.g., points in a grid array) to determine how much (or what type of) signal occurs at each locus in the grid.

[0224] As used herein, the term "distinguishable" with respect to signals refers to signals that can be distinguished from one another, e.g., by spectral properties such as fluorescent emission wavelength, color, absorbance, mass, size, fluorescence polarization properties, charge, or by the ability to interact with another moiety such as a chemical reagent, enzyme, antibody, etc.

[0225] As used herein, the term "nucleic acid detection assay" refers to any method of determining the nucleotide composition of a nucleic acid of interest.Nucleic acid detection assays include, but are not limited to, DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assays (Hologic, Inc.) and described, e.g., in U.S. Pat. Nos. 5,846,717; 5,985,557; 5,994,069; 6,001,567; 6,090,543; and 6,872,816; Lyamichev et al., Nature Biotechnology 17:292 (1999), Hall et al., Proc. Natl. Acad. Sci. USA, 97:8272 (2000), and U.S. Pat. No. 9,096,893, each of which is incorporated herein by reference in its entirety for all purposes); enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, incorporated herein by reference in their entireties); polymerase chain reaction (PCR), described above; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, 5,624,802, incorporated herein by reference in their entireties); rolling circle amplification (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960, 6,235,502, incorporated herein by reference in their entireties); variations of rolling circle amplification, known as “RAM amplification” (see, e.g., U.S. 5,942,391, incorporated herein by reference in its entirety); NASBA (e.g., U.S. Pat. No. 5,409,818, incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, incorporated herein by reference in its entirety); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, incorporated herein by reference in their entireties); cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, incorporated herein by reference in their entireties); Dade Behring signal amplification methods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, incorporated herein by reference in their entireties); ligase chain reaction (e.g., Barany Proc. Natl. Acad. Sci. USA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Pat. No. 5,288,609, incorporated herein by reference in its entirety).

[0226] In some embodiments, target nucleic acids are amplified (e.g., by PCR) and the amplified nucleic acids are detected simultaneously using an invasive cleavage assay. Assays configured for detection assays (e.g., invasive cleavage assays) in combination with amplification assays are described in U.S. Patent No. 9,096,893, which is incorporated by reference herein in its entirety for all purposes. Additional invasive cleavage detection configurations on amplification (referred to as the QuARTS method) are described in, e.g., U.S. Patent Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392, each of which is incorporated by reference herein for all purposes. As used herein, the term “invasive cleavage structure” refers to a cleavage structure comprising: i) a target nucleic acid; ii) an upstream nucleic acid (e.g., an invasive or “INVADER” oligonucleotide); and iii) a downstream nucleic acid (e.g., a probe), wherein the upstream and downstream nucleic acids anneal to contiguous regions of the target nucleic acid, and wherein an overlap is formed between the 3’ portion of the upstream nucleic acid and the duplex formed between the downstream nucleic acid and the target nucleic acid. The overlap occurs when one or more bases of the upstream and downstream nucleic acids occupy the same position relative to the target nucleic acid bases, regardless of whether the one or more overlapping bases of the upstream nucleic acid are complementary to the target nucleic acid, and regardless of whether the bases are natural or non-natural bases. In some embodiments, the 3’ portion of the upstream nucleic acid that overlaps with the downstream duplex is a non-base chemical moiety, such as an aromatic ring structure, e.g., as disclosed in U.S. Patent No. 6,090,543, incorporated by reference herein in its entirety. In some embodiments, one or more of the nucleic acids can be attached to one another, e.g., by a covalent bond (such as a nucleic acid stem loop) or by a non-nucleic acid chemical linkage (e.g., a polycarbon chain). As used herein, the term “flap endonuclease assay” encompasses both “INVADER” invasive cleavage assays and QuARTS assays as described above.

[0227] As used herein, the terms "digital PCR", "single molecule PCR" and "single molecule amplification" refer to PCR and other nucleic acid amplification methods configured to provide amplification products or signals from a single starting molecule. Typically, a sample is divided such that each portion or dilution has on average no more than a single copy of a target nucleic acid as assessed according to a Poisson distribution, for example by serial dilution or by partitioning into sufficiently small portions (e.g., in microchambers or in emulsions). Methods of single molecule PCR are described, for example, in US 6,143,496 (which relates to a method comprising dividing a sample into a plurality of chambers such that at least one chamber has at least one target; and amplifying the target to determine how many chambers have a target molecule); US 6,391,559; (which relates to assemblies for containing and dispensing fluids); and US 7,459,315 (which relates to a method that divides a sample into an assembly having sample chambers (where the sample is partitioned to the chambers by surface affinity), then seals the chambers with a solidifiable "displacing fluid"). See also US 6,440,706 and US 6,753,147 and Vogelstein et al., Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 9236-9241, August 1999. See also US 20080254474, which describes the combination of digital PCR with methylation detection.

[0228] As used herein, the term "sequencing" is used in a broad sense and can refer to any technique known in the art that allows the identification of the order of at least some consecutive nucleotides in at least a portion of a nucleic acid, including but not limited to at least a portion of an extension product or a vector insert. In some embodiments, sequencing allows for the differentiation between sequence differences between different target sequences. Exemplary sequencing techniques include targeted sequencing, single molecule real-time sequencing, electron microscope-based sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, targeted sequencing, exome sequencing, whole genome sequencing, hybridization-based sequencing, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cyclic sequencing, single base extension sequencing, solid phase sequencing, high-throughput sequencing, massively parallel signature tag sequencing, emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR) under lower denaturation temperature, multiplex PCR, reversible dye terminator sequencing, paired-end sequencing, near-term sequencing, exonuclease sequencing, ligation-based sequencing, short read sequencing, single molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse terminator sequencing, ion semiconductor sequencing, nanoball sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, miSeq (Illumina), HiSeq 2000 (Illumina), HiSeq 2500 (Illumina), Illumina Genome Analyzer (Illumina), Ion Torrent PGM TM (Life Technologies), MinION TM (Oxford Nanopore Technologies), real-time SMRT TM (cPAL TM (Complete Genomics / BGI), sequencing, MS-PET sequencing, mass spectrometry, and combinations thereof. In some embodiments, sequencing comprises the use of an instrument to detect sequencing products, such as, but not limited to, ABI 377 DNA Sequencer, ABI 310, 3100, 3100-Avant, 3730, or 3730xI Genetic Analyzer, ABI 3700 DNA Analyzer, or Applied Biosystems SOLiD TMsystems (all from Applied Biosystems), Genome Sequencer 20 System (Roche Applied Science), or mass spectrometers. In certain embodiments, sequencing comprises emulsion PCR. In certain embodiments, sequencing comprises high-throughput sequencing technology, such as, but not limited to, massively parallel signature sequencing (MPSS).

[0229] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably with respect to a chain of two or more amino acids joined together by peptide bonds. Polypeptides can be synthetic or naturally occurring and can be short (e.g., between two and about 30 amino acid residues) or can be hundreds or thousands of amino acid residues in length. Polypeptides can be composed of the 20 naturally occurring amino acids, or can include one or more non-naturally occurring amino acids (e.g., peptide nucleic acid residues that include pyrimidine or purine bases in the peptide chain backbone) or modified versions of naturally occurring amino acids (e.g., modified in the structure of a side group).

[0230] As used herein, the term "antibody" (Ab) refers to antigen-binding immunoglobulins and includes monoclonal antibodies (mAbs) and polyclonal Abs. The term further includes all modified forms of antibodies having the ability to bind to an antigen, such as fragment antibodies (fAbs) that include portions of the immunoglobulin structure.

[0231] As used herein, the terms "crowding agent" and "volume exclusion agent" are used interchangeably and refer to a compound (typically, a polymeric compound) that reduces the available fluid volume in a reaction mixture, thereby increasing the effective concentration of reactant macromolecules (e.g., nucleic acids, enzymes, etc.) as used with respect to components of a fluid reaction mixture. Crowding agents include, for example, glycerol, ethylene glycol, polyethylene glycol, ficoll, serum albumin, casein, and dextran.

[0232] As used herein, the terms "digital sequencing," "single molecule sequencing," and "next generation sequencing (NGS)" are used interchangeably and refer to determining the nucleotide sequence of individual nucleic acid molecules. Systems for sequencing individual molecules include, but are not limited to, 454 FLX TM or 454 TITANIUM TM (Roche), SOLEXA TM / Illumina Genome Analyzer (Illumina), HELISCOPE TM single molecule sequencer (Helicos Biosciences), and SOLID TMDNA sequencers (Applied Biosystems / Life Technologies) instruments) and other platforms still under development by companies such as Intelligent Biosystems and Pacific Biosciences. See also U.S. Patent No. 7,888,017 entitled "Non-invasive fetal gene screening by digital analysis" which relates to digital analysis of maternal and fetal DNA (e.g., cfDNA).

[0233] As used herein, the term "probe" or "hybridization probe" refers to an oligonucleotide (i.e., nucleotide sequence) whether naturally occurring in purified restriction digests or synthetically, recombinantly or by PCR amplification, capable of at least partial hybridization to another oligonucleotide of interest. Probes can be single-stranded or double-stranded. Probes can be used to detect, identify and isolate specific sequences. In some preferred embodiments, probes used in the present application will be labeled with a "reporter molecule" so that detection can be made in any detection system including but not limited to enzyme systems (e.g., ELISA and enzyme-based histochemical assays), fluorescent systems, radioactive systems and luminescent systems. It is not intended to limit the present application to any particular detection system or label.

[0234] As used herein, the term "MIP" refers to a molecular inversion probe (or circular capture probe). A molecular inversion probe (or circular capture probe) is a nucleic acid molecule comprising a pair of unique polynucleotide arms, one or more unique molecular tags (or unique molecular identifiers) and a polynucleotide linker (e.g., a universal primary linker). See, e.g., Figure 1 In some embodiments, a MIP can comprise more than one unique molecular tag, such as two unique molecular tags, three unique molecular tags or more. In some embodiments, the unique polynucleotide arms in each MIP are located at the 5' and 3' ends of the MIP, while the one or more unique molecular tags and the polynucleotide linker are located internally to the 5' and 3' ends of the MIP. For example, a MIP used in some embodiments of the present disclosure comprises the following components in order: a first unique polynucleotide arm - a first unique molecular tag - a polynucleotide linker - a second unique molecular tag - a second unique polynucleotide arm. In some embodiments, a MIP is a 5' phosphorylated single-stranded nucleic acid (e.g., DNA) molecule. See, e.g., WO 2017 / 020023 filed July 29, 2016 and WO 2017 / 020024 filed July 29, 2016, each of which is incorporated herein by reference for all purposes.

[0235] A unique molecular tag can be any tag that is detectable and can be incorporated into or attached to a nucleic acid (e.g., polynucleotide) and allows detection and / or identification of the nucleic acid comprising the tag. In some embodiments, the tag is incorporated into or attached to the nucleic acid during sequencing (e.g., by a polymerase). Non-limiting examples of tags include nucleic acid tags, nucleic acid indexes or barcodes, radioactive labels (e.g., isotopes), metal labels, fluorescent labels, chemiluminescent labels, phosphorescent labels, fluorophore quenchers, dyes, proteins (e.g., enzymes, antibodies or portions thereof, linkers, members of binding pairs), and the like or combinations thereof. In some embodiments, particularly sequencing embodiments, the tag (e.g., molecular tag) is a unique, known, and / or identifiable sequence of nucleotides or nucleotide analogs (e.g., nucleotides comprising nucleic acid analogs, sugars, and one to three phosphate groups). In some embodiments, the tag is six or more contiguous nucleotides. A variety of different excitation and emission spectra can obtain many fluorophore-based tags. Any suitable type and / or number of fluorophores can be used as tags. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 500 or more, 1000 or more, 10,000 or more, 100,000 or more different tags are used in the methods described herein (e.g., nucleic acid detection and / or sequencing methods). In some embodiments, one or two types of tags (e.g., different fluorescent labels) are attached to each nucleic acid in a library. In some embodiments, chromosome-specific tags are used to make chromosome counting faster or more efficient. Detection and / or quantification of labels can be performed by suitable methods, machines, or devices, non-limiting examples of which include flow cytometers, quantitative polymerase chain reaction (qPCR), gel electrophoresis, luminometers, fluorometers, spectrophotometers, suitable gene chip or microarray analysis, Western blots, mass spectrometry, chromatography, cellular fluorescence analysis, fluorescence microscopy, suitable fluorescence or digital imaging methods, confocal laser scanning microscopy, laser scanning cytometry, affinity chromatography, manual batch mode separation, electric field suspension, suitable nucleic acid sequencing methods and / or nucleic acid sequencing devices, and the like and combinations thereof.

[0236] In MIPs, unique polynucleotide arms are designed to hybridize immediately upstream and downstream of a particular target sequence (or site) in a genomic nucleic acid sample. In some embodiments, MIPs include unique molecular tags, which are short nucleotide sequences that are generated randomly. In some embodiments, unique molecular tags do not hybridize to any sequence or site located on a genomic nucleic acid fragment or in a genomic nucleic acid sample. In some embodiments, the polynucleotide linker (or master linker) in MIPs is universal across all MIPs used in embodiments of the present disclosure.

[0237] In some embodiments, MIPs are introduced to nucleic acid fragments derived from a test subject (or a reference subject) to perform capture of a target sequence or site (or a control sequence or site) located on a nucleic acid sample (e.g., genomic DNA). In some embodiments, fragmentation aids in capture of target nucleic acids by molecular inversion probes. In some embodiments, for example, when a nucleic acid sample comprises cell-free nucleic acids, fragmentation can not be required to improve capture of target nucleic acids by molecular inversion probes. As described in greater detail herein, after capture of a target sequence of interest (e.g., a locus), an enzymatic gap fill and ligation step can be performed on the captured target such that copies of the target sequence are incorporated into a circle-like structure. In some embodiments, nucleic acid analogs, for example, containing labels, haptens, and the like, can be incorporated into the filled-in segment for, for example, downstream detection, purification, or other processing steps. In some embodiments, the efficiency of capture of a target sequence on a nucleic acid fragment by MIPs can be improved by extending the hybridization and gap fill incubation period. (See, e.g., Turner E H et al. Nat Methods. 2009 Apr 6: 1-2).

[0238] In some embodiments, MIPs for capturing a target site or target sequence according to the present disclosure sequentially include the following components: a first targeting polynucleotide arm - a first unique targeting molecular tag - a polynucleotide linker - a second unique targeting molecular tag - a second targeting polynucleotide arm.

[0239] In some embodiments, MIPs for capturing a control site or control sequence in the present disclosure sequentially include the following components: a first control polynucleotide arm - a first unique control molecular tag - a polynucleotide linker - a second unique control molecular tag - a second control polynucleotide arm.

[0240] MIP technology can be used to detect or amplify specific nucleic acid sequences in complex mixtures. One of the advantages of using MIP technology is its high multiplexing capability, which allows for the capture of thousands of target sequences in a single reaction containing thousands of MIPs. Various aspects of MIP technology are described in, for example, Hardenbol et al., "Multiplexed genotyping with sequence-tagged molecular inversion probes" Nature Biotechnology 21(6):673-678 (2003); Hardenbol et al., "Highly multiplexed molecular inversion probe genotyping: Over 10,000 targeted SNPs genotyped in a single tube assay" Genome Research 15:269-275 (2005); Burmester et al., "DMET microarray technology for pharmacogenomics-based personalized medicine" Methods in Molecular Biology, 632:99-124 (2010); Sissung et al., "Clinical pharmacology and pharmacogenetics in a genomics era: the DMET platform" Pharmacogenomics, 11(1):89-103 (2010); Deeken, "The Affymetrix DMET platform and pharmacogenetics in drug development" Current Opinion in Molecular Therapeutics, 11(3):260-268 (2009);Wang et al., "High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays," BMC Medical Genomics, 2:8 (2009); Wang et al., "Analysis of molecular inversion probe performance for allele copy number determination," Genome Biology, 8(11):R246 (2007); Ji et al., "Molecular inversion probe analysis of gene copy alternations reveals distinct categories of colorectal carcinoma," Cancer Research, 66(16):7910-7919 (2006); and Wang et al., "Allele quantification using molecular inversion probes (MIP)," Nucleic Acids Research, 33(21):e183 (2005), each of which is incorporated by reference in its entirety for all purposes. See also U.S. Patent Nos. 6,858,412; 5,817,921; 6,558,928; 7,320,860; 7,351,528; 5,866,337; 6,027,889; and 6,852,487, each of which is incorporated by reference in its entirety for all purposes.

[0241] MIP technology has previously been successfully applied in other areas of research, including novel identification and subclassification of biomarkers in cancer. See, e.g., Brewster et al., "Copy number imbalances between screen-and symptom-detected breast cancers and impact on disease-free survival," Cancer Prevention Research, 4(10): 1609-1616 (2011); Geiersbach et al., "Unknown partner for USP6 and unusual SS18 rearrangement detected by fluorescence in situ hybridization in a solid aneurysmal bone cyst," Cancer Genetics, 204(4): 195-202 (2011); Schiffman et al., "Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas," Cancer Research, 70(2): 512-519 (2010); Schiffman et al., "Molecular inversion probes reveal patterns of 9p21 deletion and copy number aberrations in childhood leukemia," Cancer Genetics and Cytogenetics, 193(1): 9-18 (2009).Press et al., "Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities," BMC Cancer, 8: 17 (2008); and Deeken et al., "A pharmacogenetic study of docetaxel and thalidomide in patients with castration-resistant prostate cancer using the DMET genotyping platform," Pharmacogenomics, 10(3): 191-199 (2009), each of which is incorporated by reference in its entirety for all purposes.

[0242] MIP technology has also been applied to identify new drug-related biomarkers. See, e.g., Caldwell et al.,“CYP4F2 genetic variant alters required warfarin dose” Blood, 111(8):4106-4112 (2008); and McDonald et al.,“CYP4F2 Is a Vitamin K1 Oxidase: An Explanation for Altered Warfarin Dose in Carriers of the V433M Variant” Molecular Pharmacology, 75:1337-1346 (2009), each of which is incorporated by reference in its entirety for all purposes. Other MIP applications include drug development and safety studies. See, e.g., Mega et al.,“Cytochrome P-450 Polymorphisms and Response to Clopidogrel” New England Journal of Medicine, 360(4):354-362 (2009); Dumaual et al.,“Comprehensive assessment of metabolic enzyme and transporter genes using the Affymetrix Targeted Genotyping System” Pharmacogenomics, 8(3):293-305 (2007); and Daly et al.,“Multiplex assay for comprehensive genotyping of genes involved in drug metabolism, excretion, and transport” Clinical Chemistry, 53(7): 1222-1230 (2007), each of which is incorporated by reference in its entirety for all purposes.Other applications of MIP technology include genotyping and phenotyping database. See, e.g., Man et al., “Genetic Variation in Metabolizing Enzyme and Transporter Genes: Comprehensive Assessment in 3 Major East Asian Subpopulations with Comparison to Caucasians and Africans,” Journal of Clinical Pharmacology, 50(8):929-940 (2010), which is incorporated by reference in its entirety for all purposes.

[0243] As used herein, the term “capture” or “capturing” refers to a binding or hybridization reaction between a molecular inversion probe and its corresponding target site. In some embodiments, a circular replicon or MIP replicon is generated or formed upon capture. In some embodiments, the target site is a deletion (e.g., a partial or complete deletion of one or more exons). In some embodiments, a target MIP is designed to bind or hybridize to a naturally occurring (e.g., wild-type) genomic region of interest in which a target deletion is expected to be located. The target MIP is designed not to bind to the genomic region exhibiting the deletion. In these embodiments, binding or hybridization between the target MIP and the target deletion site is not expected to occur. The absence of such binding or hybridization is indicative of the presence of the target deletion. In these embodiments, the phrase “capture the target site” or the phrase “capture the target sequence” refers to detecting the target deletion by detecting the absence of such binding or hybridization.

[0244] As used herein, the term "MIP replicon" or "circular replicon" refers to a circular nucleic acid molecule generated by a capture reaction (e.g., a binding or hybridization reaction between a MIP and its targeted sequence). In some embodiments, the MIP replicon is a single-stranded circular nucleic acid molecule. In some embodiments, the targeting MIP captures or hybridizes to a target sequence or site. Upon the capture reaction or hybridization, a ligation / extension mixture is introduced to extend and ligate the gap region between the two targeting polynucleotide arms to form a single-stranded circular nucleotide molecule, i.e., a targeting MIP replicon. In some embodiments, the control MIP captures or hybridizes to a control sequence or site. Upon the capture reaction or hybridization, a ligation / extension mixture is introduced to extend and ligate the gap region between the two control polynucleotide arms to form a single-stranded circular nucleotide molecule, i.e., a control MIP replicon. The MIP replicon can be amplified by polymerase chain reaction (PCR) to produce multiple targeting MIP amplicons, which are double-stranded nucleic acid molecules. The MIP replicon can be particularly applied to rolling circle amplification or RCA. RCA is an isothermal nucleic acid amplification technique in which a DNA polymerase adds mononucleotides in succession to a primer annealed to a circular template, which produces long catenated single-stranded DNA containing tens to hundreds of tandem repeats (complementary to the circular template). See, e.g., M. Ali et al. "Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine" Chemical Society Reviews. 43(10): 3324-3341, which is incorporated by reference in its entirety for all purposes. See also WO 2015 / 083002, which is incorporated by reference in its entirety for all purposes.

[0245] The polymerase typically used in RCA for DNA amplification is Phi29, Bst and Vent exonuclease DNA polymerase, with Phi29 DNA polymerase being preferred for its superior processivity and strand displacement ability.

[0246] As used herein, the term "amplicon" refers to a nucleic acid generated by an amplification reaction (e.g., a PCR reaction). In some embodiments, an amplicon is a single-stranded nucleic acid molecule. In some embodiments, an amplicon is a double-stranded nucleic acid molecule. In some embodiments, the target MIP replicon is amplified using conventional techniques to produce a plurality of target MIP amplicons that are double-stranded nucleotide molecules. In some embodiments, the control MIP replicon is amplified using conventional techniques to produce a plurality of control MIP amplicons that are double-stranded nucleotide molecules.

[0247] The term "probe oligonucleotide" or "flap oligonucleotide" when used in reference to a flap assay (e.g., an INVADER invasive cleavage assay) refers to an oligonucleotide that interacts with a target nucleic acid in the presence of an invasive oligonucleotide to form a cleavage structure.

[0248] The term "invasive oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid at a position adjacent to a region of hybridization between a probe and the target nucleic acid, where the 3' end of the invasive oligonucleotide includes a portion (e.g., a chemical moiety or one or more nucleotides) that overlaps with the region of hybridization between the probe and the target. The 3' terminal nucleotide of the invasive oligonucleotide can or can not base pair with a nucleotide in the target. In some embodiments, the sequence contained by the invasive oligonucleotide at its 3' end is substantially identical to the sequence located at the 5' end of the portion of the probe oligonucleotide that anneals to the target strand.

[0249] As used herein, "flap endonuclease" or "FEN" refers to a class of un-nucleolytic enzymes that act as structure-specific endonucleases on DNA structures, typically 5' endonucleases, in which the duplex contains a single-stranded 5' overhang or flap on one of the strands that is displaced by the other strand of the nucleic acid (e.g., such that there are overlapping nucleotides at the junction between single-stranded DNA and double-stranded DNA). FENs catalyze the hydrolytic cleavage of the phosphodiester bond at the junction of single-stranded and double-stranded DNA, releasing the overhang or flap. Ceska and Savers (Trends Biochem. Sci. 1998 23:331-336) and Liu et al. (Ann. Rev. Biochem. 2004 73:589-615; incorporated by reference in its entirety) review flap endonucleases. A FEN can be a separate enzyme, a multi-subunit enzyme, or can exist as an activity of another enzyme or protein complex (e.g., a DNA polymerase).

[0250] The flap endonuclease can be thermostable. For example, the FEN-1 flap endonuclease from the Thermotoga sp. organism is typically thermostable. As used herein, the term "FEN-1" refers to a non-polymerase flap endonuclease from a eukaryotic cell or an archaeal organism. See, e.g., WO 02 / 070755 and Kaiser M.W. et al., (1999) J. Biol. Chem., 274:21387, which are incorporated by reference in their entireties for all purposes.

[0251] As used herein, the term "cleaved flap" refers to a single-stranded oligonucleotide that is the cleavage product of a flap assay.

[0252] The term "cassette" when used in reference to a flap cleavage reaction refers to an oligonucleotide or combination of oligonucleotides configured to generate a detectable signal in response to cleavage of a flap or probe oligonucleotide, e.g., in a primary or first cleavage structure formed in a flap cleavage assay. In preferred embodiments, the cassette hybridizes to a non-target cleavage product produced by cleavage of a flap oligonucleotide to form a second, overlapping cleavage structure, such that the cassette can then be cleaved by the same enzyme (e.g., a FEN-1 endonuclease).

[0253] In some embodiments, the cassette is a single oligonucleotide that includes a hairpin portion (i.e., a region in which one portion of the cassette oligonucleotide hybridizes to a second portion of the same oligonucleotide under reaction conditions to form a duplex). In other embodiments, the cassette includes at least two oligonucleotides that include complementary portions that can form a duplex under reaction conditions. In preferred embodiments, the cassette includes a label, e.g., a fluorophore. In particularly preferred embodiments, the cassette includes labeled portions that produce a FRET effect. In such embodiments, the cassette can be referred to as a "FRET cassette." See, e.g., U.S. 9,096,893, issued 08 / 04 / 15, which is incorporated by reference in its entirety for all purposes.

[0254] As used herein, the phrase "substantially non-complementary" when used in reference to a probe flap or arm means that the flap portion has sufficient non-complementarity to selectively not hybridize to a nucleic acid sequence (e.g., a target nucleic acid or amplified DNA) under specified annealing conditions or stringent conditions, which encompasses the terms "substantially non-complementary" and "completely non-complementary."

[0255] As used herein, the term "signal" refers to any detectable effect as would be caused or provided by a label or by the action or accumulation of a component or product in an assay reaction.

[0256] As used herein, the term "detector" refers to a system or component of a system, such as an instrument (e.g., a camera, a fluorometer, a charge-coupled device, a scintillation counter, a solid-state nanopore device, etc.) or a reactive medium (X-ray or camera film, a pH indicator, etc.), that can convey the presence of a signal or effect to a user or another component of the system (e.g., a computer or controller). The detector is not limited to a particular type of signal detected and can be: a photometric or spectrophotometric system (which can detect ultraviolet, visible, or infrared light, including fluorescence or chemiluminescence); a radiation detection system; a charge detection system; a system for detecting electronic signals (e.g., current or charge perturbations); a spectroscopic system, such as nuclear magnetic resonance spectroscopy, mass spectrometry, or surface-enhanced Raman spectroscopy; a system such as gel or capillary electrophoresis or gel exclusion chromatography; or other detection systems known in the art or combinations thereof.

[0257] As used herein, the term "detecting" refers to quantitatively or qualitatively identifying an analyte (e.g., DNA, RNA, or protein) in a sample, for example. As used herein, the term "detection assay" refers to a kit, test, or procedure performed for the purpose of detecting an analyte within a sample. A detection assay produces a detectable signal or effect when performed in the presence of a target analyte and includes, but is not limited to, assays associated with processes of hybridization, nucleic acid cleavage (e.g., exonuclease or endonuclease), nucleic acid amplification, nucleotide sequencing, primer extension, nucleic acid ligation, antigen-antibody binding, interaction of a primary antibody with a secondary antibody, and / or conformational changes of nucleic acids (e.g., oligonucleotides) or polypeptides (e.g., proteins or small peptides).

[0258] As used herein, the term "prenatal or pregnancy-related disease or condition" refers to any disease, disorder, or condition affecting a pregnant woman, embryo, or fetus. A prenatal or pregnancy-related condition can also refer to any disease, disorder, or condition that is directly or indirectly associated with or directly or indirectly caused by pregnancy. These diseases or conditions can include any and all birth defects, congenital conditions, or genetic diseases or conditions. Examples of prenatal or pregnancy-related diseases include, but are not limited to, Rhesus disease, hemolytic disease of the newborn, beta-thalassemia, sex determination, pregnancy determination, inherited Mendelian genetic disorders, chromosomal aberrations, fetal chromosomal aneuploidy, fetal chromosomal trisomy, fetal chromosomal monosomy, trisomy 8, trisomy 13 (Patau syndrome), trisomy 16, trisomy 18 (Edward syndrome), trisomy 21 (Down syndrome), X-linked disorders, trisomy X (XXX syndrome), monosomy X (Turner syndrome), Klinefelter syndrome, XYY syndrome, XYY syndrome, XXXY syndrome, XXYY syndrome, XYYY syndrome, XXXXX syndrome, XXXXY syndrome, XXXYY syndrome, XXYYY syndrome, fragile X syndrome, fetal growth restriction, cystic fibrosis, hemoglobinopathy, fetal death, fetal alcohol syndrome, sickle cell anemia, hemophilia, Klinefelter syndrome, dup(17)(pl l.2pl l.2) syndrome, endometriosis, Pelizaeus-Merzbacher disease, dup(22)(ql l.2ql l.2) syndrome, cat eye syndrome, cat cry syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, craniocarpal neuropathy, Smith-Magenis syndrome, neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome, DiGeorge syndrome, steroid sulfatase deficiency, Prader-Willi syndrome, Kallmann syndrome, microphthalmia with linear skin defects, adrenal hypoplasia, glycerol kinase deficiency, Pelizaeus-Merzbacher disease, testis determining factor on Y, azoospermia (factor a), azoospermia (factor b), azoospermia (factor c), 1p36 deletion, phenylketonuria, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sachs disease, Tay-Sdisease), adrenal hyperplasia signs, Fanconi anemia, spinal muscular atrophy, Duchenne muscular dystrophy, Huntington's disease, myotonic dystrophy, Robertsonian translocation, Angelman syndrome, tuberous sclerosis, ataxia telangiectasia, open spine bifida, neural tube defects, abdominal wall defects, small for gestational age, congenital cytomegalovirus, achondroplasia, Marfan's syndrome, congenital hypothyroidism, congenital toxoplasmosis, biotinidase deficiency, galactosemia, maple syrup urine disease, homocystinuria, medium-chain acyl-CoA dehydrogenase deficiency, structural birth defects, heart defects, limb abnormalities, talipes, anencephaly, anosminic brain / holoprosencephaly, hydrocephaly, anophthalmia / microphthalmia, anotia / microtia, large vessel shift, tetralogy of Fallot, left heart hypoplasia syndrome, coarctation of the aorta, cleft palate without cleft lip, cleft lip with or without cleft palate, esophageal atresia / stenosis with or without fistula, small bowel atresia / stenosis, anorectal atresia / stenosis, hypospadias, ambiguous genitalia, kidney dysplasia, cystic kidney, preaxial polydactyly, short-limb defects, diaphragmatic hernia, blindness, cataracts, visual problems, hearing loss, deafness, X-linked adrenoleukodystrophy, Rett syndrome, lysosomal diseases, cerebral palsy, autism, aglossia, albinism, ocular albinism, oculocutaneous albinism, gestational diabetes, Arnold-Chiari malformation, CHARGE syndrome, congenital diaphragmatic hernia, short toes, aniridia, hand-foot syndrome, heterochromia, Dwarnian ear, Ehlers Danlossyndrome, epidermolysis bullosa, Gorham's disease, Hashimoto's syndrome, fetal hydrops, hypotonia, Klippel-Feil syndrome, muscular dystrophy, osteogenesis imperfecta, progeria, Smith Lemli Opitz symdrom, color blindness, X-linked lymphoproliferative disease, omphalocele, gastroschisis, pre-eclampsia, eclampsia, preterm birth, premature birth, miscarriage, intrauterine growth retardation, ectopic pregnancy, hyperemesis gravidarum, morning sickness, or possible successful induction.

[0259] In some NIPT embodiments, the present technology described herein further comprises estimating a fetal fraction of the sample, wherein the fetal fraction is used to help determine whether the genetic data from the test subject is indicative of an aneuploidy. Methods for determining or calculating a fetal fraction are known in the art.

[0260] As used herein, the term "valid detection assay" refers to a detection assay that has been shown to accurately predict the association between detection of a target and a phenotype (e.g., a medical condition). Examples of a valid detection assay include, but are not limited to, a detection assay that accurately predicts a medical phenotype 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% of the time when the target is detected. Other examples of a valid detection assay include, but are not limited to, a detection assay that is suitable and / or marketed as an analyte-specific reagent (i.e., as defined by FDA regulations) or an in vitro diagnostic (i.e., approved by the FDA).

[0261] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of a reaction assay, such a delivery system includes a system that allows for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers each containing a sub-portion of the total kit components. The containers can be delivered together or separately to the intended recipient. For example, a first container can contain an enzyme for use in the assay while a second container contains oligonucleotides. The term "fragmented kit" is intended to encompass, but is not limited to, kits containing analyte-specific reagents (ASRs) that are regulated under 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, the term "fragmented kit" encompasses any delivery system that includes two or more separate containers each containing a sub-portion of the total kit components. In contrast, a "combined kit" refers to a delivery system that contains all of the components of a reaction assay in a single container (e.g., in a single box that houses each of the desired components). The term "kit" includes both fragmented kits and combined kits.

[0262] As used herein, the term "information" refers to any collection of facts or data. With respect to information stored or processed using one or more computer systems, including but not limited to the Internet, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term "information about a subject" refers to facts or data about a subject (e.g., a human, plant, or animal). The term "genomic information" refers to information about a genome, including but not limited to nucleic acid sequences, genes, allele frequencies, RNA expression levels, protein expression, phenotypes associated with genotypes, etc. "Allele frequency information" refers to facts or data about allele frequencies, including but not limited to allele identity, statistical correlations between the presence of an allele and a characteristic of a subject (e.g., a human subject), presence or absence of an allele in an individual or population, percentage likelihood of an allele being present in an individual having one or more particular characteristics, etc.

[0263] As used herein, the term "assay validation information" refers to genomic information and / or allele frequency information resulting from processing of test result data (e.g., processing by means of a computer). Assay validation information can be used, for example, to identify a particular candidate detection assay as a valid detection assay. BRIEF DESCRIPTION OF DRAWINGS

[0264] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0265] Figure 1 A schematic of a molecular inversion probe (MIP) for chromosome-specific recognition suitable for use in a large-scale multiplexed capture assay is provided.

[0266] Figure 2 A schematic of an embodiment of multiplexed chromosome-specific rolling circle amplification is provided.

[0267] Figure 3 A schematic of an embodiment of multiplexed chromosome-specific rolling circle amplification using molecular beacon probes for detection is provided.

[0268] Figure 4 A schematic of an embodiment of the present technology including circularization of cfDNA using a single-strand connecting enzyme (e.g., CircLigase TM A schematic of an embodiment of the present technology including circularization of cfDNA using a single-strand connecting enzyme (e.g., CircLigase

[0269] Figure 5Provided are schematics of embodiments of the present technology including circularizing cfDNA and using "Golden Gate Assembly" to add segments for detection (see, e.g., Engler, C, Kandzia, R., and Marillonnet, S. (2008) PLoS One 3, e3647).

[0270] Figure 6 Provided are schematics of embodiments of the present technology including circularizing cfDNA using extension ligation on unique molecular inversion probes, where embodiments using RCA are used for detection.

[0271] Figure 7 Provided are schematics of embodiments of the present technology including unique molecular inversion probes that are extended and ligated to produce circular DNA molecules, where embodiments using RCA are used for detection.

[0272] Figure 8 Provided are schematics of embodiments of the present technology including synthetic circular DNA that includes binding sites for probe binding and primer binding sites for replication, used as templates for, e.g., rolling circle amplification.

[0273] Figure 9 Provided are schematics of embodiments of the present technology including using pairs of probes configured for collisional quenching upon hybridization to strands of DNA for detection of products from RCA.

[0274] Figure 10 Provided are schematics of embodiments of the present technology including using pairs of probes configured for fluorescence resonance energy transfer (FRET) upon hybridization to strands of DNA for detection of products from RCA.

[0275] Figure 11 Provided are schematics of embodiments of the present technology including using probes configured for cleavage upon hybridization to strands of DNA, e.g., using duplex-specific nucleases such as restriction enzymes, including dyes and quenchers, for detection of products from RCA.

[0276] Figure 12 Provided are schematics of embodiments of the present technology including RCA using CID, followed by CID-specific digestion and CID-specific labeling.

[0277] Figure 13An embodiment is shown in which the MIP hybridizes to a target nucleic acid, such as cfDNA, leaving a single nucleotide gap. The gap is filled by extension to incorporate biotinylated nucleotides and closed by ligation. The circularized MIP can then bind to a streptavidin-coated surface.

[0278] Figure 14 A schematic of a primer oligonucleotide hybridized to a MIP immobilized on a surface is shown.

[0279] Figure 15 A schematic of hairpin oligonucleotides working together to form a self- assembled scaffold in the presence of a primer oligonucleotide is shown.

[0280] Figure 16 A self-assembled scaffold is shown that includes multiple labels, such as fluorescent dyes.

[0281] Figure 17 A schematic of an invasive cleavage structure is provided according to embodiments of the technology.

[0282] Figure 18 A diagram of a hairpin probe used in forming an invasive cleavage structure for a flap endonuclease assay (e.g., a determination) is provided according to embodiments of the technology.

[0283] Figure 19 A diagram of the accumulation of cleaved flap fragments in a flap endonuclease assay is provided.

[0284] Figure 20 An embodiment is shown in which cleaved biotinylated flaps are captured using immobilized complementary probes and biotin reacts with streptavidin linked to an enzyme (e.g., beta-galactosidase).

[0285] Figure 21 An embodiment of the technology is shown in which MIPs designed to target different chromosomes each require a different nucleotide to extend and ligate and in which the MIPs use nucleotides carrying different dyes or haptens for each different dNTP to extend and ligate in a chromosome-specific manner.

[0286] Figure 22 Figures A, B, and C of the present technology show an embodiment in which the MIP contains or is modified to contain an immobilization moiety or hybridizes to an oligonucleotide containing an immobilization moiety and is immobilized on a surface.

[0287] Figure 23 A schematic of a rolling circle amplification reaction is provided.

[0288] Figure 24Figures A-D provide graphs showing results from examining the effect of including biotin residues in the MIP complex on the RCA signal.

[0289] Figures 25A-25C Figures are provided showing results from varying the amount of components in a standard RCA reaction in solution.

[0290] Figure 26 Figures are provided comparing the effect of using different molecular weights of PEG on signal accumulation at the indicated percentages (w:v).

[0291] Figures 27A-27B and Figure 28 Results obtained in RCA reactions using primers bound to the surface of glass in a random manner are shown, where detection was performed using a molecular beacon probe comprising a quencher and a fluorophore.

[0292] Figure 27A Microscope images of the surface of APTES-silanized plates as described in Example 1 are shown and RCA signals with or without PEG are compared.

[0293] Figure 27B Figures are provided showing the effect of PEG on Figure 27A Figures are provided showing the effect of the number of spots and fluorescence intensity shown in Example 1.

[0294] Figure 28 Figures are provided showing the effect of different molecular weights of PEG in a 20% solution on the number of spots and fluorescence intensity on APTES-silanized plates as described in Example 1.

[0295] Figure 29A Microscope images of the surface of APTES-silanized plates as described in Example 1 are shown and RCA signals are compared for reactions hybridized for 18 hours or 1 hour before initiating the RCA reaction.

[0296] Figure 29B Figures are provided comparing the effect of hybridization time and buffer on Figure 29A Figures are provided showing the effect of the number of spots and fluorescence intensity (area) shown in Example 1.

[0297] Figure 30 Figures are provided comparing the effect of PEG 200 on standard RCA reaction conditions with or without a 2 hour hybridization time and the effect of PEG 2000 on the number of spots and fluorescence intensity (area) with a 2 hour hybridization.

[0298] Figure 31A graph comparing the effect of PEG 200 with or without a 2 hour hybridization time on standard RCA reaction conditions performed at 25°C and the effect of PEG 2000 on the number of spots and fluorescence intensity (area) with 2 hour hybridization is provided.

[0299] Figure 32 A graph comparing the effect of PEG 200 with or without a 2 hour hybridization time on standard RCA reaction conditions performed at 37°C and the effect of PEG 2000 on the number of spots and fluorescence intensity (area) with 2 hour hybridization is provided.

[0300] Figure 33 Microscope images of the surface of APTES-silanized plates as described in Example 1 are shown and RCA signal is compared for reactions including the indicated concentrations of PEG 600 performed at 37°C or 45°C.

[0301] Figure 34 A schematic of RCA molecular beacon product on a surface with or without graphene oxide is provided, where graphene oxide quenches the fluorescent background from beacons that bind non-specifically to the surface.

[0302] Figure 35 A schematic of a two-step RCA reaction as described in Example 1 is provided, in which a rolling circle reaction is initiated, molecular beacons and graphene oxide are added, and the RCA reaction is further incubated.

[0303] Figure 36 Microscope images of the surface of APTES-silanized plates as described in Example 1 are shown and RCA signal is shown for two-step reaction graphene oxide.

[0304] Figure 37 A graph comparing spot counts for RCA reactions performed in one step (no GO) or two steps (with or without GO) is provided, comparing reactions with 100 fmol of target to reactions without target.

[0305] Figure 38 A schematic of different capture complexes for applying embodiments of the present technology to detect different types of target molecules is provided.

[0306] Figure 39 A schematic of applying the present technology to detect immobilized antigens is provided.

[0307] Figure 40 A schematic of applying the present technology to detect immobilized antigen-antibody complexes is provided. DETAILED DESCRIPTION

[0308] The goal of molecular diagnostics is to achieve accurate and sensitive detection of analytes in the shortest possible time with the least amount of work and steps. One way to achieve this is through multiplex detection of the analyte in a sample, allowing multiple detection events to be performed in a single reaction vessel or solution. However, many existing diagnostic methods (including multiplex reactions) still require numerous steps, including sample preparation steps that increase the time, complexity, and cost of performing the reactions. In some embodiments, the present invention provides a solution to these problems by providing assays that can be performed directly in unpurified or untreated biological samples (e.g., blood or plasma).

[0309] In some embodiments, the techniques of the invention provided herein offer an economical method for testing samples in a digital manner (i.e., by detecting individual copies of the molecule) to count the copy number of a specific nucleic acid or protein in a sample or sample portion without using sequencing steps (e.g., digital or “next-generation” sequencing steps). The techniques of the invention can be used to measure target molecules (such as nucleic acid molecules) in any kind of sample (including, but not limited to, samples collected from subjects for diagnostic screening). Embodiments of the techniques of the invention provided herein can be used, for example, in non-invasive prenatal testing (NIPT) and other genetic analyses. Embodiments of the techniques of the invention implement one or more steps of a method for nucleic acid extraction, MIP probe design, MIP amplification / replication, and / or for measuring signals from a circularized MIP. In a preferred embodiment, the techniques of the invention provide a method for immobilizing a MIP on a surface and detecting the immobilized MIP. In a preferred embodiment, rolling circle amplification is used to detect the immobilized MIP.

[0310] In a preferred embodiment, the method of the present invention includes a target recognition event, which typically involves hybridization of a target nucleic acid (e.g., a sample of patient DNA) with another nucleic acid molecule (e.g., a synthetic probe). In a preferred embodiment, the target recognition event creates a unique product (e.g., an extended, ligated, and / or cleaved probe oligonucleotide) that then indicates the presence of the target in the reaction and the conditions under which the probe hybridizes with it.

[0311] This article describes many different “front-end” methods for recognizing target nucleic acids and generating novel products. For example, as shown in the exemplary embodiments in the figures, the present invention provides a variety of ways to generate cyclized molecules for use in “back-end” detection / readout steps (see, for example, Figures 1-3 , Figures 13-18 , Figure 34 , Figure 35 and Figures 38-40The present invention also provides a method for signaling the presence of a target nucleic acid using other probe types (such as probes that can be cleaved by valve-shaped endonucleases in the presence of the target nucleic acid) (see, for example, Figures 17-19 Each of these front-end embodiments can be used to produce unique molecules, such as cyclic or cleaved oligonucleotides.

[0312] These unique molecules can be configured to have one or more features useful for capture and / or identification in downstream back-end detection steps. Examples of molecules and features generated in the front-end reaction include cyclized MIPs or products released from flap cleavage reactions (such as single-stranded arms) having linked sequences (e.g., complete target-specific sequences formed by linking the 3' and 5' ends of a probe), added sequences (e.g., copied portions of the target template), and / or tagged nucleotides (e.g., nucleotides linked to biotin, dyes, quenchers, haptens, and / or other portions) (see, for example, Figures 17-19 In some embodiments, the MIP includes features in portions of the probe (e.g., in the main chain of the probe).

[0313] For example, in Figures 2-3 , Figures 6-7 , Figures 9-12 , Figures 15-16 , Figures 20-21 , Figure 34 , Figure 35 and Figures 38-40 Examples of back-end analysis methods for amplifying and / or detecting unique products at the front end are provided.

[0314] Although the techniques of the present invention (such as combinations of certain front-end target-dependent responses with specific back-end signal amplification methods and detection platforms) have been discussed with reference to specific embodiments (e.g., Figures 13-16 Biotin-incorporated MIPs coupled to the end of an enzyme-free hybridization chain reaction; biotin-tagged cleaved lobes (e.g. Figure 19 The enzyme-linked probe (ELIP) is coupled to the surface for capture and then catalytically hybridized with an ELIP probe that generates a fluorescent signal (e.g., ...). Figure 20 As shown herein, however, the invention is not limited to any particular combination of front-end methods and configurations and back-end methods and configurations disclosed herein, or any particular method for detecting signals from a measured product. It should be understood that those skilled in the art can readily adapt a front-end to work with alternative back-ends. For example, as... Figures 2-3 , Figures 8-7 , Figures 9-12 , Figure 21 , Figure 34 , Figure 35 and Figures 38-40 as exemplified in Figure 14 The cyclic MIP can be usedFigure 20 enzyme-linked probes to capture and detect or can alternatively be amplified in a rolling circle amplification assay. Similarly, as Figures 19-20 described in Figure 19 , cleaved flaps can be detected using hybridization chain reactions; and circularized MIP or RCA amplicons can be detected using invasive cleavage reactions as Figure 17 illustrated in

[0315] Further, although the present technology is discussed with reference to particular target nucleic acids (e.g., cell-free DNA in plasma), the present technology is not limited to any particular form of DNA or any particular type of nucleic acid or any particular type of nucleic acid variation. It will be appreciated that a skilled artisan can readily configure embodiments of the present technology for detection and counting of mutations, insertions, deletions, single nucleotide polymorphisms (SNPs), and epigenetic variations of methylation (e.g., by analyzing methylation variations of particular CpG dinucleotides resulting from treatment of DNA with an agent that converts unmethylated cytosines to uracils, thereby producing detectable sequence variations that reflect cytosine methylation variations in the target DNA).

[0316] In some embodiments, assays are performed in multiplex. In some embodiments, multiplexed assays can be performed under conditions that allow different loci to achieve more similar levels of amplification.

[0317] Figure 1 A schematic of a molecular inversion probe (MIP) is provided. The MIP contains first and second targeting polynucleotide arms that are complementary to adjacent or proximal regions on a target nucleic acid to be detected, with a polynucleotide linker or "backbone" connecting the two arms (see Figure 1 ).

[0318] In the presence of a complementary target nucleic acid, the MIP can be circularized to form a MIP replicon suitable for detection. In some embodiments, the MIP is simply ligated using a nick repair enzyme (e.g., T4 DNA ligase), while in some embodiments, sealing the probe to form a circle includes additionally modifying the probe to create ligatable nicks (e.g., overlapping cleavages between the ends), filling gaps between the ends using a nucleic acid polymerase, etc.

[0319] As used herein, a target site or sequence refers to a portion or region of a nucleic acid sequence sought to be sorted out from other nucleic acids in a sample having other sequences that are of significance in determining the presence or absence of a genetic disorder or condition (e.g., the presence or absence of a mutation, polymorphism, deletion, insertion, aneuploidy, etc.). As used herein, a control site or sequence refers to a site having a known or normal copy number of a particular control gene. In some embodiments, a targeting MIP comprises, in order, the following components: a first targeting polynucleotide arm - a first unique targeting molecular tag - a polynucleotide linker - a second unique targeting molecular tag - a second targeting polynucleotide arm. In some embodiments, a target population of targeting MIPs is used in the methods of the present disclosure. In the target population, the pairs of first and second targeting polynucleotide arms in each of the targeting MIPs are identical and are substantially complementary to first and second regions, respectively, flanking a target site in a nucleic acid. See, e.g., WO 2017 / 020023 and WO 2017 / 020024, each of which is incorporated herein by reference in its entirety.

[0320] In some embodiments, each of the targeting polynucleotide arms is between 18 and 35 base pairs in length. In some embodiments, each of the targeting polynucleotide arms is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 base pairs in length or any size ranging between 18 and 35 base pairs. In some embodiments, each of the control polynucleotide arms is between 18 and 35 base pairs in length. In some embodiments, each of the control polynucleotide arms is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 base pairs in length or any size ranging between 18 and 35 base pairs. In some embodiments, each of the targeting polynucleotide arms has a melting temperature between 57°C and 63°C. In some embodiments, each of the targeting polynucleotide arms has a melting temperature of 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, or 63°C or any size ranging between 57°C and 63°C. In some embodiments, each of the control polynucleotide arms has a melting temperature between 57°C and 63°C. In some embodiments, each of the control polynucleotide arms has a melting temperature of 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, or 63°C or any size ranging between 57°C and 63°C. In some embodiments, each of the targeting polynucleotide arms has a GC content between 30% and 70%. In some embodiments, each of the targeting polynucleotide arms has a GC content of 30-40%, or 30-50%, or 30-60%, or 40-50%, or 40-60%, or 40-70%, or 50-60%, or 50-70%, or any size ranging between 30% and 70% or any specific percentage between 30% and 70%. In some embodiments, each of the control polynucleotide arms has a GC content between 30% and 70%. In some embodiments, each of the control polynucleotide arms has a GC content of 30-40%, or 30-50%, or 30-60%, or 40-50%, or 40-60%, or 40-70%, or 50-60%, or 50-70%, or any size ranging between 30% and 70% or any specific percentage between 30% and 70%.

[0321] In some embodiments, the polynucleotide adapter is not substantially complementary to any genomic region of the sample or subject. In some embodiments, the polynucleotide adapter is between 30 and 40 base pairs in length. In some embodiments, the polynucleotide adapter is 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 base pairs in length or any interval between 30 and 40 base pairs. In some embodiments, the polynucleotide adapter has a melting temperature between 60 °C and 80 °C. In some embodiments, the polynucleotide adapter has a melting temperature of 60 °C, 65 °C, 70 °C, 75 °C, or 80 °C or any interval between 60 °C and 80 °C or any specific temperature between 60 °C and 80 °C. In some embodiments, the polynucleotide adapter has a GC content between 40% and 60%. In some embodiments, the polynucleotide adapter has a GC content of 40%, 45%, 50%, 55%, or 60% or any interval between 40% and 60% or any specific percentage between 40% and 60%.

[0322] In some embodiments, the target MIP replicon is generated by: i) hybridizing first and second targeting polynucleotide arms to first and second regions, respectively, flanking a target site in a nucleic acid; and ii) following hybridization, using a ligation / extension mix to extend and ligate the gap region between the two targeting polynucleotide arms to form a single-stranded circular nucleic acid molecule.

[0323] In certain embodiments, the methods described herein are used to detect a deletion or insertion or duplication of an exon. In some embodiments, the target site (or sequence) is a deletion or insertion or duplication in a gene of interest or a genomic region of interest. In some embodiments, the target site is a deletion or insertion or duplication in one or more exons of a gene of interest. In some embodiments, the multiple exons targeted are contiguous. In some embodiments, the multiple exons targeted are non-contiguous. In some embodiments, the first and second targeting polynucleotide arms of the MIP are designed to hybridize upstream and downstream of a deletion (or insertion or duplication) in a gene or a deleted (or inserted or duplicated) genomic region (e.g., one or more exons) or a genomic region of interest. In some embodiments, the first or second targeting polynucleotide arm of the MIP comprises a sequence that is substantially complementary to a genomic region of a gene of interest encompassing the target deletion or duplication site (e.g., an exon or portion of an exon).

[0324] Circular DNA molecules, such as ligated MIPs, are suitable substrates for amplification using rolling circle amplification (RCA). In certain embodiments of RCA, a rolling circle replication primer hybridizes to a circular nucleic acid molecule (e.g., a ligated MIP) or a circularized cfDNA. A DNA polymerase that uses strand displacement (e.g., the (Phi29), Bst large fragment, and Klenow fragment) to the primers results in long single-stranded DNA molecules containing repeats of nucleic acid sequences complementary to the MIP circular molecules.

[0325] In some embodiments, a ligation-mediated rolling circle amplification (LM-RCA) involving a ligation step prior to replication is used. In the ligation step, a probe hybridizes to its complementary target nucleic acid sequence (if present) and the ends of the hybridized probe are joined by ligation to form a covalently closed single-stranded nucleic acid. Following ligation, a rolling circle replication primer hybridizes to the probe molecule to initiate rolling circle replication as described above. Typically, LM-RCA comprises mixing an empty probe with a target sample, thereby producing a probe-target sample mixture, and incubating the probe-target sample mixture under conditions that promote hybridization between the empty probe and the target sequence; mixing a ligase enzyme with the probe-target sample mixture, thereby producing a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the empty probe to form an amplified target circle (ATC, also referred to as an RCA replicon). Mixing a rolling circle replication primer (RCRP) with the ligation mixture, thereby producing a primer-ATC mixture, incubating the primer-ATC mixture under conditions that promote hybridization between the amplified target circle and the rolling circle replication primer. Mixing a DNA polymerase enzyme with the primer-ATC mixture, thereby producing a polymerase-ATC mixture, incubating the polymerase-ATC mixture under conditions that promote replication of the amplified target circle, wherein replication of the amplified target circle results in formation of a concatemeric sequence DNA (TS-DNA) (i.e., a long chain of single-stranded DNA containing a concatemer of sequences complementary to the amplified target circle).

[0326] In the embodiments illustrated in Figure 2 , the circularized molecules A, B, C, and D consist of MIP specific for chromosome 13, 8, 21, or a reference chromosome (e.g., MIP specific for Chr. 1). The sequence of the MIP encloses the empty complementary region of the targeted chromosome, and the backbone of the MIP contains unique sequences for hybridization of probes containing specific fluorescent dyes (FITC, ALEXA, Dylight, Cyan, Rhodamine dyes, quantum dots, etc.). Step 1 includes hybridization of the MIP to cfDNA, single base pair extension (or longer extension), and ligation to circularize the extended MIP. Step 2 includes rolling circle amplification of the circularized MIP, such that the sequence required for hybridization of the fluorescently labeled oligonucleotide is amplified. A*, B*, C*, D* are the complements of the MIP sequences. Step 3 includes hybridization of the fluorescently labeled probes to the rolling circle products. In the embodiments illustrated in Figure 3 , detection of the RCA products is facilitated by molecular probes rather than fluorescent dye-labeled oligonucleotides.

[0327] There are multiple ways to immobilize MIPs to a surface (e.g., a bead or glass surface). For example, this can be accomplished by priming rolling circle amplification with a modified oligonucleotide that includes a conjugable moiety. Groups used to modify the priming oligonucleotide include, but are not limited to, thiols, amines, azides, alkynes, and biotin, such that the modified oligonucleotide can be immobilized using, for example, appropriate reactions as outlined in Meyer et al., “Advances in DNA-mediated immobilization” Current Opinions in Chemical Biology 18:8:8-15 (2014), which is incorporated by reference in its entirety for all purposes.

[0328] Imaging of fluorescent dye-incorporated MIPs can be accomplished by using methods that include immobilizing MIPs to a surface (slide or bead) (e.g., using modification of the MIP backbone to contain modified bases that can be immobilized using appropriate reactions as outlined above and in Meyer et al. (Id.) and detected using antibodies). Once immobilized to a surface, antibodies against the incorporated tag can be used to form antibody-MIP complexes that can be imaged with a microscope. In some embodiments, antibodies can be conjugated to enhance or amplify the detectable signal from the complex. For example, conjugation of β-galactosidase to antibodies allows detection in single molecule arrays (“SIMOA”) using a process described by Quanterix, in which each complex is immobilized on a bead such that any bead has no more than one labeled immunocomplex, and the beads are partitioned to an array of picowell-sized wells such that each well contains at most one bead. By adding resorcinol β-galactopyranoside, the β-galactosidase on the immobilized immunocomplex catalyzes the production of resorcinol that fluoresces. Upon visualization, the fluorescence emitted in wells with immobilized individual immunocomplexes can be detected and counted. See, e.g., Quanterix Whitepaper 1.0, Scientific Principle of Simoa (Single Molecule Array) Technology, 1-2 (2013); and Quanterix Whitepaper 6.0, Simoa Technology for Ultra-Sensitive Multiplexed Immunoassay of Protein Biomarkers, 1-2 (2013). TM Practical Application of Simoa TMHD-1 Analyzer for Ultrasensitive Multiplex Immunodetection of Protein Biomarkers), 1-3 (2015), each of which is incorporated herein by reference for all purposes. In some embodiments, antibody MIP complexes can be detected directly, for example, using a solid-state nanopore with antibodies labeled with poly(ethylene glycol) of various molecular weights as described in Morin et al., "Nanopore-Based Target Sequence Detection" PLoS ONE, DOI: 10.1371 / journal.pone.0154426 (2016), which is incorporated herein by reference.

[0329] Figure 4 Embodiments of the present technology are provided that include circularizing circulating cfDNA (ccfDNA) for detection using single-strand ligases (e.g., CircLigase TM A schematic of an embodiment of the present technology that includes circularizing cfDNA and adding segments for detection using "Golden Gate Assembly" (see, e.g., Engler, C, Kandzia, R., and Marillonnet, S. (2008) PLoS ONE 3, e3647) is illustrated in FIG. 1. Figure 5 A schematic of an embodiment of the present technology that includes circularizing cfDNA and adding segments for detection using "Golden Gate Assembly" (see, e.g., Engler, C, Kandzia, R., and Marillonnet, S. (2008) PLoS ONE 3, e3647) is illustrated in FIG. 1.

[0330] Figure 6Another method of detecting ccfDNA is demonstrated. In this example, a plasma sample is processed to purify ccfDNA as previously described. In step 1, the ccfDNA is heat denatured and treated with T4 polynucleotide kinase to create 5' phosphorylated and 3' hydroxyl end DNA fragments. Prior to heat denaturation and T4 polynucleotide kinase treatment, additional DNA repair can be used to repair the DNA, such as with T4 DNA polymerase. A complementary oligonucleotide with a 3' protected end, so it will not be extended by the polymerase, is hybridized to the ccfDNA. This complementary oligonucleotide is composed of chromosome specific regions A and C and a universal sequence B. The ccfDNA is extended and ligated to complete a circular DNA molecule. The circularized ccfDNA is purified from the oligonucleotide and RCA is used by annealing the oligonucleotide to the universal sequence B. After RCA, a fluorescently labeled probe is hybridized to the rolling circle product.

[0331] Figure 7 Another method of detecting ccfDNA is demonstrated. A plasma sample is processed to purify ccfDNA as previously described. In step 1, the ccfDNA is heat denatured. A complementary oligonucleotide with a phosphorylated 5' primer protected end is hybridized to the ccfDNA. This complementary oligonucleotide is composed of chromosome specific regions A and C and a universal sequence B. Both the ccfDNA and the complementary oligonucleotide are extended. However, only the complementary oligonucleotide has a 5' phosphate to allow completion of a circular DNA molecule. The circularized complementary oligonucleotide is amplified by rolling circle amplification using a primer complementary to the universal sequence B. After rolling circle amplification, a fluorescently labeled probe is hybridized to the rolling circle product.

[0332] Figure 8 A schematic of a synthetic circular DNA used as a template for rolling circle amplification and including a binding rolling circle primer binding site and two probe binding sites, and optionally a binding moiety (e.g., biotin), is shown.

[0333] Figure 9 Provided are methods including using for detecting products from RCA, e.g., with Figure 8A schematic of an embodiment of the present technology including the use of a probe pair configured for collisional quenching upon hybridization to a strand of DNA similar to the circular DNA shown in FIG. 1) that is configured for fluorescence resonance energy transfer (FRET) as described above. In this embodiment, dye-labeled probes in solution are not quenched and produce a signal. Probes hybridized to the target proximal to the quencher-labeled probe are quenched, reducing the fluorescent signal. As the amount of RCA product increases, fluorescence decreases.

[0334] Figure 10 A schematic of an embodiment of the present technology including the use of a probe pair configured for fluorescence resonance energy transfer (FRET) as described above for detecting product from RCA.

[0335] Figure 11 A schematic of an embodiment of the present technology including the use of a probe including a dye and a quencher configured to be cleaved upon hybridization to a strand of DNA, for example using a duplex-specific nuclease such as a restriction enzyme, for detecting product from RCA.

[0336] As Figure 12 An embodiment of the present technology including the use of a chromosome-specific identifier sequence (CID) for RCA, followed by CID-specific digestion of non-targeted chromosomes and CID-specific labeling for targeted CIDs, as illustrated in FIG. 2. CIDs are amplified by RCA, but maintain their individual single-molecule identity. CID amplification increases the fluorescent signal from individual target molecules. Sequences from chromosomes that have not been analyzed are doubly suppressed by enzymatic digestion and the use of labels that are specific only to the chromosomes being analyzed.

[0337] In some embodiments, MIPs can be detected using non-enzymatic methods of signal amplification. For example, in some embodiments, MIPs are immobilized on a surface and detected using methods such as "hybridization chain reaction" (HCR) or the like, for example as described by RM Dirks et al., Proc Natl Acad Sci U S A 101(43): 15275-15278 (2004) and U.S. Patent No. 8,105,778, each of which is incorporated herein by reference. Figures 13-16 An exemplary configuration using HCR for signal amplification is shown.

[0338] Figure 13 An embodiment in which a MIP hybridizes to a target nucleic acid, for example cfDNA, leaving a single nucleotide gap is shown. The gap is filled by extension to incorporate biotinylated nucleotides and closed by ligation. The circularized MIP can then be detected using, for example Figure 14Streptavidin-coated surfaces are shown in the middle to bind, and after washing away any unbound MIP, the backbone of the bound MIP hybridizes to the initiator oligonucleotide. In preferred embodiments, a spacer, such as a hexaethylene glycol spacer of 18 atoms, is included between the initiator sequence and the backbone binding sequence. Preferably, the footprint of the MIP binding region is chosen to have high T m (e.g., about 79°C) to proceed with stable binding. As discussed above, binding tags other than biotin (such as amine, thiol, azide, or hapten) can be used to tag the MIP and immobilize to an appropriate reactive surface.

[0339] Figure 15 Examples of hairpin oligonucleotides used to form a self-assembled scaffold in HCR are shown. One or both oligonucleotides include at least one label, such as a fluorophore. In preferred embodiments, the dyes are positioned to provide a large enough separation in the assembled scaffold to prevent quenching effects. For example, in some embodiments, as shown in Figure 14 the dyes are located at opposite ends of the hairpin. As shown in Figure 16 Once the reaction is initiated by hybridization of the initiator oligonucleotide bound to the MIP backbone, the HCR hairpins unfold and hybridize in long chains, producing a scaffold that includes a large number of labels.

[0340] Flap endonuclease reactions (e.g., Invader assay) can be used for specific, quantitative detection of chromosomes. In Figures 17-20 an exemplary embodiment is shown. Figure 17 An invader oligonucleotide and a probe oligonucleotide hybridized to a target region of a chromosome are shown. The 3' end of the invader oligonucleotide overlaps the 5' end of the region of the probe oligonucleotide that is complementary to the target region. In this embodiment, the probe oligonucleotide includes a 5' flap including a biotin moiety and a 3' tail including a label (e.g., a fluorophore). A flap endonuclease (e.g., FEN-1 nuclease) recognizes the overlapping invader cleavage structure and cleaves the probe in a highly specific, structure-dependent manner, releasing the 5' flap. In preferred embodiments, as Figure 19 schematically shown in 3 4

[0341] In preferred embodiments, as Figure 18 ​​As shown, the probe oligonucleotides used comprise hairpin structures in which the 5' lobes and 3' tails of the probe hybridize to each other. A fluorophore or another moiety (e.g., 2,4-dinitrophenyl) can be used as a hapten, allowing the uncut probe and / or the 3' moiety of the cut probe to be removed from the reaction using an antibody against the hapten used for capture.

[0342] Cleavage lobes from a valve-shaped endonuclease reaction can be detected in a variety of ways. In a preferred embodiment, such as... Figure 20 As shown, the cleaved lobes are captured using immobilized complementary probes, and biotin reacts with streptavidin linked to the detectable portion. In the illustrated embodiment, streptavidin is coupled to β-galactosidase, and a fluorescent signal is generated by providing a non-fluorescent halogen-β-galactopyranoside, catalyzed by β-galactosidase to produce D-galactose and the fluorescent dye halogen. A femtoliter array and Poisson statistics are used to generate digital readout variants, and individual hybridization events can be detected using this enzymatic signal amplification. See, for example, DM Rissin and DR Walt, Digital Concentration Readout of Single Enzyme Molecules Using Femtoliter Arrays and Poisson Statistics. Nano Letters 6(3):520-523 (2006); Quanterix White Paper 1.0, Scientific Principles of Simoa (Single Molecules Array) Technology, 1-2 (2013); Quanterix White Paper 6.0, Simoa for Ultrasensitive Multiplex Immunoassay of Protein Biomarkers. TM Practical Applications of the HD-1 Analyzer, 1-3 (2015), each of which is incorporated herein by reference for all purposes. In some preferred embodiments, kinetic readout is used, i.e., signals are collected from the array at two time points.

[0343] Figure 21In the embodiment illustrated in FIG. 1, A, B, C, and D consist of MIPs specific for chromosome 13, 8, 21, or a reference chromosome (e.g., 1). The sequence of the MIPs that enclose the gap is complementary to the region of the chromosome targeted and is designed to contain a single nucleotide gap. Step 1: Fill in this gap with dNTPs conjugated to a hapten (e.g., a fluorescent dye, biotin, etc.). Filling in the gap introduces a different hapten into the MIP targeting each of the different specific chromosomes. For example, addition of A only completes the MIP targeting chromosome 21, T completes the MIP targeting chromosome 18, G completes the MIP targeting chromosome 13, and C completes the MIP targeting a reference chromosome (e.g., chromosome 1). This method labels the four different MIPs with four unique haptens. A pool of MIPs targeting each chromosome that requires a specific dNTP to complete a single extension and ligation is used to increase the number of capture events. Step 2 includes incubating the MIPs containing the haptens with labeled antibodies specific for each hapten. The label can include, for example, a fluorescent dye, quantum dot, or other fluorescent particle. Step 3 includes an optional step of exposing the immunocomplexes including the hapten-targeting primary antibodies to labeled secondary antibodies against the primary antibodies, thereby amplifying the fluorescent signal.

[0344] As Figure 21 As illustrated in FIG. 1, in this embodiment of the technology, the MIPs designed to target different chromosomes each require a different nucleotide to extend and ligate and wherein the MIPs use nucleotides carrying different dyes for each different dNTP to extend and ligate in a chromosome-specific manner. For example, in preferred embodiments, CY2, CY3, CY5, and CY7 are used. The MIPs with dye tags can be detected using antibodies specific for each different dye (and by extension, each different chromosome to be detected). The signal can be amplified by using secondary antibodies. For example, CY2 primary rabbit antibodies bind to the target MIPs and secondary goat anti-rabbit antibodies bind to the primary antibodies to amplify the signal, etc.

[0345] As discussed above, a number of different fluorescent labeling systems can be applied in embodiments of the technology. In some embodiments, fluorescent dyes (e.g., fluorescein, Texas Red, TAMRA, Cy3, Cy5) can be used, for example, attached to nucleotide analogs incorporated into the oligonucleotides or extension products. In some embodiments, fluorescent particles, for example, nanoparticles, nanocrystals, quantum dots, silica (e.g., mesoporous silica nanoparticles), polymer beads (e.g., latex) can be used.

[0346] There are many options for detecting and quantifying fluorescent signals from embodiments of the inventive technology described herein above. Detection can be based on measuring, for example, physicochemical, electromagnetic, electrical, optoelectrical, or electrochemical properties or characteristics of immobilized molecules and / or target molecules. Two factors relevant to single molecule detection of molecules on a surface are achieving sufficient spatial resolution to resolve individual molecules and distinguishing the desired single molecules from background signals (e.g., with probes that are not specifically bound to the surface). Exemplary methods for detecting single molecule related signals are found, for example, in WO 2016 / 134191, which is incorporated herein by reference in its entirety for all purposes. In some embodiments, assays are configured for standard SBS microplate detection, for example, in a SpectraMax microplate reader or other plate reader. While this approach generally requires low variation fluorescence (multi-well, multiple measurements), this format can be multiplexed and read on multiple different fluorescence channels. Additionally, the throughput of the format is very high.

[0347] Embodiments can also be configured for detection on a surface (e.g., a glass surface, a gold surface, or a carbon (e.g., diamond) surface). In some embodiments, signal detection is accomplished by any method for detecting electromagnetic radiation (e.g., light), such as a method selected from far-field optical methods, near-field optical methods, epi-fluorescence spectroscopy, confocal microscopy, two-photon microscopy, optical microscopy, and total internal reflection microscopy, with the target molecule labeled with an electromagnetic radiation emitter. Other microscopy methods, such as atomic force microscopy (AFM) or other scanning probe microscopy (SPM), are also applicable. In some embodiments, it can not be necessary to label the target. Alternatively, a label that can be detected by SPM can be used. In some embodiments, signal detection and / or measurement includes surface reading by counting fluorescent clusters using an imaging system such as an ImageXpress imaging system (Molecular Devices, San Jose, CA) and similar systems.

[0348] Embodiments of the present technology can be configured for detection using many other systems and instrument platforms (e.g., bead assays (e.g., Luminex), array hybridization, NanoString nCounter single molecule counting device). See, e.g., GK Geiss et al., Direct multiplexed measurement of gene expression with color-coded probe pairs; Nature Biotechnology 26(3):317-25 (2008), U.S. Patent Application 2018 / 0066309 Al published August 3, 2018 (PN Hengen et al., inventors of Nanostring Technologies, Inc.), and the like.

[0349] In a Luminex bead assay, color-coded beads pre-coated with analyte-specific capture antibodies for molecules of interest are added to a sample. Multiple analytes can be detected simultaneously in the same sample. The analyte-specific antibodies capture the analyte of interest. A biotinylated detection antibody that is also specific for the analyte of interest is added, allowing antibody-antigen sandwich formation. Phycoerythrin (PE)-conjugated streptavidin is added, and the beads are read on a dual-laser flow-based detection instrument. The beads are read on a dual-laser flow-based detection instrument (e.g., Luminex 200 TM or The analyzer). One laser classifies the beads and determines the analyte to be detected. The second laser determines the magnitude of the PE-derived signal, which is proportional to the amount of bound analyte.

[0350] NanoString nCounter is a single molecule counting device for the digital quantification of hundreds of different genes in a single multiplexed reaction. The present technology uses molecular "barcodes" (each of which is color-coded to correspond to a single probe for a gene (or other nucleic acid) of interest and is linked thereto) in combination with solid phase hybridization and automated imaging and detection. See, e.g., Geiss et al. (Id.), which describes the use of unique pairs of capture and reporter probes constructed for the detection of each nucleic acid of interest. In the described embodiments, the probes are mixed together with nucleic acids (e.g., unpartitioned cfDNA) or total RNA from a sample in a single solution phase hybridization reaction. Hybridization results in the formation of tripartite structures composed of the target nucleic acid bound to its specific reporter and capture probes, and unhybridized reporter and capture probes are removed, e.g., by affinity purification. When biotin immobilization tags are used, the hybridization complexes are exposed to an appropriate capture surface (e.g., a streptavidin-coated surface). After capture on the surface, an applied electric field extends in the same direction in solution and orients each complex. The complexes are then immobilized in the elongated state and imaged. Thus, each target molecule of interest can be identified and counted by the color code generated by the ordered fluorescent segments present on the reporter probe and counted to count the target molecules.

[0351] Figure 22 FIGS. A, B, and C of the present technology are illustrated in which the MIPs, including the immobilization moiety or linked thereto, are immobilized on a surface. While not limited to any particular embodiment for incorporating a unique signature indicative of target recognition into the circularized MIP molecule, the embodiments of Figure 22 Embodiments of the present technology are illustrated using embodiments that use one or more nucleotides of the target nucleic acid, including using a polymerase to replicate the target nucleic acid, to extend the linear MIP, followed by ligation to circularize the extended probe.

[0352] In Figure 22In the embodiment shown in FIG. A, in step 1, MIPs are hybridized to target DNA and then extended by DNA polymerase in the presence of modified dNTPs such that the immobilization moiety is incorporated into each MIP during extension. The MIPs are then ligated to themselves to complete the circularized probe. The modified dNTPs can include, but are not limited to, dNTPs that include reactive chemicals such as amine groups or thiol groups or other bindable features such as biotin or antibody hapten. In step 2, the circularized MIPs are exposed to a surface under conditions in which the immobilization features of the MIPs interact with the surface to bind the MIPs. Such surfaces include, but are not limited to, derivatized or non-derivatized glass, silica, diamond, gold, agarose, plastic, ferromagnetic materials, alloys, etc., and can be in any form, for example, a glass slide, a sample well, a channel, a bead, a particle, and / or a nanoparticle, any of which can be porous or non-porous.

[0353] In Figure 22 In the embodiment shown in FIG. B, in step 1, MIPs are hybridized to target DNA and ligated to circularize. In the embodiment shown, the MIPs are extended by DNA polymerase to fill in sequence gaps prior to ligation, while in other embodiments, the MIPs can be designed to simply hybridize to the target nucleic acid and ligate to circularize in the manner of, for example, padlock probes, without the use of a polymerization step. See, for example, M. Nilsson et al., “Padlock probes: circularizing oligonucleotides for localized DNA detection”. Science 265(5181): 2085-2088 (1994). In step 2, the circular MIPs are hybridized to a complementary oligonucleotide that contains an immobilization moiety as described above (e.g., a reactive amine, a reactive thiol group, biotin, a hapten, etc.). In step 3, the hybridized MIP complex of MIPs and oligonucleotide including the immobilization moiety are exposed to a surface under conditions in which the immobilization features of the MIP complex interact with the surface to bind the MIP complex. As described above, the surface includes, but is not limited to, derivatized or non-derivatized glass, silica, diamond, gold, agarose, plastic, ferromagnetic materials, alloys, etc., and can be in any form, for example, a glass slide, a sample well, a channel, a bead, a particle, and / or a nanoparticle, any of which can be porous or non-porous.

[0354] In Figure 22In the embodiment shown in Figure C, in step 1, the MIP containing the immobilized portion constructed into the probe backbone is hybridized with DNA, extended by DNA polymerase, and ligated to circularize the probe. As in the embodiment described above in Figure B, the MIP can be designed to hybridize and ligate to the target nucleic acid without using a polymerization step. In step 2, the cyclized MIP containing the immobilized portion is exposed to a surface under conditions where the surface interacts with the immobilization features of the MIP to bind the MIP. As described above, the surface includes, but is not limited to, derivatized or underivatized glass, silica, diamond, gold, agarose, plastics, ferromagnetic materials, alloys, etc., and can be in any form, such as a glass slide, sample well, channel, bead, particle, and / or nanoparticle, any of which can be porous or non-porous.

[0355] exist Figure 22 In each of the embodiments shown herein, once the MIP has been immobilized to the surface, labeling and / or signal amplification (e.g., fluorescent labeling and / or fluorescent signal amplification) and detection can be performed using any of the various back-end analysis methods discussed herein. Suitable methods for amplifying and / or detecting unique immobilized MIP products include, but are not limited to, the NanoString nCounter technology described above and Figures 2-3 , Figures 6-7 , Figures 9-12 , Figures 15-16 and Figures 20-21 The method is illustrated in the figure. In some embodiments, the labeling and / or signal amplification (e.g., fluorescent labeling and / or fluorescent signal amplification) is performed before the MIP has been fixed to the surface.

[0356] In preferred embodiments, a back-end process configured for single-molecule visualization is used. For example, as described above, the Quanterix platform uses an array of nanoparticle-sized pores that capture beads with no more than one tagged complex, wherein signals from the captured complexes are visualized using a halogen-β-galactopyranoside / β-galactosidase reaction to generate fluorescent halogen. Visualization of the array allows detection of signals from each individual complex. In some preferred embodiments, solid-state nanopore devices are used, such as those described by Morin et al. (see “Nanopore-Based Target Sequence Detection”, PLOS ONE 11(5):e0154426(2016)). Solid-state nanopores are nanoscale openings formed in a solid film that separates two aqueous volumes

[23] . A voltage clamp amplifier applies a voltage across the film while measuring the ion current passing through the opening ( Figure 1a). When a single charged molecule such as double stranded DNA is captured and driven through a pore by electrophoresis, the measured current shift and the depth (delta I) and duration of the shift are used to characterize the event. (Morin et al., supra). While DNA can be detected individually using this system, unique tags (e.g., different sizes of polyethylene glycol (PEG)) can be attached to highly sequence-specific probes (e.g., peptide nucleic acid probes, PNA) to give any particular DNA-PNA-PEG complex a unique signature tag that is indicative of the target nucleic acid detected in the front end of the assay.

[0357] In the embodiments shown in Figure 23 , a complex is formed that includes an oligonucleotide primer and a circular probe such as a MIP or a tethered padlock probe. Extension of the primer in a rolling circle amplification reaction produces a long chain of single stranded DNA containing a concatemer of sequences complementary to the circular probe. The RCA product binds to a plurality of molecular beacon probes having a fluorophore and a quencher. Hybridization of the beacons separates the quencher from the fluorophore, allowing fluorescence from the beacons to be detected. Accumulation of the RCA product can be monitored in real time by measuring an increase in fluorescence intensity indicative of the beacons binding to increasing amounts of product over the course of the reaction.

[0358] Real time quantification of the accumulating fluorescence in the reaction was used to examine the effect of the attached biotin moiety on the MIP or on the primer. Figure 24 Figures A-D of the accompanying drawings show results obtained from examining the effect of including a biotin residue in the circularized MIP only (A), in the RCA primer only (B), in both (C), and not in both (D) on the RCA signal. In this experiment, the MIP contained the following sequence:

[0359]

[0360] (circularized).

[0361] In the above biotinylated MIP, the boxed "T" shows the site of attachment of the biotin in the biotin-containing MIP (Integrated DNA Technologies, "Internal Biotin dT"). The biotinylated primer includes a biotin attached at the terminal '5' phosphate (Integrated DNA Technologies, "5' Biotin-TEG"). Rolling circle reactions were performed at 37°C for one hour according to the "Standard Rolling Circle Reaction" procedure described below in Example 1. These data show that the presence of biotin in the circularized MIP inhibits RCA, while the presence of biotin on the primer does not inhibit the reaction.

[0362] Figures 25A-25C Results are shown of varying the amount of components in a standard RCA reaction in solution.Figure 25A A comparison was made between using 5 units and 25 units of Phi29 polymerase in each reaction, and it was shown that the higher concentration of polymerase consistently produced higher signals under the conditions tested. Figure 25B The effect of using different concentrations of molecular beacon probes ("beacons") was shown; Figure 25C The effect of using different concentrations of Phi29 polymerase and molecular beacon probes was compared to the effect of using 200 mM or 800 mM total dNTPs on standard reactions. Based on these data, further testing was performed on reactions adjusted to include 1000 nM beacons, 800 mM dNTPs, and 2000 nM phi 29 polymerase (80 units).

[0363] The effect of adding different concentrations of PEG and using different sizes of PEG on enhanced RCA conditions (E-RCA, see Example 1 below) was examined. Figure 26 The effect of using different sizes of PEG (200 and 8000) at the indicated percentages (w:v) in E-RCA conditions was compared. Under the conditions tested for this example, PEG 200 provided superior results at all concentrations tested, with 20% PEG 200 providing the best results. In contrast, PEG 8000 significantly reduced the efficiency of RCA. Based on these data, further testing was performed on RCA reactions including at least 20% w:v PEG 200.

[0364] As discussed above, when performing single molecule detection on a surface, it is preferable to minimize the spot size of the signal from any individual bound molecule, such that separation between spots is ensured. The effect of using PEG 200 on spot size and the number of detected spots was examined. Assays were performed using the E-RCA conditions described below with or without 20% w:v PEG 200, incubated for 140 minutes. The results are shown in Figures 27A-27B and Figure 28 The results are shown in Figure 27A The presence of PEG was shown to reduce spot size, thereby enhancing the measurement of fluorescent signal from individual spots. Figure 27B The effect of PEG on the number and fluorescence intensity of spots shown in Figure 27A was shown, and it was shown that the addition of PEG increased the number of detectable spots while reducing the size of the detected spots.

[0365] The effect of using different molecular weight PEG in a 20% solution on spot count and spot size in reactions performed on APTES silanized plates was examined. Reactions were performed on APTES treated surfaces as described in “One-step rolling circle amplification on surfaces” in Example 1, with modifications to the PEG component as indicated in Figure 28 Figure 28 It was shown that spot number was maximized and spot size was minimized when the PEG used was less than 1000, preferably between 200 and 800, more preferably 600 average molecular weight.

[0366] The length of hybridization time was examined prior to initiating the RCA reaction. Figure 29A Microscope images of the surface of APTES silanized plates as described in Example 1 were shown, and RCA signal was compared for hybridization for 18 hours or 1 hour prior to initiating the RCA reaction in TBS buffer or RCA buffer. Enhanced RCA was performed with 20% PEG 600 for 140 minutes as described above. Figure 29B Plots comparing the effect of hybridization time and buffer on Figure 29A The number of spots and fluorescence intensity (area) shown in Figures

[0367] Figure 30 , 31 and 32 provide plots comparing the effect of PEG 200 with or without a 2 hour hybridization time, and PEG 2000 replacing PEG 200 with a 2 hour hybridization on standard RCA reaction conditions, enhanced RCA (E-RCA) conditions, and E-RCA conditions with other changes. Reactions in each plot were performed at the same temperature, with the reaction in Figure 30 , 31 and 32, the reactions were performed at 30°C, 25°C, and 37°C, respectively.

[0368] ​The number of spots and fluorescence intensity (area) were assessed for each condition. These data show that a reaction temperature of 37°C in the presence of PEG 200 produced the best combination of high spot count and small spot size. The effect of using a higher RCA reaction temperature to vary the concentration of beacon probe was also examined. Reactions containing 1000 nM, 2000 nM, 4000 nM, or 8000 nM molecular beacon probe were performed at 37°C or 42°C, and the reactions showed a significant increase in the number of spots counted at the higher temperature (data not shown). While the present technology is not limited to any particular mechanism of action, these data suggest that performing the reaction at a higher temperature (e.g., 42°C or higher) produces more RCA product and more bound beacon probe.

[0369] The effect of elevated temperature in the presence of different concentrations of PEG 600 was further examined. Figure 33 Microscope images of the surface of an APTES-silanized plate as described in Example 1 were shown and RCA signals were compared for reactions including the indicated concentrations of PEG 600 performed at 37°C or 45°C. These data show that a 45°C reaction produced a significantly higher spot count, and 10% to 15% w:v PEG 600 produced the best combination of spot count and spot size at 45°C.

[0370] The effect of adding graphene oxide to the RCA surface binding reaction was examined. A two-step RCA procedure as described in Example 2 and as schematically shown in Figure 35 Figure 36 Microscope images of the surface of an APTES-silanized plate as described in Example 1 were shown and RCA signals were shown for the two-step reaction graphene oxide. The negative control contained no input target and shows background from the molecular beacon probe. Figure 37 A graph comparing spot count for RCA reactions performed in one step (no GO) or two steps (with or without GO) is provided, comparing reactions with 100 fmol target to reactions without target. These data show that the use of GO significantly reduces the number of background spots in the no target control reactions, improving the signal:background results in the assay.

[0371] Experiment

[0372] Example 1

[0373] This example provides an example of a workflow for analyzing DNA, e.g., cfDNA, from a sample such as a blood sample.

[0374] Sample Collection

[0375] ​Blood is collected from patients at standard draw volumes. 10 mL of blood is stored in Streck blood collection tubes or alternatively EDTA containing blood collection tubes. The samples are shipped to the laboratory at ambient temperature and processed as follows:

[0376] • Centrifuge blood at 2000 x g for 20 minutes at room temperature to obtain the plasma fraction from the blood.

[0377] • Transfer the plasma to a new sterile nuclease-free polypropylene tube and centrifuge at 3220 x g for 30 minutes.

[0378] Free DNA (cfDNA) purification

[0379] Free DNA is purified from the plasma using standard methods, for example using the MagMAX Free DNA Isolation Kit (ThermoFisher Scientific, Cat# A29319).

[0380] Assay plate preparation

[0381] Glass-bottomed microtiter plates are treated to immobilize oligonucleotides that prime the rolling circle amplification of circularized MIPs. Several methods can be used (see, for example, E. J. Devor et al., “Strategies for Attaching Oligonucleotides to Solid Supports” in Integrated DNA Technologies (2005), which is incorporated by reference herein in its entirety for all purposes).

[0382] 1) Acid pre-wash

[0383] For each method, the glass-bottomed plates are first acid washed as follows:

[0384] (a) Add 100 μΐ of 0.5 N sulfuric acid to each well.

[0385] (b) Add foil seal to the plate.

[0386] (c) Incubate the plate at 37 °C for 2 hours by spinning at 300 RPM.

[0387] (d) Remove the well contents.

[0388] (e) Wash the wells twice with 100 μΐ of molecular grade water.

[0389] (f) Wash the wells twice with 100 μΐ of 95% ethanol.

[0390] 2) 3-aminopropyltriethoxysilane (APTES) silanization and streptavidin-biotin primer immobilization:

[0391] (a) Prepare 2% APTES by adding 200 pL of 99% APTES (Sigma-Aldrich, Catalog # 440140), 500 pL of molecular grade water, and 9.3 ml of 95% ethanol.

[0392] (b) Vortex the solution and pipette 100 pL into each well.

[0393] (c) Incubate for 15 minutes at room temperature.

[0394] (d) Remove the well contents.

[0395] (e) Wash the wells twice with 100 pL of 95% ethanol.

[0396] (f) Remove the last wash.

[0397] (g) Incubate the plate for 24 hours at 37°C.

[0398] Primer immobilization

[0399] (h) Add 1 nanogram of streptavidin in 100 pL of Tris-buffered saline to the amine-functionalized glass plate.

[0400] (i) Incubate for 1 hour at room temperature.

[0401] (j) Wash each well three times with 100 pL of TBS.

[0402] (k) Add 100 pL of 1 mM biotinylated oligonucleotide solution.

[0403] (l) Incubate for 1 hour at room temperature.

[0404] (m) Wash each well three times with 100 pL of TBS.

[0405] 3) Acrydite primer immobilization

[0406] (a) Prepare 4% acrydite by adding 400 pL of 99% acrydite (3-(trimethoxysilyl)propyl methacrylate; Sigma-Aldrich, Catalog # 440159), 1 mL of molecular grade water, and 18.6 mL of 100% ethanol.

[0407] (b) Add 100 pL of 4% acrydite solution to each well.

[0408] (c) Incubate for 15 minutes at room temperature.

[0409] (d) Remove 4% Acrydite solution.

[0410] (e) Wash each well four times with 100 μL of 100% ethanol per wash.

[0411] (f) Incubate the plate at 37°C for 24 hours.

[0412] (g) Prepare the acrydite primer solution by adding:

[0413] (i) 250 μL 5x TRIS-borate EDTA (TBE) buffer,

[0414] (ii) 500 μL 40% acrylamide,

[0415] (iii) 17.5 μL 10% ammonium persulfate,

[0416] (iv) 5 μL tetramethyl ethylene diamine (TEMED),

[0417] (v) 25 μL 100 μM oligonucleotide primer including '5' acrydite (or acrydite-phosphoramidite) (vi) 1.7 mL of molecular grade water.

[0418] (h) Add 25 μL of the acrydite primer solution to each well and gently stir the plate to cover the well bottoms.

[0419] (i) Incubate at room temperature for 30 minutes.

[0420] (j) Wash the wells four times with 100 μL of 0.5x TBE, discarding the first three washes and leaving the last wash in the well, then proceed with the RCA assay.

[0421] Primers can be immobilized by other methods (e.g., as described by Devor et al. (supra).

[0422] Molecular inversion probe pools

[0423] Probe pools are used to capture specific loci in a DNA sample, such as a cfDNA sample, and generate circularized MIPs for rolling circle amplification. NIPT assays typically include molecular inversion probe pools. In preferred embodiments, a NIPT assay includes about 5,000-10,000 molecular inversion probes.

[0424] • Generate targeted MIPs to target features to be investigated by the assay (e.g., chromosomes 13, 18, 21, X, Y, and CHR22q11.2).

[0425] • Approximately 10,000 unique MIPs are generated for each feature.

[0426] • MIPs are mixed together to create a probe pool, where each probe has a custom concentration.

[0427] MIP capture and ligation of cfDNA

[0428] • The MIP pool is added to purified cfDNA in the following reaction.

[0429] o 2 μΐ of AMP Ligase Buffer (10x), 1 μΐ of MIP probe pool, 16 μΐ of cfDNA preparation, and 1 μΐ of AMP Ligase (80 units).

[0430] o The reaction is incubated at 98°C for 2 minutes and cooled at 1 degree per minute until the reaction reaches 45°C, then held at 45°C for 2 hours.

[0431] Molecular Beacon Probes

[0432] Examples of molecular beacon probes that can be used in the present technology are as follows:

[0433] 1) 5'Alexa 405-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-dabcyl 3'

[0434] 2) 5'Alexa 488-CC TCAATG CTG CTG CTG TAC TAC mGmAmG mG-dabcyl 3'

[0435] 3) 5'Alexa 594-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-BHQ2 3'

[0436] 4) 5'Alexa 647-CCTCAGCGCTGCCTATTCGAACTmGmAmGmG-BHQ2 3'

[0437] 5) 5'Alexa 750-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-BHQ3 3'

[0438] Standard Rolling Circle Amplification Assay Conditions

[0439] o For a 100 μΐ RCA solution, combine on ice

[0440] ■MIP Probe-Target DNA Preparation (e.g., entire MIP capture / cfDNA preparation described above, approximately 20 μΐ)

[0441] ■10X Phi29 buffer at 10 pL, final concentration IX

[0442] 1X Phi29 DNA polymerase reaction buffer

[0443] - 50 mM Tris-HCl

[0444] - 10 mM MgCl2

[0445] - 10 mM (NH4)2S04

[0446] - 4 mM DTT

[0447] - (pH 7.5 at 25 °C)

[0448] ■200 pM dNTP

[0449] ■5 units of Phi29 DNA polymerase

[0450] ■100 nM Beacon probe

[0451] ■Molecular grade water up to 100 pL

[0452] o Incubate at 30-37 °C for the reaction time (e.g., 90-120 min).

[0453] Enhanced RCA (E-RCA) conditions:

[0454] o For 100 pL of enhanced RCA solution, combine on ice

[0455] ■MIP probe-target DNA preparation (e.g., entire MIP capture / cfDNA preparation described above, approximately 20 pL);

[0456] ■10X Phi29 buffer at 10 pL, final concentration IX

[0457] ■800 pM dNTP

[0458] ■80 units of Phi29 DNA polymerase

[0459] ■1000 nM Beacon probe

[0460] ■Molecular grade water up to 100 pL

[0461] o Incubate at 30-37 °C for the reaction time (e.g., 90-120 min).

[0462] One-step enhanced rolling circle amplification on surface

[0463] • Prepare rolling circle amplification (RCA) solution

[0464] o For 100 μL RCA solution, combine on ice

[0465] ■MIP probe - target DNA preparation (e.g., entire MIP capture / cfDNA preparation described above, approximately 20 μL);

[0466] ■10 μL of 10X Phi29 buffer, final concentration 1X

[0467] 1X Phi29 DNA polymerase reaction buffer

[0468] - 50 mM Tris-HCl

[0469] - 10 mM MgCl2

[0470] - 10 mM (NH4)2SO4

[0471] - 4 mM DTT

[0472] - (pH 7.5 at 25 °C)

[0473] ■4 μL of 10 mM dNTPs, total dNTP final concentration 0.4 mM;

[0474] ■50 μL of filtered 30% PEG 600;

[0475] ■0.5 μL of 100 μM molecular beacons, final concentration 0.5 μM

[0476] ■8 μL of Phi29 polymerase (10 units / μL); and

[0477] ■22.5 μL of molecular grade water

[0478] o Mix solution, e.g., by vortexing, and pipette onto treated glass surface including bound primers, then seal plate;

[0479] o Incubate plate on flat-bottomed heating block of thermal mixer with thermal lid at 45 °C for 90 minutes;

[0480] o Remove well contents and wash wells twice with 100 μL of 1X TBS; discard wash solutions;

[0481] o Add 100 μL of 1X TBS and image in microscope as described below.

[0482] Image samples with IXM4 microscope (Molecular Devices, San Jose, CA)

[0483] Images are typically captured using a 20x, 40x or 60x objective lens.

[0484] • Place the plate in the IXM4 microscope and image as follows:

[0485] o The plate is automatically exposed to ensure a wide dynamic range of fluorescence intensity values (the maximum range of the camera used, such as 16-bit images).

[0486] o Subdivide each well of the plate into approximately 100 images.

[0487] For high-throughput assays, an automated microscope can be used.

[0488] Image analysis

[0489] • Analyze the images as follows:

[0490] o Determine the relative fluorescence intensity in images without sample (negative control).

[0491] o Determine the threshold by multiplying the average relative fluorescence intensity from the negative control by three.

[0492] o In each channel, count the spots above the threshold.

[0493] Variations of one-step protocol

[0494] Crowding reagent (e.g., PEG) addition: Prepare a 30% solution in molecular grade water; filter with a filter with a pore size of 0.2 pm. Add PEG to the RCA reaction, adjusting the water added to the RCA to maintain consistent volume.

[0495] Beacons: Add the desired concentration, adjusting the water added to the RCA to maintain consistent volume.

[0496] dNTPs: Add the desired concentration, adjusting the water added to the RCA to maintain consistent volume.

[0497] Graphene oxide: Perform the 2-step reaction as described in Example 2, adding graphene oxide and labeled probes.

[0498] Example 2

[0499] Detection using two-step rolling circle amplification on surfaces with graphene oxide

[0500] Prepare rolling circle amplification (RCA) solution on ice:

[0501] o For 100 pL RCA solution (without molecular beacons), combine:

[0502] ■MIP probe - target DNA preparation (e.g., entire MIP capture / cfDNA preparation described above, approximately 20 μL);

[0503] ■10 μL of 10X Phi29 buffer, final concentration 1X

[0504] 1X Phi29 DNA polymerase reaction buffer

[0505] - 50 mM Tris-HCl

[0506] - 10 mM MgCl2

[0507] - 10 mM (NH4)2SO4

[0508] - 4 mM DTT

[0509] - (pH 7.5 at 25 °C)

[0510] ■4 μL of 10 mM dNTPs, total dNTP final concentration 0.4 mM;

[0511] ■50 μL of filtered 30% PEG 600;

[0512] ■8 μL of Phi29 polymerase (10 units / μL); and

[0513] ■23 μL of molecular grade water

[0514] o Mix solution by vortexing and pipette onto treated glass surface, then seal plate;

[0515] o Incubate plate on flat-bottomed heating block of thermal mixer with heat lid at 45 °C for 90 minutes;

[0516] o Remove well contents and wash wells three times with 100 μL of 1X TBS; discard wash solutions;

[0517] o Add 50 μL of graphene oxide-molecular beacon solution including:

[0518] ■5 μL of 10X Phi 29 buffer, final concentration 1X

[0519] ■0.5 μL of 100 μM molecular beacon, final concentration 0.5 μM

[0520] ■5 μL of 2 mg / mL graphene oxide solution, final concentration 0.2 mg / mL;

[0521] ■Molecular grade water to 50 μL

[0522] o Incubate the reaction at 37°C for 60 minutes

[0523] o Wash three times with 100 μΙ_ 1 x TBS;

[0524] o Wash once with 100 μΙ_ 1 x TBS containing 5% w:v Tween 20:

[0525] o Wash twice with 100 μΙ_ of 1 X TBS; discard wash solutions;

[0526] o Add 100 μΙ_ of 1 X TBS and image in the microscope as described above.

[0527] It will be readily apparent that each of the disclosed front-end target recognition systems provided in relation to the disclosure herein can be configured to generate a detectable signal for use with any of the back-end instruments and systems described above.

[0528] Additional References

[0529] 1. F. Dahl et al., Imaging single DNA molecules for high precision NIPT. Nature Scientific Reports 8:4549 (2018) pp. 1-8

[0530] 2. R. M. Dirks et al., Triggered amplification by hybridization chain reaction. Proc. Natl. Acad. Sci. USA 101 (43): 15275-15278 (2004)

[0531] 3. T. J. Morin et al., Nanopore-Based Target Sequence Detection. PLoS ONE 11(5): e0154426 (2016)

[0532] 4. M. Nilsson et al., Real-time monitoring of rolling-circle amplification using a modified molecular beacon design, Nucleic Acids Research 30(14): e66 (2002)

[0533] 5. J. R. Epstein et al., High-Density Fiber-Optic Genosensor Microsphere Array Capable of Zeptomole Detection Limits, Anal. Chem. 74: 1836-1840 (2002)

[0534] 6. D. M. Rissin and DR Walt, Digital Concentration Readout of Single Enzyme Molecules Using Femtoliter Arrays and Poisson Statistics, Nano Letters 6(3): 520-523 (2006)

[0535] 7. R. Roy et al., A Practical Guide to Single Molecule FRET Nat Methods 5(6): 507-516 (2008)

[0536] 8. Z. Li et al., Detection of Single-Molecule DNA Hybridization Using Enzymatic Amplification in an Array of Femtoliter-Sized Reaction Vessels, J. Am. Chem. Soc. 130: 12622-12623 (2008)

[0537] 9. W. Zhang et al., Automated Multiplexing Quantum Dots in Situ Hybridization Assay for Simultaneous Detection of ERG and PTEN Gene Status in Prostate Cancer, The Journal of Molecular Diagnostics 15(6):754-764 (2013)

[0538] 10. Quanterix Whitepaper 1.0, Scientific Principle of Simoa (Single Molecule Array) Technology, 1-2 (2013)

[0539] 11. Quanterix Whitepaper 6.0, Simoa for Ultrasensitive Multiplex Immunodetection of Protein Biomarkers, 1-3 (2015) TM Practical Application of Simoa HD-1 Analyzer for Ultrasensitive Multiplex Immunodetection of Protein Biomarkers, 1-3 (2015) TM Practical Application of Simoa HD-1 Analyzer for Ultrasensitive Multiplex Immunodetection of Protein Biomarkers, 1-3 (2015)

[0540] 12. H. Matsui et al., Molecular and Biochemical Characterization of a Serine Proteinase Predominantly Expressed in the Medulla Oblongata and Cerebellar White Matter of Mouse Brain, J. Biol. Chem. 275(15): 11050-11057 (2000)

[0541] 13. C. M. Van der Loos et al., Multiple immunoenzyme staining techniques: Use of fluoresceinated, biotinylated and unlabelled monoclonal antibodies. J. Immunol. Methods 117:45-52 (1989)

[0542] 14. J. Hagen et al., Hapten-Anti-Hapten Technique for Two-Color IHC Detection of Phosphorylated EGFR and H2AX Using Primary Antibodies Raised in the Same Host Species; Signal Transduction Immunohistochemistry: Methods and Protocols, Methods in Molecular Biology, Vol. 1554: 155-160 (Alexander E. Kalyuzhny (ed.) Molecular Biology ) (2019)

[0543] 15. G. K. Geiss et al., Direct multiplexed measurement of gene expression with color-coded probe pairs; Correction of authors' affiliations at Nature Biotechnology 26(3):317-25 (March 2008) and 26(6):1 (June 2008)

[0544] 16. P. N. Hengen et al., U.S. Patent Application Serial No. 15 / 729,421, published as U.S. Patent Publication 2018 / 0066309 Al on August 3, 2018 (Nanostring Technologies, Inc.)

[0545] 17. M. Nilsson et al. "Padlock probes: circularizing oligonucleotides for localized DNA detection". Science 265(5181): 2085-2088 (1994)

[0546] 18. P.-J. J. Huang and J. Liu "Molecular Beacon Lighting up on Graphene Oxide" Analytical Chemistry 84: 4192-4198 (2012)

[0547] 19. Y. Phillip et al., "Common Crowding Agents Have Only a Small Effect on Protein-Protein Interactions" Biophysical Journal 97: 875-885 (2009)

[0548] 20. L. M. Dominak et al., "Polymeric Crowding Agents Improve Passive Biomacromolecule Encapsulation in Lipid Vesicles" Langmuir 26(16): 13195-13200 (2010)

[0549] 21. B. Schweitzer et al., "Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection" Proceedings of the National Academy of Sciences of the United States of America 97(18): 10113-10119 (2000)

[0550] 22. C. Hong et al., "Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide" Analytical Chemistry 88:2999-3003 (2016)

[0551] 23. E. J. Devor et al., "trategies for Attaching Oligonucleotides to Solid Supports" Integrated DNA Technologies (2005)

[0552] 24. WO 2015 / 083002 "Multiplex Detection of Nucleic Acids"

[0553] All literature and similar materials cited in this application, including but not limited to, publications, books, treatises, and internet web pages, whether or not specifically identified herein, are expressly incorporated by reference herein in their entirety for the purpose of providing a clear and enabling understanding of the various embodiments of the present technology presented herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong. In the event that there is a plurality of definitions for a term herein, those in this disclosure control.

[0554] Various modifications and variations of the described compositions, methods, and uses of the described technology will be apparent to those skilled in the art without departing from the scope and spirit of the described technology. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the claimed technology is not intended to be limited to these specific embodiments. Indeed, various modifications of the described modes of carrying out the technology that are obvious to those skilled in molecular biology, molecular diagnostics, nucleic acid structure, biochemistry, medical science, or related fields are intended to be within the scope of the claims.

Claims

1. A method comprising: a) forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid, wherein the primer is bound to a surface of a solid support; b) exposing the at least one complex to a reaction mixture comprising a DNA polymerase, one or more deoxynucleotide triphosphates (dNTPs), and at least 12% polyethylene glycol (PEG), wherein the PEG has an average molecular weight of between 400 and 800, under conditions wherein the primer in the complex is extended on the circularized nucleic acid by the DNA polymerase to form an extended primer.

2. The method of claim 1, further comprising detecting the extended primer.

3. The method of claim 2, wherein extension of the primer in the complex on the circularized nucleic acid is in a rolling circle amplification (RCA) reaction, wherein the extended primer comprises a RCA product.

4. The method of claim 3, wherein detecting the extended primer comprises: i) hybridizing at least one probe to the RCA product; and ii) detecting at least one hybridized probe; wherein detection of a hybridized probe at a locus on the surface of the solid support indicates formation of a complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid at that locus on the surface of the solid support.

5. The method of claim 4, wherein the at least one probe comprises a labeled probe, and wherein detecting the at least one hybridized probe comprises detecting at least one hybridized labeled probe.

6. The method of claim 5, wherein the labeled probe comprises a fluorescent label, and wherein detecting the at least one hybridized labeled probe comprises detecting fluorescence.

7. The method of claim 6, wherein detecting fluorescence comprises fluorescence microscopy.

8. The method of claim 4, wherein the at least one probe comprises a plurality of labeled probes, wherein a plurality of RCA products hybridized to the plurality of labeled probes are immobilized in a dispersed manner on the surface of the solid support at a locus; wherein at least a portion of the plurality of RCA products can be individually detected at the locus on the surface of the solid support by detecting hybridized labeled probes.

9. The method of claim 8, wherein the hybridized labeled probes each comprise the same label.

10. The method of claim 8, wherein the dispersion of RCA products is irregular.

11. The method of claim 8, wherein the label on the plurality of labeled probes comprises a fluorescent label, and wherein RCA products that can be individually detected at a locus on the surface of the solid support can be detected by detecting fluorescence.

12. A composition comprising: a) a plurality of complexes bound to a silanized surface of a solid support, each complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid, wherein the primer in the complex is covalently linked to the silanized surface of the solid support in a dispersed manner; and b) a plurality of RCA products, each RCA product comprising an extended primer hybridized to a circularized nucleic acid, wherein the RCA products are immobilized on the silanized surface of the solid support in a dispersed manner. b) a reaction mixture in contact with the plurality of complexes, the reaction mixture comprising a DNA polymerase enzyme, one or more dNTPs, and at least 12% PEG, wherein the PEG has an average molecular weight of between 400 and 800.

13. The composition of claim 12, wherein the dispersion is a random dispersion.

14. The composition of claim 12, wherein the reaction mixture further comprises a labeled probe.

15. The composition of claim 14, wherein the labeled probe comprises a fluorescent label.

16. The composition of claim 12, wherein the primers are covalently attached to the solid support in a random dispersion.

17. The composition of claim 12, wherein the PEG has an average molecular weight of 600.

18. The composition of claim 12, wherein the composition comprises at least 16% (w:v) PEG.

19. The composition of claim 12, wherein the composition comprises at least 18% (w:v) PEG.

20. The composition of claim 12, wherein the composition comprises at least 20% (w:v) PEG.

Images