Methods, systems, and compositions for counting nucleic acid molecules

Abstract

To provide a method for detecting and quantifying variations in numbers of molecules, particularly variations in gene dosage, due to gene duplication or to trisomy of one or more chromosomes that are normally found in diploid pairs, without digital sequencing.SOLUTION: The present invention provides 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 solid support surface; and (b) exposing the at least one complex to a reaction mixture comprising DNA polymerase, one or more deoxynucleoside triphosphates (dNTPs), and at least 12% polyethylene glycol (PEG), wherein the PEG has an average molecular weight between 400 and 800, under conditions in which the primer in the complex is extended on the circularized nucleic acid by the DNA polymerase to form an extended primer.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 651,676, filed Apr. 2, 2018, and U.S. Provisional Application No. 62 / 660,699, filed Apr. 20, 2018, each of which is incorporated herein by reference.

[0002] The present invention relates to compositions and methods for determining the copy number of individual molecules, such as nucleic acid molecules, without using digital sequencing. For example, the technology can be used to analyze variations in the copy number of a particular nucleic acid sequence that can arise from variations in, for example, chromosome number, gene copy number, expression level, etc. The technology finds particular use in genetic screening for prenatal testing, particularly non-invasive prenatal testing (NIPT). NIPT aims to analyze cell-free DNA (cfDNA) of a fetus circulating in the blood of a woman carrying the fetus in her uterus. Analysis of cell-free DNA in maternal blood can be used to assess the health of the fetus. The technology herein relates to methods, systems, and kits for detecting and quantifying variations in the number of molecules, particularly variations in gene dosage, that are due to, for example, gene duplications or variations from a normal euploid complement of chromosomes, such as trisomies of one or more chromosomes typically found in diploid pairs.

Background Art

[0003] Detection of the presence or variation in the number of molecules in a sample is a useful way to characterize the sample and the source of the sample. For example, variation in gene dosage is a clinically important indicator of disease state in, for example, the subject from whom the sample was taken. Variation in gene dosage can occur due to errors in DNA replication, can occur in germ line cells, leading to congenital defects and even fetal death, or often cause cancer in somatic cells. These replication abnormalities can cause deletions or duplications of parts of genes, full-length genes and their surrounding regulatory regions, megabase-length portions of chromosomes, or entire chromosomes. Analysis of other biomolecules is also clinically important. For example, variation in the amount of RNA or protein may indicate changes in the expression of genes associated with disease state. Although embodiments of the techniques provided herein are discussed in relation to specific applications, such as the measurement of DNA, the techniques are not limited to those applications and can be readily adapted to the analysis of many different types of molecules or moieties that can bind to partner molecules in a specific manner, such as antigen and antibody, nucleic acid and complementary nucleic acid, nucleic acid structures (e.g., stem-loop, bulge nucleotide, flap, promoter sequence) and proteins that bind to such structures, lecithin and carbohydrate, protein and protein binding partner, protein and lipid (e.g., SH2 domain and lipid), etc.

[0004] Chromosomal abnormalities can affect either the number or the structure of chromosomes. A condition in which a cell, tissue, or individual lacks one or more whole chromosomes or chromosomal segments, or has more than the normal euploid complement of chromosomes, can be termed aneuploidy. Germline replication errors resulting from chromosomal nondisjunction lead to either monosomy (one copy of an autosome instead of the usual two or just one sex chromosome) or trisomy (three copies). Such events, when they do not result in obvious fetal death, typically give rise to a variety of disorders that are often recognized as syndromes, such as trisomy 21 and Down syndrome, trisomy 18 and Edwards syndrome, and trisomy 13 and Patau syndrome. Structural chromosomal abnormalities that affect part of a chromosome occur due to chromosome breakage and result in deletions, inversions, translocations, or duplications of large chunks of genetic material. These events are often as devastating as the gain or loss of whole chromosomes and cause disorders such as Prader-Willi syndrome (del15q11-13), retinoblastoma (del13ql4), cri-du-chat syndrome (del5p), and other disorders listed in U.S. Patent No. 5,888,740, which is hereby incorporated by reference in its entirety.

[0005] Major chromosomal abnormalities are detected in approximately 1 in 140 live births and at much higher rates in fetuses that do not reach term or are stillborn. Hsu (1998) Prenatal diagnosis of chromosomal abnormalities through amniocentesis. In: Milunsky A, editor. 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 live births. Hsu; Staebler et al. Although not as common as trisomy 21, trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome) occur in 1 in 5,500 and 1 in 17,200 live births, respectively. Hsu. A wide variety of congenital defects, growth retardation, and intellectual disabilities are seen in children with chromosomal aneuploidy, which pose lifelong challenges for families and society. Jones (2006) Smith’s recognizable patterns of human malformation. Philadelphia: Elsevier Saunders. There are various prenatal tests that can indicate a high risk of fetal aneuploidy, including invasive diagnostic tests such as amniocentesis or chorionic villus sampling, which are the current gold standard but carry a non - negligible risk of fetal loss.American College of Obstetricians and Gynecologists(2007)ACOG Practice Bulletin No.88,December 2007.Invasive prenatal testing for aneuploidy.Obstet Gynecol 110:1459-1467.Therefore, more reliable, non-invasive tests for fetal aneuploidy have long been sought. The most promising of these tests are based on the detection of fetal DNA in maternal plasma. It has been demonstrated that ultra-parallel sequencing of libraries generated from maternal plasma can reliably detect abnormalities in chromosome 21. See, for example, Chiu et al., Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci USA 105:20458-20463(2008), Fan et al., Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci USA 105:16266-16271(2008). See also U.S. Patent No. 7,888,017.

[0006] Current methods for quantifying fluctuations in molecule numbers that rely on next-generation sequencing (NGS), such as performing aneuploidy screening, are time-consuming, expensive, and often require extensive bioinformatics analysis. SUMMARY OF THE INVENTION

[0007] The present invention provides compositions, methods, and systems for detecting and characterizing a sample by counting specific molecules that may be present in the sample (e.g., small molecules, haptens, proteins, antibodies, lipids, carbohydrates, and nucleic acids, such as genes or other DNA molecules or fragments, and / or RNA, such as messenger RNA, microRNA and other non-coding RNA). The technology finds use, for example, in monitoring gene expression, measuring the abundance of non-coding RNA, and analyzing genetic mutations including, but not limited to, changes in gene dosage such as aneuploidy. In preferred embodiments, the technology provides a method for detecting and thus counting single copies of target molecules comprising nucleic acids without using "next generation" sequencing (NGS) such as that described by Chiu et al. and Fan et al. above, or single molecule amplification techniques that rely on differentiating amplification reactions of individual target molecules in distinct physical compartments such as microvessels or emulsion droplets.

[0008] Generally, these compositions, methods, and systems provide improved means for detecting features of single nucleotides, including whole chromosomes, chromosomal arms, microscopic deletions and duplications, submicroscopic deletions and duplications, and genomic deletions and duplications of various sizes, as well as single nucleotide polymorphisms, deletions, and insertions. In certain embodiments, the methods of the present disclosure can be used to detect subchromosomal genetic damage, such as microdeletions. Exemplary uses of the method include prenatal and pediatric aneuploidy diagnosis, testing for products of conception or early miscarriage risk, non-invasive prenatal testing (both qualitative and quantitative genetic testing, such as detection of Mendelian diseases, insertions / deletions, and chromosomal imbalances), preimplantation genetic testing, tumor characterization, postnatal testing including cytogenetics, and monitoring the effects of mutagens.

[0009] In some embodiments, the technology herein provides a method for characterizing nucleic acids, preferably DNA, more preferably circulating cell-free DNA derived from blood or plasma, in a sequence-specific and quantitative manner. In preferred embodiments, single copies of DNA are detected and counted without using polymerase chain reaction or DNA sequencing methods. Embodiments of the technology provide methods, compositions, and systems for detecting target DNA using a method for amplifying a signal indicative of the presence of the target DNA in a sample. In preferred embodiments, the signal detectable from a single target molecule is amplified to the extent that the signal derived from a single target molecule is detectable and identifiable, independent of signals from other targets and other copies of the target molecule, and in such a manner.

[0010] In some embodiments, the technology is a method for counting target molecules on a solid support, comprising forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, the primer being attached to the solid support, and detecting the formation of at least one complex in a process comprising: i) extending the primer in the complex in a rolling circle amplification (RCA) reaction to form an RCA product; ii) hybridizing a plurality of labeled probes to the RCA product; and iii) detecting the hybridized labeled probes, wherein the hybridized labeled probes indicate the presence of the target molecule on the solid support. In some embodiments, the solid support comprises a silane-treated surface, preferably a surface comprising glass.

[0011] In some embodiments, the present technology is a method for counting target molecules on a solid support, comprising: a) providing a silane-treated surface comprising at least one of an acrylic group and a reactive amine group; b) forming a plurality of complexes on the glass surface, the plurality of complexes comprising at least one of an RCA product comprising a plurality of hybridized labeled probes and a double-stranded scaffold product comprising a plurality of ligated labeled scaffold oligonucleotides, the formation of the complexes indicating the presence of target molecules on the glass surface, and forming the plurality of complexes comprising exposing the glass surface to a solution comprising graphene oxide; and c) counting the plurality of complexes. In some embodiments, the silane-treated surface is glass. In certain preferred embodiments, the silane-treated surface comprises a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate.

[0012] The surface is not limited to any particular form. For example, in any of the above embodiments, the solid support may comprise a surface within 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.

[0013] In some embodiments of the present technology, the primer of any of the above embodiments is directly bound to the solid support, preferably covalently bound 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, the single or plural 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 attached to the solid support.

[0014] In any of the embodiments described herein, forming one or more complexes can include exposing a solid support to a solution containing a crowding agent. In some embodiments, the crowding agent includes polyethylene glycol (PEG), preferably at least 2-10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18%-20% PEG. In certain preferred embodiments, the PEG has an average molecular weight of 200-8000, preferably 200-1000, preferably 400-800, preferably 600.

[0015] In any of the above embodiments, forming one or more complexes can include including exposing a solid support to a solution containing graphene oxide. In a preferred embodiment, the solid support is exposed to graphene oxide prior to the step of detecting the hybridized labeled probe. In a particularly preferred embodiment, the solid support is exposed to a solution containing a mixture of the labeled probe and graphene oxide. In some embodiments, the solid support or glass surface exposed to the solution containing graphene oxide is washed with a solution containing a surfactant before detection or counting. In certain preferred embodiments, the surfactant includes Tween20.

[0016] The present technology is used, for example, for the detection of many different types of molecules including those schematically illustrated in FIG. 38. In some embodiments, the target molecule is a nucleic acid, preferably DNA from a sample derived from a subject, preferably a blood sample or a blood product sample. In certain preferred embodiments, the DNA is cell-free DNA from a blood sample or a blood product sample. In some embodiments, the cell-free DNA includes maternal DNA and / or fetal DNA from a maternal blood sample.

[0017] Any of the embodiments described hereinabove may include forming an RCA product in a process that includes extending a primer onto a circularized nucleic acid probe in a reaction mixture, the reaction mixture including Phi29 DNA polymerase at least 0.2 units / μL, preferably at least 0.8 units / μL, and dNTPs at least 400 μM, preferably at least 600 μM, more preferably at least 800 μM in total. In some embodiments, forming an RCA product that includes a plurality of hybridized labeled probes includes forming an RCA product that further includes more than 100 nM molecular beacon probe in the reaction mixture, preferably at least 1000 nM molecular beacon probe in the reaction mixture.

[0018] In certain embodiments of the technology provided herein, a plurality of RCA products hybridized to a labeled probe are dispersed and immobilized on a solid support, and at least a portion of the plurality of RCA products are individually detectable by detection of the label. In some embodiments, the dispersion of the RCA products is irregular, and in some embodiments, the dispersion of the RCA products is within an addressable array.

[0019] In any of the embodiments described herein, the complex immobilized on the surface may include at least one polypeptide, such as an antibody, and / or the complex may include at least one specifically bindable molecule selected from heptane, lectin, and lipid.

[0020] In some embodiments, at least one labeled probe of the technology described herein includes a fluorescent label, and 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 a preferred embodiment, at least one labeled probe is a molecular beacon probe.

[0021] In some embodiments of the present technology, a plurality of RCA products hybridize to a labeled probe that contains all the same label, and in some embodiments, a plurality of RCA products hybridize to a labeled probe that contains two or more different labels, preferably two or more different fluorescent dyes.

[0022] Embodiments of the present technology are not limited to any particular means of detecting or counting complexes bound to a surface. In some embodiments, detecting or counting includes detecting fluorescence. In certain preferred embodiments, detecting or counting includes fluorescence microscopy, and in some embodiments, detecting or counting includes flow cytometry.

[0023] In some embodiments of the present technology, forming the RCA product includes incubating the reaction mixture at at least 37 degrees, preferably at least 42 degrees, preferably at least 45 °C. In certain embodiments, the reaction mixture contains PEG, preferably at least 2-10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18%-20% PEG.

[0024] The technology also provides a composition for carrying out the method. In some embodiments, the technology provides a composition comprising a silane-treated surface bound to a plurality of complexes, each complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a solid support and the reaction mixture comprises Phi29 DNA polymerase at least 0.2 units / μL, preferably at least 0.8 units / μL, a buffer, dNTPs at least 400 μM, preferably at least 600 μM, more preferably at least 800 μM in total, and PEG, preferably at least 2-10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18%-20% PEG. In some embodiments, the PEG has an average molecular weight of 200-8000, preferably 200-1000, preferably 400-800, 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.

[0025] In some embodiments of the composition, the primers are randomly dispersed and bound to the solid support, and in some embodiments, the primers are bound to the solid support within an addressable array. In certain embodiments, the primer is covalently bound to the solid support, and in some embodiments, the primer comprises a biotin moiety and the solid support comprises avidin, preferably streptavidin. In some embodiments, the complex comprises 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 some embodiments, the antigen or hapten is covalently attached to the solid support.

[0026] In some embodiments of the compositions of the present specification, the 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 specifically bindable molecule selected from heptane, lectin, and lipid.

[0027] Embodiments of the above compositions may include a silane-treated surface bound to a plurality of complexes, each of which contains an RCA product comprising a plurality of hybridized labeled probes, and a solution containing graphene oxide. In some embodiments, the silane-treated surface is glass. In some preferred embodiments, the silane-treated surface includes a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate, preferably a glass surface.

[0028] In some embodiments, the solution containing graphene oxide further comprises a molecular beacon probe, preferably a molecular beacon probe with a concentration greater than 100 nM, preferably at least 1000 nM of the molecular beacon probe.

[0029] In some embodiments of the present composition, the solution containing graphene oxide comprises a buffer containing MgCl 2 . In certain embodiments, the buffer containing MgCl 2 is a Phi29 DNA polymerase buffer.

[0030] The technology provided herein is not limited to any particular use or application. In some embodiments, the technology is found in use, preferably in connection with non-invasive prenatal testing, in the analysis of chromosomal abnormalities, such as aneuploidy. For example, some embodiments of the use of the technology include obtaining a maternal sample that contains genetic material from both the mother and the fetus, and measuring a plurality of target nucleic acids, the target nucleic acids including a specific sequence associated with a first chromosome, the first chromosome being suspected of being mutated (e.g., in gene dosage or chromosome number) in the fetal material, and the target nucleic acids further including a specific sequence associated with a second chromosome that is not suspected of being mutated in the fetal material. The method includes analyzing the amount of the target nucleic acid associated with the first chromosome and the amount of the target nucleic acid associated with the second chromosome in the sample, and determining whether the amount of the target nucleic acid associated with the first chromosome is sufficiently different from the amount of the target nucleic acid associated with the second chromosome to indicate a chromosomal or gene dosage mutation in the fetus. In preferred embodiments, the target nucleic acids associated with the first and second chromosomes are present in the genetic material of both the mother and the fetus, and are nucleic acids of the mother and the fetus that are not specific to either one. In preferred embodiments, the maternal sample is cell-free DNA from maternal blood. Statistical methods for analyzing chromosomal abnormalities based on the measurement of the amount of DNA in a sample, including determining fetal DNA abnormalities when fetal DNA is a very small fraction of the total DNA in the maternal sample, are known in the art. See, for example, U.S. Patent No. 6,100,029, which is incorporated herein by reference. Definitions To facilitate understanding of the present invention, some terms and phrases are defined below: Unless the context clearly indicates otherwise, throughout this specification and the claims, the following terms take the meanings explicitly associated with them in this specification. As used herein, the phrase "in one embodiment" may, but does not necessarily, refer to the same embodiment. Further, as used herein, the phrase "in another embodiment" may, but does not necessarily, refer to a different embodiment. Thus, as described below, various embodiments of the present invention can be readily combined without departing from the scope or spirit of the present invention.

[0031] Further, as used herein, the term "or" is an inclusive "or" operator and, unless the context clearly indicates otherwise, is equivalent to the term "and / or". The term "based on" is not exclusive and allows for being based on additional factors not recited, unless the context clearly indicates otherwise. Further, throughout this specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "within" includes "within" and "on".

[0032] As used in the claims of this application, the transitional phrase "consisting essentially of" limits the claim to the specified materials or steps and those that do not materially affect the "basic and novel characteristics(s)" of the claimed invention, 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" the recited elements may contain contaminants that are present but are not enumerated at a level that would change the function of the composition compared to a pure composition, i.e., a composition "consisting of" the recited components.

[0033] As used herein, the terms "subject" and "patient" refer to any living organism, including plants, microorganisms, and animals (e.g., mammals such as dogs, cats, livestock, and humans).

[0034] The term "sample" as used herein and in the claims is used in its broadest sense. On the one hand, it means to include a specimen or a culture (e.g., a microbiological culture). On the other hand, it means to include both biological samples and environmental samples. A sample can include specimens of synthetic origin. A biological sample can be an animal including a human, a fluid, a solid (e.g., feces), or a tissue, as well as liquid and solid food and feed products and materials such as dairy products, vegetables, meat, and meat by-products, and waste. A biological sample can be obtained from domestic animals of all kinds of families, as well as feral or wild animals including, but not limited to, ungulates, bears, fish, rabbits, rodents, etc.

[0035] Environmental samples include environmental substances such as surface substances, soil, water, and industrial samples, as well as samples obtained from food and dairy processing equipment, devices, facilities, instruments, disposable and non-disposable articles. These examples should not be construed as limiting the types of samples applicable to the present invention.

[0036] As used herein, the term "target" refers to a molecule that is required to be selected from other molecules for evaluation, measurement, or other characterization. For example, a target nucleic acid can be selected from other nucleic acids in a sample by, for example, probe binding, amplification, isolation, capture, etc. When used in hybridization-based detection, for example, in connection with the polymerase chain reaction, "target" refers to the region of the nucleic acid to which the primer used in the polymerase chain reaction binds. When used in an assay where the target DNA is not amplified, for example, in capture by molecular inversion probes (MIPs), the target includes the site bounded by hybridization of the target-specific arm of the MIP so that the MIP can be ligated and the presence of the target nucleic acid can be detected.

[0037] The term "source of target nucleic acid" refers to any sample containing nucleic acid (RNA or DNA). Particularly preferred sources of target nucleic acid are biological samples including, but not limited to, blood, plasma, serum, saliva, urine, feces, gastrointestinal fluid, cerebrospinal fluid, pleural fluid, milk, lymph, sputum, and semen.

[0038] As used herein, the term "gene dosage" refers to the copy number of a gene, gene region, chromosome, or fragment or portion thereof. Normal individuals have 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 on the X or Y chromosome, or when the gene sequence is present in a pseudogene.

[0039] As used herein, the term "aneuploidy" refers to the state in which a cell, tissue, or individual does not have a normal euploid complement of chromosomes or, in addition thereto, has one or more whole chromosomes or chromosomal segments.

[0040] As used herein, the "sensitivity" of a given assay (or set of assays used together) is the percentage of samples that report a particular form or variant, such as a mutant gene duplication, chromosomal duplication, that exceeds a threshold that distinguishes samples showing a mutant phenotype (e.g., cancer cells, aneuploidy) from samples showing a normal or wild-type phenotype (e.g., non-cancerous, euploid). In some embodiments, "positive" is defined as a clinically confirmed variant that reports an assay result associated with the presence of a detected disease or condition, and false negative is defined as a clinically confirmed variant that reports an assay result associated with the absence of a disease or condition. Thus, the sensitivity value reflects the probability that a given diagnostic assay performed on a sample of a known variant or disease will yield a result indicating the presence of the variant or disease. As defined herein, the clinical relevance of the calculated sensitivity value represents an estimate of the probability of detecting the presence of a condition when a given assay is applied to a subject having the clinical condition. Using the techniques described herein, it may be possible to achieve a particular level of accuracy without the need to generate sequence reads. Accuracy may refer to sensitivity, may refer to specificity, or may refer to some combination thereof. Desired levels of accuracy are 90% - 95%, may be 95% - 98%, may be 98% - 99%, may be 99% - 99.5%, may be 99.5% - 99.9%, may be 99.9% - 99.99%, may be 99.99% - 99.999%, or may be 99.999% - 100%. Levels of accuracy above 95% may sometimes be referred to as high accuracy.

[0041] As used herein, the "specificity" of a given assay (or set of assays used together) refers to the percentage of normal samples that report assay results associated with the presence of the detected disease or condition, and false positives are defined as clinically confirmed normal samples that report assay results 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 results indicating a variation or the presence of a disease. As defined herein, the clinical relevance of the calculated specificity value represents an estimate of the probability of detecting the absence of a condition when a given marker is applied to a subject not having the clinical condition.

[0042] The term "gene" refers to a DNA sequence that includes the control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., ribosomal RNA or transfer RNA), a polypeptide, or a precursor. The RNA or polypeptide may be encoded by the full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or function is retained.

[0043] As used herein, the term "gene region" refers to a gene, its exons, its introns, and the regions upstream and downstream and adjacent thereto, for example, 5' and 3' of 5 to 10 kilobases of the transcription start site and the transcription termination site, respectively.

[0044] As used herein, the term "gene sequence" refers to the sequence of a gene, its introns, and the regions upstream and downstream and adjacent thereto, for example, 5' and 3' of 5 to 10 kilobases of the transcription start site and the transcription stop site, respectively.

[0045] As used herein, the term "chromosome-specific" refers to a sequence found only in that particular type of chromosome.

[0046] As used herein, the term "hybridization" is used with respect to the formation of complementary nucleic acid pairs. Hybridization and the strength of hybridization (i.e., the strength of the association between nucleic acids) are affected by such factors as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the T of the hybrid formed. The method of "hybridization" involves annealing a nucleic acid to another complementary nucleic acid, i.e., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acids containing complementary sequences to find each other and anneal through base-pairing interactions is a well-recognized phenomenon. Since the "hybridization" process was first observed by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960), this process has been refined and has become an essential tool in modern biology. m and the like.

[0047] As used herein, the term "oligonucleotide" is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 10 - 15 nucleotides, and even more preferably at least about 15 - 30 nucleotides. The exact size depends on many factors, which also depend 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.

[0048] Mononucleotides are reacted to produce oligonucleotides in such a way that the 5' phosphate of one mononucleotide pentose ring attaches in one direction via a phosphodiester bond to the adjacent 3' oxygen of its neighboring mononucleotide. Thus, the end of an oligonucleotide where the 5' phosphate is not attached to the 3' oxygen of a mononucleotide pentose ring is termed the "5' end", and the 3' oxygen is attached to the 5' phosphate of the subsequent mononucleotide pentose ring is not termed the "3' end". As used herein, a nucleic acid sequence can be said to have a 5' end and a 3' end even if it is internal to a larger oligonucleotide. The first region along a nucleic acid strand is upstream of another region if, when moving along the nucleic acid strand in the 5' to 3' direction the 3' end of the first region is before the 5' end of the second region.

[0049] If two different non - overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence and the 3' end of one oligonucleotide faces the 5' end of the other, the former may be termed the "upstream" oligonucleotide and the latter the "downstream" oligonucleo It may also be referred to as a chide. Similarly, when two overlapping oligonucleotides hybridize to the same linear complementary nucleic acid sequence and the 5' end of the first oligonucleotide 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 may be referred to as the "upstream" oligonucleotide, and the second oligonucleotide may be referred to as the "downstream" oligonucleotide.

[0050] The term "primer" refers to an oligonucleotide that can act as a starting point for synthesis when placed under conditions where primer extension is initiated, for example, in the presence of nucleotides and an appropriate nucleic acid polymerase. Oligonucleotide "primers" may be naturally occurring, may be made using molecular biology methods such as purification of restriction digests, or may be synthetically generated. In preferred embodiments, the primer is composed of DNA or contains DNA.

[0051] Primers are selected to be "substantially" complementary to the strand of a particular sequence of the template. For primer extension to occur, the primer must be complementary enough to hybridize to the template strand. The primer sequence does not need to reflect the exact sequence of the template. For example, non-complementary nucleotide fragments may be attached to the 5' end of the primer and the remaining portion of the primer sequence may be substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed in the primer as long as the primer sequence has sufficient complementarity to the template sequence to hybridize and thereby form a template-primer complex for the synthesis of the primer extension product.

[0052] As used herein, the term "sequence variation" refers to a difference in nucleic acid sequence between two nucleic acids. For example, a wild-type structural gene and a variant of this wild-type structural gene may differ in sequence due to the presence of a single base substitution and / or deletion or insertion of one or more nucleotides. These two forms of the structural gene are said to be in a different order from each other. A second variant of the structural gene may exist. This second variant is said to differ in sequence from both the wild-type gene and the first variant of the gene.

[0053] As used herein, the term "nucleotide analog" refers to modified or unnatural nucleotides including, but not limited to, analogs having altered stacking interactions such as 7-deazapurines (i.e., 7-deaza-dATP and 7-deaza-dGTP); base analogs having 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 (e.g., "K" 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, modified forms of deoxyribonucleotides and 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 based on purine or pyrimidine ring systems, and other heterocyclic bases.

[0054] 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 arrangement of the base portions of each nucleotide, whether a base pair, single strand, or mismatch, is not an element in the definition of a continuous strand. The backbone of a continuous strand is not limited to the ribose-phosphate or deoxyribose-phosphate compositions found in naturally occurring unmodified nucleic acids. The nucleic acids of the present invention can include modifications in the structure of the backbone that include, but are not limited to, phosphorothioate residues, phosphonate residues, 2'-substituted ribose residues (e.g., 2'-O-methylribose), and residues containing alternative sugars (e.g., arabinose).

[0055] As used herein, the term "continuous duplex" refers to a region of double-stranded nucleic acid in which there are no breaks in the progression of base pairs within the duplex (i.e., the base pairs along the duplex are not distorted to correspond to gaps, bulges, or mismatches within the region of the continuous duplex). As used herein, this term refers only to the arrangement of base pairs within the duplex and not to the continuity of the backbone portion of the nucleic acid strand. A double-stranded nucleic acid having uninterrupted base pairing but having a nick in one or both strands is within the definition of a continuous duplex.

[0056] The term "duplex" refers to the state of a nucleic acid in which the base portions of the nucleotides on one strand are bound to their complementary bases arranged on a second strand via hydrogen bonds. The condition of being in duplex form reflects the state of the bases of the nucleic acid. Through base pairing, the strands of a nucleic acid also generally assume a double-helical tertiary structure having a major groove and a minor groove. Assuming a helical form is potentially included in the act of duplexing.

[0057] The term "template" refers to a nucleic acid strand upon which a complementary copy is constructed from nucleoside triphosphates via the activity of a template-dependent nucleic acid polymerase. Within a duplex, the template strand is conventionally depicted and described as the "bottom" strand. Similarly, the non-template strand is often depicted and described as the "top" strand.

[0058] When applied to polynucleotides, the term "substantial identity" means that a polynucleotide has at least 85 percent sequence identity, preferably at least 90 - 95 percent sequence identity, and more usually at least 99 percent sequence identity when compared to a reference sequence over a comparison window of at least 20 nucleotide positions, often over a window of at least 25 - 50 nucleotides, and the percentage of sequence identity is calculated by comparing the polynucleotide sequence to the reference sequence, which may include deletions or additions that are less than or equal to 20% of the total of the reference sequence over the comparison window. The reference sequence may be, for example, a splice variant of the full-length sequence or a subset of a larger sequence.

[0059] When applied to polypeptides, the term "substantial identity" means that when optimally aligned by programs such as GAP or BESTFIT using the default gap weight, two peptide sequences share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, the positions of non-identical residues differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, amino acid groups having aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; amino acid groups having aliphatic hydroxyl side chains are serine and threonine; amino acid groups having amide-containing side chains are asparagine and glutamine; amino acid groups having aromatic side chains are phenylalanine, tyrosine, and tryptophan; amino acid groups having basic side chains are lysine, arginine, and histidine; and amino acid groups having sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0060] As used herein, the term "label" refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and that can be attached to a nucleic acid or protein. Labels include, but are not limited to, dyes; 32Radioactive labels such as P; binding moieties such as biotin; haptens such as digoxigenin; luminescent, phosphorescent or fluorescence-generating moieties; mass tags; and fluorescent dyes, alone or in combination with moieties that can suppress (“quench”) or shift the emission spectrum by fluorescence resonance energy transfer (FRET). FRET is a distance-dependent interaction between two molecules in an electronically excited state (e.g., two dye molecules, or a dye molecule and a non-fluorescent quencher molecule), where excitation is transferred from the donor molecule to the acceptor molecule without the 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 that has an absorption spectrum that overlaps the emission spectrum of the donor and can absorb some or most of the energy emitted from the donor when in the vicinity of the donor group (typically 1-100 nm). When the acceptor is a fluorophore, it generally re-emits at a third, even longer wavelength, and when it is a chromophore or quencher, it releases the energy absorbed from the donor without the emission of a photon. In some embodiments, a detectable change in the emission from the donor dye (e.g., when the acceptor moiety is close or far away) is detected. In some embodiments, a detectable change in the 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 so that the emissions from the dyes can be distinguished from each other (e.g., spectrally resolved).

[0061] In some embodiments, the donor dye is used in combination with a plurality of acceptor moieties. In preferred embodiments, the donor dye is used in combination with a non-fluorescent quencher and an acceptor dye, such that when the donor dye is in the vicinity of 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), the 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), which is incorporated herein by reference.

[0062] Labels can provide a signal detectable by fluorescence (e.g., simple fluorescence, FRET, time-resolved fluorescence, fluorescence polarization, etc.), radiation, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, mass characteristics or behavior affected by mass (e.g., MALDI time-of-flight mass spectrometry), etc. The label may be a charged moiety (positive or negative charge) or alternatively may be charge-neutral. The label can comprise or consist of a nucleic acid or protein sequence as long as the sequence containing the label is detectable.

[0063] In some embodiments, the label comprises particles for detection. In preferred embodiments, the particles are phosphor particles. In particularly preferred embodiments, the phosphor particles are upconverting phosphor particles (see, for example, 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 particles. The phosphor particles can be detected by any suitable method including, but not limited to, upconverting phosphor technology (UPT) where an upconverting phosphor converts low energy infrared (IR) radiation to high energy visible light. While the present invention is not limited to any particular mechanism, in some embodiments, UPT upconverts infrared light to visible light by multi-photon absorption and subsequent emission of dopant-dependent phosphorescence. See, for example, U.S. Patent No. 6,399,397, issued June 4, 2002 to Zarling et al., which is hereby incorporated by reference in its entirety; 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 hereby incorporated by reference in its entirety.

[0064] As used herein, the terms "solid support" or "support" refer 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 metals, glass, quartz, plastics, silicon, wafers, 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 support need not be flat. The support includes any kind of shape including spherical (e.g., beads).

[0065] As used herein, the term "bead" refers to a small solid support that can move around when in solution (e.g., it has dimensions smaller than the dimensions of the enclosure or container in which the solution is present). In some embodiments, the beads can sediment out of the solution if the solution is not mixed (e.g., by shaking, thermal mixing, vortexing), while in other embodiments, the beads can be colloidal suspended in the solution. In some embodiments, the beads are completely or partially spherical or cylindrical. However, the beads are not limited to any particular three-dimensional shape.

[0066] The material attached to the solid support can attach to any part of the solid support (e.g., inside or outside a porous solid support material, or to a flat part of a non-flat support or vice versa). In preferred embodiments of the present technology, biomolecules such as nucleic acid or protein molecules are attached to the solid support. A biological substance is "attached" to the solid support if it is immobilized on the solid support via chemical or physical interactions. In some embodiments, the attachment is by 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, such as a change in temperature, ionic change, addition or removal of a chelating agent, or other changes in the solution conditions to which the surface and the binding molecule are exposed.

[0067] In some embodiments, a target molecule, e.g., a biological substance, is attached to the solid support via a "spacer molecule" or "linker group". Such a spacer molecule is a molecule having a first part that attaches to the biological substance and a second part that attaches to the solid support. The spacer molecule typically includes a chain of atoms, e.g., carbon atoms that provide additional distance between the first and second parts. Thus, the spacer molecule, when attached to the solid support, allows separation between the solid support and the biological substance while being attached to both.

[0068] As used herein, the terms “array” and “microarray” refer to a surface or container that includes a plurality of pre-defined loci addressable for analysis of loci, e.g., to determine the results of an assay. Analysis at a locus within the array is not limited to any particular type of analysis and includes, for example, analysis, inhibition, or alteration (e.g., intensity or wavelength) for detection of atoms, molecules, chemical reactions, luminescence, or fluorescence emission indicative of the results at that locus. Examples of pre-defined loci include a grid or any other pattern, and the loci to be analyzed are determined by their known positions within the array pattern. For example, microarrays are generally reviewed 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 attached (e.g., in a grid or other regular pattern) to a surface, and a container that includes a plurality of defined reaction loci (e.g., wells) where a molecule or signal generating reaction can be detected. In some embodiments, for example, as described above for SIMOA technology, the array includes a patterned distribution of wells that receive beads. 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.

[0069] As used herein, the term "irregular distribution" when used with respect to a site on a solid support or surface refers to the distribution of loci on or within a surface in a non-array format. While providing an approximate average distance between molecules on the surface, a solution of a particular concentration providing any pattern on the surface is applied to sites that are not pre-defined or addressable, or by means of applying the solution (e.g., inkjet printing), molecules can be irregularly distributed on the surface. In such embodiments, analysis of the surface can include finding the loci of the molecules by detecting signals wherever the signals can appear (e.g., scanning the entire surface to detect fluorescence somewhere on the surface). This is in contrast to analyzing the surface or container only at predetermined loci (e.g., points of a grid array) to identify signals and determining the amount (or type) of signal appearing at each locus within the grid.

[0070] As used herein, the term "distinct" with respect to signals refers to signals that can be distinguished from one another, for example, by spectral properties such as fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization characteristics, charge, etc., or by the ability to interact with another moiety such as a chemical reagent, enzyme, antibody, etc.

[0071] As used herein, the term "nucleic acid detection assay" refers to any method for 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., including the INVADER assay (Hologic, Inc.), e.g., U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000) and U.S. Patent No. 9,096,893 (each of which is hereby incorporated by reference in its entirety for all purposes); the enzyme mismatch cleavage method (e.g., U.S. Patent Nos. 6,110,684, 5,958,692, 5,851,770 of Variagenics, each of which is hereby incorporated by reference in its entirety); the polymerase chain reaction (PCR) described above; the branched hybridization method (e.g., U.S. Patent Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802 of Chiron, each of which is hereby incorporated by reference in its entirety); rolling circle amplification (e.g., U.S. Patent Nos. 6,210,884, 6,183,960, and 6,235,502, each of which is hereby incorporated by reference in its entirety); a variation of rolling circle amplification called "RAM amplification" (see, e.g., US5,942,391, which is hereby incorporated by reference in its entirety); NASBA (e.g., U.S. Patent No. 5,409,818, which is hereby incorporated by reference in its entirety); molecular beacon technology (e.g., U.S. Patent No. 6,150,097, which is hereby incorporated by reference in its entirety); E sensor technology (U.S. Patent Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573 of Motorola, each of which is hereby incorporated by reference in its entirety); cycling probe technology (e.g., U.S. Patent Nos. 5,403,711, 5,011,769, and 5,660,988, each of which is hereby incorporated by reference in its entirety); the Dade Behring signal amplification method (e.g., U.S. Patent Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, each of which is hereby incorporated by reference in its entirety); ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and the sandwich hybridization method (e.g., U.S. Patent No. 5,288,609, which is hereby incorporated by reference in its entirety).

[0072] In some embodiments, the target nucleic acid is amplified (e.g., by PCR), and the amplified nucleic acid is simultaneously detected using an invasive cleavage assay. Assays configured to perform a detection assay (e.g., an invasive cleavage assay) in combination with an amplification assay are described in U.S. Patent No. 9,096,893, which is hereby incorporated by reference in its entirety for all purposes. An additional amplification and invasive cleavage detection configuration, referred to as the QuARTS method, is described in, for example, U.S. Patent Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392 (each of which is hereby incorporated by reference in its entirety for all purposes). As used herein, the term "invasive cleavage structure" refers to a cleavage structure that includes 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), where the upstream and downstream nucleic acids anneal to contiguous regions of the target nucleic acid and 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. An overlap occurs when one or more bases from the upstream and downstream nucleic acids occupy the same position relative to the target nucleic acid bases, regardless of whether the overlapping bases of the upstream nucleic acid are complementary to the target nucleic acid and regardless of whether those bases are natural or non-natural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps the downstream duplex is a non-base chemical moiety, such as an aromatic ring structure, as disclosed in U.S. Patent No. 6,090,543, which is hereby incorporated by reference in its entirety. In some embodiments, one or more of the nucleic acids can be attached to each other via a covalent bond, such as a nucleic acid stem-loop, or via a non-nucleic acid chemical bond (e.g., a multi-carbon chain). As used herein, the term "flap endonuclease assay" includes, as described above, the "INVADER" invasive cleavage assay and the QuARTS assay.

[0073] 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, the sample is partitioned into sufficiently small portions (e.g., microchambers or emulsions) such that, on average, no more than a single copy of the target nucleic acid is present, for example by serial dilution or when each portion or dilution is evaluated according to a Poisson distribution. Methods of single molecule PCR include, for example, those described in US6,143,496, which relate to methods that include partitioning 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 the target molecule; US6,391,559, which relates to an assembly for containing and partitioning a fluid; and US7,459,315, which relates to a method of partitioning a sample into an assembly having sample chambers that partition the sample by surface affinity and then seal the chambers with a curable "displacement fluid". See also US6,440,706 and US6,753,147, as well as Vogelstein, et al., Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9236-9241, August 1999. See also US20080254474, which describes a combination of digital PCR combined with methylation detection.

[0074] As used herein, the term "sequencing" is used in a broad sense and is not limited, but can refer to any technique known in the art that enables the identification of the order of at least some consecutive nucleotides in at least a portion of a nucleic acid, including at least a portion of an extension product or vector insert. In some embodiments, sequencing can distinguish sequence differences between different target sequences.Exemplary sequencing technologies include target sequencing, single molecule real-time sequencing, electron microscopy-based sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy chain termination sequencing, target sequencing, exon sequencing, whole genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, ultraparallel signature sequencing, emulsion PCR, co-amplification-PCR (COLD-PCR) at lower denaturation temperatures, multiplex PCR, sequencing by reversible dye terminators, paired-end sequencing, short read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reversible terminator sequencing, ion semiconductor sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, miSeq (Illumina), HiSeq2000 (Illumina), HiSeq2500 (Illumina), Illumina Genome Analyzer (Illumina), Ion Torrent PGMτ (trademark) (Life Technologies), MinION (trademark) (Oxford Nanopore Technologies), real-time SMRT (trademark) technology (Pacific Biosciences), Probe-Anchor Ligation (cPAL (trademark)) (Complete Genomics / BGI), SOLiD (registered trademark) sequencing, MS-PET sequencing, mass spectrometry, and combinations thereof.In some embodiments, sequencing includes, but is not limited to, detecting sequencing products using, for example, an ABI PRISM® 377 DNA Sequencer, ABI PRISM® 310, 3100, 3100-Avant, 3730, or 3730xI Genetic Analyzer, ABI PRISM® 3700 DNA Analyzer, or an Applied Biosystems SOLiD™ System (all made by Applied Biosystems), Genome Sequencer 20 System (Roche Applied Science), or an instrument including a mass spectrometer. In certain embodiments, sequencing includes emulsion PCR. In certain embodiments, sequencing includes high-throughput sequencing techniques, such as, but not limited to, massively parallel signature sequencing (MPSS).

[0075] 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. A polypeptide may be synthetic or naturally occurring, and may be short, for example, 2 to about 30 amino acid residues, or may be hundreds or thousands of amino acid residues in length. A polypeptide may be composed of the 20 major naturally occurring amino acids or may include one or more non-natural amino acids, such as peptide nucleic acid residues containing pyrimidine or purine bases in the peptide chain backbone, or modified forms of natural amino acids (e.g., having an altered side chain structure).

[0076] As used herein, the term “antibody” (Ab) refers to an antigen-binding immunoglobulin and includes monoclonal antibodies (mAbs) and polyclonal Abs. The term further includes all modified forms of antibodies having the ability to bind an antigen, such as fragment antibodies (fAbs) including a portion of the immunoglobulin structure.

[0077] As used herein, the terms "crowding agent" and "volume exclusion agent" when used with respect to components of a fluid reaction mixture are used interchangeably and refer to a compound, generally a polymeric compound, that reduces the available fluid volume in the reaction mixture, thereby increasing the effective concentration of reactant macromolecules (e.g., nucleic acids, enzymes, etc.). Crowding reagents include, for example, glycerol, ethylene glycol, polyethylene glycol, ficoll, serum albumin, casein, and dextran.

[0078] 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, 454FLX™ or 454TITANIUM™ (Roche), SOLEXA™ / Illumina Genome Analyzer (Illumina), HELISCOPE™ Single Molecule Sequencer (Helicos Biosciences), and SOLID™ DNA Sequencer (Life Technologies / Applied Biosystems) instruments, as well as other platforms still under development by companies such as Biosystems and Pacific Biosystems. See also U.S. Patent No. 7,888,017, entitled "Non-invasive fetal genetic screening by digital analysis", regarding digital analysis of maternal and fetal DNA, e.g., cfDNA.

[0079] As used herein, the term "probe" or "hybridization probe" refers to an oligonucleotide (i.e., a nucleotide sequence) that can hybridize at least in part with another oligonucleotide of interest, whether it occurs naturally as a purified restriction enzyme digest or is produced synthetically, recombinantly, or by PCR amplification. The probe can be single-stranded or double-stranded. The probe is useful for the detection, identification, and isolation of a specific sequence. In some preferred embodiments, the probe used in the present invention is labeled with a "reporter molecule" and thus is detectable by any detection system including, but not limited to, enzymes (e.g., ELISA and enzyme-based histochemical assays), fluorescence, radioactivity, and luminescence systems. The present invention is not intended to be limited to any particular detection system or label.

[0080] 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 that includes a pair of unique polynucleotide arms, one or more unique molecular tags (or unique molecular identifiers), and a polynucleotide linker (e.g., a universal backbone linker). For example, see FIG. 1. In some embodiments, the MIP can include two or more unique molecular tags such as two unique molecular tags, three unique molecular tags, or more. In some embodiments, the unique polynucleotide arms of each MIP are located at the 5' and 3' ends of the MIP, while the unique molecular tag(s) and polynucleotide linker are located internal to the 5' and 3' ends of the MIP. For example, the present disclosure The MIPs used in some of the embodiments shown herein sequentially include the following components: 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, the MIP is a 5'-phosphorylated single-stranded nucleic acid (e.g., DNA) molecule. See, for example, WO2017 / 020023 filed Jul. 29, 2016 and WO2017 / 020024 filed Jul. 29, 2016 (each of which is hereby incorporated by reference in its entirety for all purposes).

[0081] An inherent molecular tag can be any tag that is detectable and can be incorporated into or attached to a nucleic acid (e.g., a polynucleotide), enabling the detection and / or identification of the nucleic acid containing 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, radiolabels (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), etc., or combinations thereof. In some embodiments, particularly in sequencing embodiments, the tag (e.g., a molecular tag) is a unique, known, and / or identifiable sequence of nucleotides or nucleotide analogs (e.g., nucleic acid analogs, nucleotides containing a sugar and one to three phosphate groups). In some embodiments, the tag is six or more contiguous nucleotides. A number of fluorophore-based tags with diverse different excitation and emission spectra are available. Any suitable type and / or number of fluorophores can be used as tags. In some embodiments, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, thirty or more, fifty or more, one hundred or more, five hundred or more, one thousand or more, ten thousand or more, one hundred thousand or more different tags are utilized 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.The detection and / or quantification of tags can be carried out by suitable methods, machines or devices, and non-limiting examples thereof include flow cytometry, quantitative polymerase chain reaction (qPCR), gel electrophoresis, luminometer, fluorometer, spectrophotometer, suitable gene chip or microarray analysis, Western blot, mass spectrometry, chromatography, cell fluorescence analysis, fluorescence microscopy, suitable fluorescence or digital imaging methods, confocal laser scanning microscopy, laser scanning cytometry, affinity chromatography, manual batch mode separation, electrokinetic suspension, suitable nucleic acid sequencing methods and / or nucleic acid sequencing devices, etc., as well as combinations thereof.

[0082] In MIP, the unique polynucleotide arms are designed to hybridize immediately upstream and downstream of a specific target sequence (or site) in a genomic nucleic acid sample. In some embodiments, the MIP contains unique molecular tags that are short nucleotide sequences generated randomly. In some embodiments, the unique molecular tags do not hybridize to any sequence or site located on the genomic nucleic acid fragment or in the genomic nucleic acid sample. In some embodiments, the polynucleotide linker (or backbone linker) in MIP is universal in all MIPs used in the embodiments of the present disclosure.

[0083] In some embodiments, the MIP is introduced into nucleic acid fragments derived from a test subject (or reference subject) and performs capture of a target sequence or site (or control sequence or site) located on a nucleic acid sample (e.g., genomic DNA). In some embodiments, fragmentation serves to capture the target nucleic acid by molecular inversion probes. In some embodiments, for example, when the nucleic acid sample consists of cell-free nucleic acids, fragmentation may not be necessary to improve the capture of the target nucleic acid by molecular inversion probes. As described in more detail herein, after capture of the target sequence of interest (e.g., locus), the captured target may be subjected to enzymatic gap filling and ligation steps such that copies of the target sequence are incorporated into a circular structure. In some embodiments, for example, nucleic acid analogs including labels, haptens, etc. may be incorporated into the fill section for use in downstream detection, purification, or other processing steps. In some embodiments, the capture efficiency of the MIP for the target sequence on the nucleic acid fragment can be improved by extending the hybridization and gap filling incubation periods. (See, e.g., Turner E H, et al., Nat Methods. 2009 Apr. 6:1-2.).

[0084] In some embodiments, the MIPs used in accordance with the present disclosure to capture target sites or target sequences sequentially include the following components: A first target polynucleotide arm - a first unique target molecular tag - a polynucleotide linker - a second unique target molecular tag - a second target polynucleotide arm.

[0085] In some embodiments, the MIPs used in the present disclosure to capture control sites or control sequences 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.

[0086] The MIP technology can be used to detect or amplify specific nucleic acid sequences in complex mixtures. One of the advantages of using the MIP technology is its high multiplexing ability, which enables the capture of thousands of target sequences in a single reaction containing thousands of MIPs. Various aspects of the MIP technology are described, for example, in 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 hereby 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 hereby incorporated by reference in its entirety for all purposes).

[0087] The MIP technology has previously been successfully applied to other research areas, including the novel identification and subclassification of cancer biomarkers. For example, 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.See, “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 hereby incorporated by reference in its entirety for all purposes).

[0088] The MIP technology has also been applied to the identification of biomarkers related to new drugs. For example, see 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 herein by reference in its entirety for all purposes). Other MIP applications include drug development and safety studies. For example, see 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 herein by reference in its entirety for all purposes). Further applications of the MIP technology include the creation of genotype and phenotype databases.For example, see 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 hereby incorporated by reference in its entirety for all purposes).

[0089] As used herein, the terms “capture” or “capturing” refer to the binding or hybridization reaction between a molecular inversion probe and its corresponding target site. In some embodiments, upon capture, a circular replicon or MIP replicon is generated or formed. In some embodiments, the target site is a deletion (e.g., a partial or complete deletion of one or more exons). In some embodiments, the target MIP is designed to bind or hybridize to a naturally occurring (e.g., wild-type) genomic region of interest where the target deletion is predicted to be located. The target MIP is designed not to bind to the genomic region showing the deletion. In these embodiments, it is expected that no binding or hybridization will occur between the target MIP and the target site of the deletion. The absence of such binding or hybridization indicates the presence of the target deletion. In these embodiments, the phrase “capturing the target site” or the phrase “capturing the target sequence” refers to the detection of the target deletion by detecting the absence of such binding or hybridization.

[0090] As used herein, the terms "MIP replicon" or "circular replicon" refer to circular nucleic acid molecules generated via a capture reaction (e.g., a binding or hybridization reaction between an MIP and its target sequence). In some embodiments, the MIP replicon is a single-stranded circular nucleic acid molecule. In some embodiments, the target MIP captures or hybridizes to a target sequence or site. After the capture reaction or hybridization, a ligation / extension mixture is introduced to extend and ligate the gap region between two target polynucleotide arms to form a single-stranded circular nucleotide molecule, i.e., the target MIP replicon. In some embodiments, the control MIP captures or hybridizes to a control sequence or site. After the capture reaction or hybridization, a ligation / extension mixture is introduced to extend and ligate the gap region between two control polynucleotide arms to form a single-stranded circular nucleotide molecule, i.e., the control MIP replicon. The MIP replicon may be amplified via polymerase chain reaction (PCR) to generate multiple target MIP amplicons that are double-stranded nucleic acid molecules. The MIP replicon finds particular use in rolling circle amplification, or RCA. RCA is an isothermal nucleic acid amplification technique in which DNA polymerase continuously adds single nucleotides to a primer annealed to a circular template, resulting in a long concatemer of single-stranded DNA containing dozens to hundreds of tandem repeats (complementary to the circular template). See, for example, 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 hereby incorporated by reference in its entirety for all purposes). See also WO2015 / 083002 (which is hereby incorporated by reference in its entirety for all purposes).

[0091] Polymerases typically used in RCA for DNA amplification are Phi29, Bst, and Vent exo - DNA polymerases, with Phi29 DNA polymerase being preferred from the perspective of excellent processivity and strand displacement ability.

[0092] As used herein, the term "amplicon" refers to a nucleic acid generated via an amplification reaction (e.g., a PCR reaction). In some embodiments, the amplicon is a single - stranded nucleic acid molecule. In some embodiments, the amplicon is a double - stranded nucleic acid molecule. In some embodiments, a target MIP replicon is amplified using conventional techniques to generate a plurality of target MIP amplicons that are double - stranded nucleotide molecules. In some embodiments, a control MIP replicon is amplified using conventional techniques to generate a plurality of control MIP amplicons that are double - stranded nucleotide molecules.

[0093] The term "probe oligonucleotide" or "flap oligonucleotide", when used in relation to a flap assay (e.g., an INVADER 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.

[0094] The term "invasive oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid at a position adjacent to the hybridization region between a probe and the target nucleic acid, and the 3' end of the invasive oligonucleotide contains a portion (e.g., a chemical moiety, or one or more nucleotides) that overlaps with the hybridization region between the probe and the target. The 3' - terminal nucleotide of the invasive oligonucleotide may or may not form a base pair with a nucleotide in the target. In some embodiments, the invasive oligonucleotide contains, at its 3' end, a sequence substantially identical to the sequence located at the 5' end of a portion of the probe oligonucleotide that anneals to the target strand.

[0095] As used herein, the term "flap endonuclease" or "FEN" refers to a class of nucleolytic enzymes, typically 5' nucleases, that act as structure-specific endonucleases on DNA structures having a duplex with a single-stranded 5' overhang or flap in one of the strands replaced by another strand of nucleic acid (such as where there are overlapping nucleotides at the junction between single-stranded and double-stranded DNA). FEN catalyzes the hydrolysis of the phosphodiester bond at the junction of single-stranded and double-stranded DNA, releasing the overhang or flap. Flap endonucleases have been reviewed by Ceska and Savers (Trends Biochem. Sci. 1998 23:331-336) and Liu et al (Annu. Rev. Biochem. 2004 73:589-615), which are hereby incorporated by reference in their entirety. FEN can be an individual enzyme, a multi-subunit enzyme, or may exist as an activity of another enzyme or protein complex (such as a DNA polymerase).

[0096] Flap endonucleases can be thermostable. For example, FEN-1 flap endonucleases from archival thermophilic organisms are typically thermostable. As used herein, the term "FEN-1" refers to non-polymerase flap endonucleases from eukaryotic or archaeal organisms. See, for example, WO02 / 070755 and Kaiser M.W., et al. (1999) J. Biol. Chem., 274:21387, which are hereby incorporated by reference in their entirety for all purposes.

[0097] As used herein, the term "cleaved flap" refers to single-stranded oligonucleotides that are cleavage products of a flap assay.

[0098] The term "cassette", when used with respect to the 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, for example, 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 generated by cleavage of the flap oligonucleotide to form a second overlapping cleavage structure, such that the cassette can then be cleaved by the same enzyme, for example, FEN-1 endonuclease.

[0099] In some embodiments, the cassette is a single oligonucleotide that includes a hairpin portion (i.e., a region where a 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, such as a fluorophore. In particularly preferred embodiments, the cassette includes a labeled moiety that provides a FRET effect. In such embodiments, the cassette may be referred to as a "FRET cassette". See, for example, US9,096,893, issued August 4, 2015, which is hereby incorporated by reference in its entirety for all purposes.

[0100] As used herein, the phrase "substantially non-complementary" when used with respect to a probe flap or arm means that the flap portion is sufficiently non-complementary that it does not selectively hybridize to a nucleic acid sequence, such as a target nucleic acid or amplified DNA, under the specified annealing conditions or stringent conditions, and includes the terms "substantially non-complementary" and "completely non-complementary".

[0101] As used herein, the term "signal" refers to any detectable effect caused by or provided by a label or by the action or accumulation of a component or product in an assay reaction.

[0102] As used herein, the term "detector" refers to a system or component of a system, such as an instrument (e.g., a camera, a fluorescence measurement device, a charge-coupled device, a scintillation counter, a solid nanopore device, etc.) or a reactive medium (e.g., an X-ray film or a camera film, a pH indicator, etc.), that can communicate the presence of a signal or effect to a user or another component of the system (e.g., a computer or a controller). The detector is not limited to a particular type of signal being detected and can be a photometric or spectrophotometric system capable of detecting ultraviolet, visible, or infrared light, including fluorescence or chemiluminescence; a radiation detection system; a charge detection system; a system for detecting electrical signals, such as perturbations in current or charge; a spectroscopic system such as nuclear magnetic resonance spectroscopy, mass spectrometry, or surface-enhanced Raman spectroscopy; a system such as gel electrophoresis or capillary electrophoresis or gel exclusion chromatography; or other detection systems known in the art, or combinations thereof.

[0103] As used herein, the term "detection" refers to, for example, quantitatively or qualitatively identifying an analyte (e.g., DNA, RNA, or protein) in a sample. As used herein, the term "detection assay" refers to a kit, test, or procedure performed for the purpose of detecting an analyte in a sample. A detection assay generates a detectable signal or effect when performed in the presence of the target analyte and includes, but is not limited to, assays incorporating hybridization, nucleic acid cleavage (e.g., exonuclease or endonuclease), nucleic acid amplification, nucleotide sequencing, primer extension, nucleic acid ligation, antigen-antibody binding, the interaction of a primary antibody and a secondary antibody, and / or the process of structural changes in nucleic acids (e.g., oligonucleotides) or polypeptides (e.g., proteins or small peptides).

[0104] As used herein, the term "prenatal or pregnancy-related disease or condition" refers to any disease, disorder, or condition that affects a pregnant woman, embryo, or fetus. Prenatal or pregnancy-related conditions may also refer to any disease, disorder, or condition that is directly or indirectly related to or results from pregnancy. These diseases or conditions may include any congenital defect, congenital condition, or genetic disease or condition. Examples of prenatal or pregnancy-related diseases include, but are not limited to, simian virus disease, neonatal hemolytic disease, beta-thalassemia, sex determination, pregnancy determination, hereditary Mendelian genetic diseases, chromosomal abnormalities, fetal chromosomal aneuploidy, fetal chromosomal trisomy, fetal chromosomal monosomy, trisomy 8, trisomy 13 (Patau syndrome), trisomy 16, trisomy 18 (Edwards syndrome), trisomy 21 (Down syndrome), X chromosome-related diseases, trisomy X (XXX syndrome), monosomy X (Turner syndrome), XXY 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)(p11.2p1.2) syndrome, endometriosis, Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2) syndrome, cat eye syndrome, cri du chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, neuropathy with liability to pressure palsy, 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, sex-determining region Y, azoospermia (factor a), azoospermia (factor b), azoospermia (factor c), 1p36 deletion, phenylketonuria, Tay-Sachs disease, adrenal hyperplasia, Fanconi anemia, spinal muscular atrophy, Duchenne muscular dystrophy, Huntington disease, myotonic dystrophy, Robertsonian translocation, Angelman syndrome, tuberous sclerosis,Ataxia telangiectasia, open spina bifida, neural tube defect, intrauterine growth restriction, congenital cytomegalovirus, achondroplasia, Marfan syndrome, congenital hypothyroidism, congenital toxoplasmosis, biotinidase deficiency, galactosemia, maple syrup urine disease, homocystinuria, medium-chain acyl-CoA dehydrogenase deficiency, structural congenital defect, heart defect, limb abnormality, clubfoot, anencephaly, arhinencephaly / holoprosencephaly, hydrocephalus, anophthalmia / microphthalmia, anotia / microtia, transposition of the great vessels, tetralogy of Fallot, hypoplastic left heart syndrome, coarctation of the aorta, cleft palate without cleft lip, cleft lip with or without cleft palate, esophageal atresia / stricture with or without fistula, small intestine atresia / stricture, anorectal atresia / stricture, hypospadias, ambiguous sex, renal agenesis, polycystic kidney, preaxial polydactyly, limb reduction defect, diaphragmatic hernia, blindness, cataract, visual impairment, hearing loss, deafness, X-linked adrenoleukodystrophy, Rett syndrome, lysosomal disorder, cerebral palsy, autism, aglossia, leukoderma, ocular leukoderma, palpebral leukoderma, gestational diabetes, Arnold-Chiari malformation, CHARGE syndrome, congenital diaphragmatic hernia, brachydactyly, aniridia, cleft foot and cleft hand, heterochromia, Darwin ear, Ehlers-Danlos syndrome, epidermolysis bullosa, Gorham disease, Hashimoto's syndrome, fetal hydrops, hypotonia, Klippel-Feil syndrome, muscular dystrophy, osteogenesis imperfecta, progeria, Smith-Lemli-Opitz syndrome, color weakness, X-linked lymphoproliferative disorder, umbilical hernia, gastroschisis, preeclampsia, eclampsia, preterm birth, premature labor, miscarriage, intrauterine growth retardation, ectopic pregnancy, hyperemesis gravidarum, morning sickness, or the possibility of inducing normal labor.

[0105] In some NIPT embodiments, the techniques described herein further include estimating the fetal fraction of a sample, which is used to assist in determining whether the genetic data from a test subject exhibits aneuploidy. Methods for determining or calculating the fetal fraction are known in the art.

[0106] As used herein, the term "valid detection assay" refers to a detection assay that has been shown to accurately predict the relationship between the detection of a target and a phenotype (e.g., a medical condition). Examples of valid detection assays include, but are not limited to, detection assays that accurately predict a medical phenotype with a probability of 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% when the target is detected. Other examples of valid detection assays include, but are not limited to, detection assays that are qualified and / or sold as analyte-specific reagents (i.e., as defined by FDA regulations) or in vitro diagnostics (i.e., as approved by the FDA).

[0107] 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 enables the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in suitable containers) and / or auxiliary materials (e.g., buffers, instructions for performing the assay) from one location to another. For example, a kit includes one or more enclosures (e.g., boxes) that contain the relevant reaction reagents and / or auxiliary materials. As used herein, the term "subdivided kit" refers to a delivery system that includes two or more separate containers, each of which contains a portion of the total kit components. The containers may be delivered together or separately to the intended recipient. For example, the first container may contain an enzyme for use in the assay, and the second container may contain oligonucleotides. The term "subdivided kit" is intended to include, but is not limited to, kits that contain analyte-specific reagents (ASRs) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system that includes two or more separate containers, each of which contains a portion of the total kit components, is included within the term "subdivided kit". In contrast, a "combination kit" refers to a delivery system that includes all of the components of a reaction assay within a single container (e.g., within a single box that contains each of the desired components). The term "kit" includes both subdivided kits and combination kits.

[0108] As used herein, the term "information" refers to any collection of facts or data. With respect to information stored or processed using a computer system(s), including but not limited to the Internet, this term refers to any data stored in any form (e.g., analog, digital, optical, etc.). As used herein, the term "information related to a subject" refers to facts or data related to a subject (e.g., a human, plant, or animal). The term "genomic information" refers to information related to a genome, including but not limited to nucleic acid sequences, genes, allele frequencies, RNA expression levels, protein expression, phenotypes related to genotypes, etc. "Allele frequency information" refers to facts or data related to allele frequencies, including but not limited to the identity of alleles, the statistical correlation between the presence of alleles and the characteristics of a subject (e.g., a human subject), the presence or absence of alleles in an individual or population, the percentage likelihood of the presence of alleles in an individual having one or more specific characteristics, etc.

[0109] As used herein, the term "assay validation information" refers to genomic information and / or allele frequency information resulting from the processing (e.g., computer-based processing) of test result data. Assay validation information can be used, for example, to identify a particular candidate detection assay as a valid detection assay.

Brief Description of the Drawings

[0110] This patent or application file includes at least one drawing made in color. Copies of this patent or patent application publication that include color drawings are provided by applying to the Patent Office and paying the necessary fee.

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[0111] [Figures 27A - 27B and Figures 28A - 28B] show the results achieved in an RCA reaction carried out using primers bound to the glass surface in an irregularly dispersed state, and a molecular beacon probe containing a quencher and a fluorophore was used for detection.

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DETAILED DESCRIPTION OF THE INVENTION

[0112] The goal of molecular diagnostics has been to achieve accurate and sensitive detection of analytes in as short a time as possible using minimal effort and steps. One way this is achieved is through multiplex detection of analytes in a sample that allows multiple detection events in a single reaction vessel or solution. However, many existing diagnostic methods involving multiplex reactions still require many steps, including sample preparation steps that increase the time, complexity, and cost of performing the reaction. The present invention provides a solution to these problems, in some embodiments, by providing an assay that can be performed directly in an unpurified or untreated biological sample (e.g., blood or plasma).

[0113] In some embodiments, the techniques provided herein provide an economical way to assay a sample in a digital manner, i.e., by detecting individual copies of molecules, to count the copy number of a particular nucleic acid or protein in a sample or a portion of the sample, without using a sequencing step (e.g., a digital or “next-generation” sequencing step). The techniques are used to measure target molecules such as nucleic acid molecules in any type of sample, including but not limited to samples collected from a subject for diagnostic screening. Embodiments of the techniques provided herein are used, for example, in non-invasive prenatal testing (NIPT) and other genetic analyses. Embodiments of the techniques implement one or more steps of nucleic acid extraction, MIP probe design, MIP amplification / replication, and / or methods for measuring signals from circularized MIPs. In preferred embodiments, the techniques provide a method for immobilizing MIPs on a surface and detecting the immobilized MIPs. In preferred embodiments, the immobilized MIPs are detected using rolling circle amplification.

[0114] In preferred embodiments, the methods of the techniques include a target recognition event, typically including hybridization of a target nucleic acid, e.g., a sample of a patient's DNA, to another nucleic acid molecule, e.g., a synthetic probe. In preferred embodiments, the target recognition event creates conditions under which a unique product is generated (e.g., an extended, ligated, and / or cleaved probe oligonucleotide), where the product indicates that the target was present during the reaction and that the probe hybridized thereto.

[0115] Several different "front-end" methods for recognizing a target nucleic acid and generating a new product are described herein. For example, as shown in the exemplary embodiments of the figures, the present technology provides several ways to generate circularized molecules for use in the "back-end" detection / readout step (see, e.g., FIGS. 1-3, FIGS. 13-18, FIG. 34, FIG. 35, and FIGS. 38-40). The technology also provides methods for signaling the presence of a target nucleic acid using other probe types, such as probes that can be cleaved by flap endonucleases in the presence of the target nucleic acid (see, e.g., FIGS. 17-19). Each of these front-end embodiments can be used to generate characteristic molecules, such as circular or cleaved oligonucleotides.

[0116] These characteristic molecules can be configured to have one or more characteristics useful for capture and / or identification in a downstream back-end detection step. Examples of molecules and characteristics generated in the front-end reaction include appended sequences (e.g., a complete target-specific sequence formed by ligation of the 3' and 5' ends of a probe) having a bound sequence, appended sequences (e.g., a copy portion of the target template), and / or tagged nucleotides (e.g., nucleotides attached to biotin, dyes, quenchers, haptens, and / or other moieties), such as circularized MIPs, or products such as single-stranded arms released from a flap cleavage reaction (see, e.g., FIGS. 17-19). In some embodiments, the MIP includes a feature as part of the probe, e.g., in the backbone of the probe.

[0117] Examples of back-end analysis methods for amplifying and / or detecting the unique products of the front-end are provided, for example, in FIGS. 2-3, FIGS. 6-7, FIGS. 9-12, FIGS. 15-16, FIGS. 20-21, FIG. 34, FIG. 35, and FIGS. 38-40.

[0118] This technology is discussed with reference to specific embodiments such as combinations of specific front - end target - dependent reactions and specific back - end signal amplification methods and detection platforms (e.g., MIPs incorporating biotin in FIGS. 13 - 16 combined with an enzyme - free hybridization chain reaction back - end; biotin - tagged cleavage flaps as in FIG. 19 combined with capture on a surface and subsequent hybridization to an enzyme - bound probe that catalytically generates a fluorescent signal as shown in FIG. 20), but the invention is not limited to any specific combination of the front - end and back - end methods and configurations disclosed herein, or to any specific method for detecting signals from assay products. One of ordinary skill in the art will understand that a front - end can be readily adapted to work with alternative back - ends. For example, the circularized MIP of FIG. 14 may be captured and detected using the enzyme - bound probe of FIG. 20, or alternatively, amplified in a rolling circle amplification assay exemplified in FIGS. 2 - 3, FIGS. 8 - 7, FIGS. 9 - 12, FIG. 21, FIGS. 34, 35, and FIGS. 38 - 40. Similarly, the cleaved flap as shown in FIG. 19 may be detected using a hybridization chain reaction as illustrated in FIGS. 19 - 20, and the circularized MIP or RCA amplicon may be detected using an invasive cleavage reaction as shown in FIG. 17.

[0119] Furthermore, this technology is discussed with respect to specific target nucleic acids, such as cell - free DNA in plasma, but the invention is not limited to any specific form of DNA, or any specific type of nucleic acid, or variations in any specific type of nucleic acid. One of ordinary skill in the art should understand that mutations, insertions, deletions, single - nucleotide polymorphisms (SNPs), and epigenetic variations such as methylation (e.g., conversion of unmethylated cytosine to uracil, creating a detectable sequence variation that reflects methylation variations of cytosine in the target DNA by analysis of DNA treated with a reagent) can be readily configured in embodiments of this technology to detect and count variations in specific CpG dinucleotides.

[0120] In some embodiments, the assay is performed in a multiplexed manner. In some embodiments, the multiplexed assay can be performed under conditions that allow different loci to reach more similar levels of amplification.

[0121] Figure 1 provides a schematic of a molecular inversion probe (MIP). The molecular inversion probe includes first and second target polynucleotide arms that are complementary to adjacent or proximal regions of the target nucleic acid to be detected, and a polynucleotide linker or "backbone" connects the two arms (see Figure 1).

[0122] In the presence of the complementary target nucleic acid, the MIP can be circularized to form an 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, closing the probe to form a circle involves further modification of the probe to create a ligatable nick, such as cleavage of the overlap between the ends, filling of the gap between the ends using a nucleic acid polymerase, etc.

[0123] As used herein, a target site or sequence refers to a part or region of a nucleic acid sequence that is sought to be selected from other nucleic acids in a sample having other sequences, which is useful for determining the presence or absence of a genetic disorder or condition (e.g., the presence or absence of mutations, polymorphisms, deletions, insertions, 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 target MIP comprises, in order, the following components: a first target polynucleotide arm - a first unique target molecular tag - a polynucleotide linker - a second unique target molecular tag - a second target polynucleotide arm. In some embodiments, a target population of target MIPs is used in the methods of the present disclosure. In the target population, the pair of the first and second target polynucleotide arms in each of the target MIPs is the same and is substantially complementary to the first and second regions, respectively, within the nucleic acid adjacent to the target site. See, for example, WO2017 / 020023 and WO2017 / 020024, each of which is incorporated herein by reference in its entirety.

[0124] In some embodiments, the length of each of the target polynucleotide arms is 18 to 35 base pairs. In some embodiments, the length of each of the target polynucleotide arms is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 base pairs, or any size range from 18 to 35 base pairs. In some embodiments, the length of each of the control polynucleotide arms is 18 to 35 base pairs. In some embodiments, the length of 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, or any size range from 18 to 35 base pairs. In some embodiments, each of the target polynucleotide arms has a melting temperature of 57°C to 63°C. In some embodiments, each of the target 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 range from 57°C to 63°C. In some embodiments, each of the control polynucleotide arms has a melting temperature of 57°C to 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 range from 57°C to 63°C. In some embodiments, each of the target polynucleotide arms has a GC content of 30% to 70%. In some embodiments, each of the target polynucleotide arms has a GC content of 30 to 40%, or 30 to 50%, or 30 to 60%, or 40 to 50%, or 40 to 60%, or 40 to 70%, or 50 to 60%, or 50 to 70%, or any size range from 30% to 70%, or a specific percentage from 30% to 70%. In some embodiments, each of the control polynucleotide arms has a GC content of 30% to 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 range of 30%-70%, or any specific percentage of 30%-70%.

[0125] In some embodiments, the polynucleotide linker is not substantially complementary to any genomic region of the sample or subject. In some embodiments, the polynucleotide linker has a length of 30-40 base pairs. In some embodiments, the polynucleotide linker has a length of 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 base pairs, or any interval of 30-40 base pairs. In some embodiments, the polynucleotide linker has a melting temperature of 60°C-80°C. In some embodiments, the polynucleotide linker has a melting temperature of 60°C, 65°C, 70°C, 75°C, or 80°C, or any interval of 60°C-80°C, or any specific temperature within 60°C-80°C. In some embodiments, the polynucleotide linker has a GC content of 40%-60%. In some embodiments, the polynucleotide linker has a GC content of 40%, 45%, 50%, 55%, or 60%, or any interval of 40%-60%, or any specific percentage within 40%-60%.

[0126] In some embodiments, the target MIP replicon is generated by: i) hybridizing a first and a second target polynucleotide arm to first and second regions of a nucleic acid adjacent to the target site, respectively; and ii) after hybridization, using a ligation / extension mixture to extend and ligate the gap region between the two target polynucleotide arms to form a single-stranded circular nucleic acid molecule.

[0127] In certain embodiments, the methods described herein are used to detect exon deletions, insertions, or duplications. In some embodiments, the target site (or sequence) is a deletion, insertion, or duplication in a gene of interest or a genomic region of interest. In some embodiments, the target site is a deletion, insertion, or duplication in one or more exons of a gene of interest. In some embodiments, multiple exons of the target are contiguous. In some embodiments, multiple exons of the target are non - contiguous. In some embodiments, the first and second target polynucleotide arms of the MIP are designed to hybridize upstream and downstream of a deletion (or insertion, or duplication) in a gene or genomic region of interest or a genomic region (e.g., one or more exons) that has been deleted (or inserted, or duplicated). In some embodiments, the first or second target polynucleotide arm of the MIP contains a sequence substantially complementary to a genomic region of a gene of interest that encompasses the target deletion or duplication site (e.g., an exon or partial exon).

[0128] 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 circularized cfDNA. Extension of the primer using a strand - displacing DNA polymerase (e.g., φ29 (Phi29), Bst Large Fragment, and the Klenow fragment of E.coli Pol I DNA polymerase) results in a long single - stranded DNA molecule containing repeats of a nucleic acid sequence complementary to the MIP circular molecule.

[0129] In some embodiments, ligation-mediated rolling circle amplification (LM-RCA) is utilized, which includes a ligation operation prior to replication. In the ligation operation, if present, the probe hybridizes to its complementary target nucleic acid sequence, and the ends of the hybridized probes are joined by ligation to form a covalently closed single-stranded nucleic acid. After ligation, the rolling circle replication primer hybridizes to the probe molecule and initiates rolling circle replication as described above. Generally, LM-RCA involves mixing an open circular probe with a target sample to produce a probe-target sample mixture, incubating the probe-target sample mixture under conditions that promote hybridization between the open circular probe and the target sequence, mixing a ligase with the probe-target sample mixture to produce a ligation mixture, incubating the ligation mixture under conditions that promote ligation of the open circular 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 to produce a primer-ATC mixture and incubating it under conditions that promote hybridization between the amplified target circle and the rolling circle replication primer. Mixing a DNA polymerase with the primer-ATC mixture to produce a polymerase-ATC mixture and incubating it under conditions that promote replication of the amplified target circle, and the replication of the amplified target circle results in the formation of tandem sequence DNA (TS-DNA), i.e., a long single-stranded DNA containing a concatemer of sequences complementary to the amplified target circle.

[0130] In the embodiment shown in FIG. 2, the circularized molecules A, B, C, and D consist of MIPs specific to a reference chromosome such as chromosome 13, 8, 21, or Chr. 1. The sequence of the MIP surrounding the gap complements the region of the target chromosome, and the backbone of the MIP contains a unique sequence for hybridizing with a probe containing a specific fluorescent dye (FITC, ALEXA, Dylight, cyanine, rhodamine dye, quantum dots, etc.). Step 1 includes hybridizing the MIP to cfDNA, single nucleotide extension (or longer extension), and ligation to circularize the extended MIP. Step 2 includes rolling circle amplification of the circularized MIP such that the sequences required to hybridize to the fluorescently labeled oligonucleotide are amplified. A * , B * , C * , D * is a complement of the MIP sequence. Step 3 includes hybridizing the fluorescently labeled probe to the rolling circle product. In the embodiment shown in FIG. 3, the detection of the RCA product is facilitated by a molecular probe instead of a fluorescent dye-labeled oligonucleotide.

[0131] There are multiple ways to immobilize the MIP on a surface (e.g., beads or glass surface). For example, this can be achieved by initiating rolling circle amplification with a modified oligonucleotide containing a bindable moiety. Groups useful for modification of the initiating oligonucleotide include, but are not limited to, thiol, amino, azide, alkyne, and biotin, and the modified oligonucleotide can be immobilized using appropriate reactants as outlined, for example, in Meyer et.al., “Advances in DNA-mediated immobilization” Current Opinions in Chemical Biology, 18:8:8-15 (2014) (which is hereby incorporated by reference in its entirety for all purposes).

[0132] Imaging of MIPs incorporating fluorescent dyes can be achieved by using a method that includes immobilizing the MIP on a surface (glass slide or bead), for example, immobilizing it using appropriate reactants as outlined above and as described by Meyer et al. above, and using modifications of the MIP backbone that include modified bases that can be detected using an antibody. Once immobilized on the surface, an antibody-MIP complex can be formed that can be imaged by a microscope using an antibody against the incorporated tag. In some embodiments, the antibody may be conjugated to enhance or amplify the detectable signal from the complex. For example, conjugation of β-galactosidase to the antibody enables detection in a single molecule array (“SIMOA”) using the process described by Quanterix, where each complex is immobilized on a bead such that any bead has only one labeled immune complex, and the beads are dispensed into an array of femtoliter-sized wells such that each well contains at most one bead. Addition of resorufin-β-galactopyranoside causes β-galactosidase on the immobilized immune complex to catalyze the production of fluorescent resorufin. Upon visualization, the fluorescence emitted from the wells having the immobilized individual immune complexes can be detected and counted. See, for example, Quanterix Whitepaper 1.0, Scientific Principle of Simoa (Single Molecule Array) Technology, 1-2 (2013), and Quanterix Whitepaper 6.0, Practical Application of Simoa™ HD-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, the antibody-MIP complex may be directly detected using a solid nanopore having an antibody labeled with various molecular weights of poly(ethylene glycol), as described, for example, in Morin et.al., “Nanopore-Based Target Sequence Detection” PLOS One, DOI:10.1371 / journal.pone.0154426(2016), which is incorporated herein by reference.

[0133] Figure 4 provides a schematic of an embodiment of the present technology that involves circularizing circulating cfDNA (ccfDNA) using a single-stranded ligase (e.g., CircLigase™ thermostable RNA ligase) to create a “native circle” for detection. Once created, the circular ccfDNA can be detected using several different methods, including several RCA methods. For example, as shown in Figure 5, one embodiment of the present technology involves 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).

[0134] Figure 6 shows a further method for detecting ccfDNA. In this embodiment, as previously described, a plasma sample is processed to purify ccfDNA (see, e.g., M. Fleischhacker, et al., Methods for isolation of cell-free plasma DNA strongly affect DNA yield, Clin Chim Acta. 2011 Nov 20;412(23-24):2085-8). In step 1, the ccfDNA is heat denatured and treated with T4 polynucleotide kinase to create 5’ phosphorylated and 3’ hydroxyl-ended DNA fragments. Additional DNA such as T4 DNA polymerase Repair can be used to repair DNA prior to heat denaturation and T4 polynucleotide kinase treatment. A complementary oligonucleotide with a protected 3’ end is hybridized to ccfDNA (so as not to be extended by polymerase). This complementary oligonucleotide consists of chromosome-specific regions A and C, as well as 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 can be used by annealing the oligonucleotide to the universal sequence B. After RCA, a fluorescently labeled probe is hybridized to the rolling circle product.

[0135] Figure 7 shows another method for detecting ccfDNA. A plasma sample is processed to purify ccfDNA as described above. Step 1, heat denature the ccfDNA. A complementary oligonucleotide with a phosphorylated 5 prime protected end is hybridized to the ccfDNA. This complementary oligonucleotide consists of chromosome-specific regions A and C, as well as 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 the 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.

[0136] Figure 8 shows a schematic of a synthetic circular DNA that is useful as a template for rolling circle amplification and includes a rolling circle primer binding site and two probe binding sites, as well as an optional binding moiety (e.g., biotin).

[0137] Figure 9 provides a schematic of an embodiment of the present technology that involves the use of a probe pair configured for collision quenching when hybridized to a DNA strand for the detection of products from RCA, such as circular DNA like that shown in Figure 8. In this embodiment, the dye-labeled probe in solution is not quenched and generates a signal. A probe that hybridizes to a target near the quencher-tagged probe is quenched, thereby reducing the fluorescence signal. As the amount of the RCA product increases, the fluorescence decreases.

[0138] Figure 10 provides a schematic of an embodiment of the present technology that involves the use of a probe pair configured for fluorescence resonance energy transfer (FRET) as described above when hybridized to a DNA strand for the detection of products from RCA.

[0139] Figure 11 provides a schematic of an embodiment of the present technology that involves the use of a probe containing a dye and a quencher that is configured to be cleaved using a double-strand specific nuclease, such as a restriction enzyme, when hybridized to a DNA strand for the detection of products from RCA.

[0140] As shown in Figure 12, one embodiment of the present technology involves the use of RCA of chromosome-specific identifier sequences (CIDs), followed by CID-specific digestion of non-target chromosomes, and CID-specific labeling of the target CIDs. The CIDs are amplified by RCA, but their individual single-molecule IDs are maintained. CID amplification increases the fluorescence signal from individual target molecules. The sequences of unanalyzed chromosomes are doubly suppressed by enzymatic digestion and the use of labels specific only to the chromosomes being analyzed.

[0141] In some embodiments, MIPs may 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 triggered “hybridization chain reaction” (HCR) as described in, for example, RM Dirks, et al., Proc. Natl. Acad. Sci. USA 101(43):15275-15278(2004), and U.S. Patent No. 8,105,778, each of which is incorporated herein by reference. FIGS. 13-16 show exemplary configurations using HCR for signal amplification.

[0142] FIG. 13 shows an embodiment 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 then binds to a streptavidin-coated surface as shown in FIG. 14, and after washing away any unbound MIP, the backbone of the bound MIP hybridizes to the initiation oligonucleotide. In a preferred embodiment, a spacer, such as an 18-atom hexaethylene glycol spacer, is included between the initiation sequence and the backbone binding sequence. Preferably, the footprint of the MIP binding region is selected to have a high T m (e.g., about 79° C.) for stable binding. As described above, binding tags can be used to tag MIPs using biotin such as amine groups, thiol groups, azides, or haptens and immobilize them on a suitable reactive surface.

[0143] Figure 15 shows an example of a hairpin oligonucleotide used in HCR to form a self-assembled scaffold. One or both of the oligonucleotides contain at least one label, such as a fluorophore. In a preferred embodiment, the dyes are arranged to provide a sufficiently large spacing in the assembled scaffold to prevent quenching effects. For example, in some embodiments, as shown in Figure 14, the dyes are arranged at both ends of the hairpin. As shown in Figure 16, when the reaction is initiated by hybridization to the starting oligonucleotide bound to the MIP backbone, the HCR hairpins unfold and hybridize to long strands, creating scaffolds containing multiple labels.

[0144] Flap endonuclease reactions (e.g., Invader assay) can be used for specific and quantitative detection of chromosomes. Exemplary embodiments are shown in Figures 17-20. Figure 17 shows an Invader oligonucleotide and a probe oligonucleotide hybridized to a target region of a chromosome. The 3' end of the invasive oligonucleotide overlaps with the 5' end of a region of the probe oligonucleotide complementary to the target region. In this embodiment, the probe oligonucleotide contains a 5' flap containing a biotin moiety and a 3' tail containing a label, such as a fluorophore. A flap endonuclease, such as FEN-1 nuclease, recognizes the overlapping invasive cleavage structure, cleaves the probe in a very specific structure-dependent manner, and releases the 5' flap. In a preferred embodiment, as schematically shown in Figure 19, the reaction is carried out isothermally resulting in linear signal amplification, providing 10 3 ~10 4 cleaved probes per target in 1-3 hours.

[0145] In a preferred embodiment, the probe oligonucleotide used contains a hairpin structure in which the 5' flap and 3' tail of the probe hybridize to each other, as shown in Figure 18. The non-cleaved probe and / or the 3' portion of the cleaved probe are captured by a hapt A fluorophore or another moiety, such as 2,4-dinitrophenyl, may be used as a hapten so that it can be removed from the reaction using an antibody against it.

[0146] The cleaved flaps from the flap endonuclease reaction can be detected in several ways. In a preferred embodiment, the cleaved flaps are captured using immobilized complementary probes, and biotin reacts with streptavidin conjugated to a detectable moiety as shown in FIG. 20. In the embodiment shown, streptavidin is conjugated to β-galactosidase, and a fluorescent signal is generated by providing non-fluorescent resorufin-β-galactopyranoside, which is catalyzed by β-galactosidase to produce D-galactose and the fluorescent dye resorufin. Using a femtoliter array and Poisson statistics to generate a digital readout format, such enzyme signal amplification can be used to detect a single hybridization event. 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 Whitepaper 1.0, Scientific Principle of Simoa (Single Molecule Array) Technology, 1-2(2013); and Quanterix Whitepaper 6.0, Practical Application of Simoa (trademark) HD-1 Analyzer for Ultrasensitive Multiplex Immunodetection of Protein Biomarkers, 1-3(2015) (each of which is hereby incorporated by reference for all purposes). In certain preferred embodiments, a kinetic readout, i.e., collection of signals from the array at two time points, is used.

[0147] In the embodiment shown in FIG. 21, A, B, C, and D consist of MIPs specific to reference chromosomes such as chromosome 13, 8, 21, or 1. The sequences of the MIPs surrounding the gap complement the region of the target chromosome and are designed to contain a single nucleotide gap. Step 1: This gap is filled with dNTPs conjugated to haptens such as fluorescent dyes, biotin. When the gap is filled, different haptens are introduced into the MIPs targeting each of the different specific chromosomes. For example, adding A completes only 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 such as chromosome 1. In this approach, these four different MIPs are labeled 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 involves incubating the hapten-containing MIPs with labeled antibodies specific to 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 immune complex containing the primary antibody targeting the hapten to a labeled secondary antibody against the primary antibody, thereby amplifying the fluorescent signal.

[0148] As shown in FIG. 21, in this embodiment of the present technology, MIPs designed to target different chromosomes each require different nucleotides for extension and ligation, and the MIPs are extended and ligated in a chromosome-specific manner using nucleotides carrying different dyes for different dNTPs. For example, in a preferred embodiment, CY2, CY3, CY5, and CY7 are used. The MIPs with dye tags can be detected using specific antibodies for each different dye (and also, by extension, for each different chromosome being detected). The signal can be amplified by using a secondary antibody. For example, a CY2 primary rabbit antibody binds to the target MIP, and a secondary goat anti-rabbit antibody binds to the primary antibody to amplify the signal, etc.

[0149] As described above, many different fluorescent labeling systems find use in embodiments of the present technology. In some embodiments, fluorescent dyes (e.g., fluorescein, Texas Red, TAMRA, Cy3, Cy5) can be used and attached, for example, to oligonucleotides or nucleotide analogs incorporated into extension products. In some embodiments, fluorescent particles, e.g., nanoparticles, nanocrystals, quantum dots, silica (e.g., mesoporous silica nanoparticles) polymeric beads (e.g., latex) can be used.

[0150] There are many options for the detection and quantification of fluorescence signals from the embodiments of the above technology. Detection can be based, for example, on physicochemical, electromagnetic, electrical, optoelectronic or electrochemical properties, or the measurement of the characteristics of immobilized molecules and / or target molecules. Two factors related 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, for example, from probes non-specifically bound to the surface. Exemplary methods for detecting single molecule-related signals can be found, for example, in WO2016 / 134191 (which is hereby incorporated by reference in its entirety for all purposes). In some embodiments, the assay is configured for standard SBS microplate detection, for example, in a SpectraMax microplate reader or other plate reader. This method typically requires low-dispersion fluorescence (multiple wells, multiple measurements), but this format can be multiplexed and read in multiple different fluorescence channels. Further, this format has a very high throughput.

[0151] Embodiments can also be configured for detection on a surface, for example, a glass, gold, or carbon (e.g., diamond) surface. In some embodiments, signal detection is performed 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, and the target molecule is labeled with an electromagnetic emitter. Other microscopy methods, such as atomic force microscopy (AFM) or other scanning probe microscopy (SPM), are also suitable. In some embodiments, it may not be necessary to label the target. Alternatively, labels detectable by SPM may be used. In some embodiments, signal detection and / or measurement includes surface reading by counting fluorescence clusters using an imaging system, such as an ImageXpress imaging system (Molecular Devices, San Jose, CA) and similar systems.

[0152] Embodiments of the present technology can be configured for use in many other systems and equipment platforms, such as bead assays (e.g., Luminex), array hybridization, and detection using the NanoString nCounter single molecule counting device. See, for example, GK Geiss, et al., Direct multiplexed measurement of gene expression with color-coded probe pairs; Nature Biotechnology 26(3):317-25(2008), US Patent Application Publication No. 2018 / 0066309A1 (published March 8, 2018) (PN Hengen, et.Al., Invent., Nanostring Technogies, Inc.), etc.

[0153] In a Luminex bead assay, color-coded beads pre-coated with an analyte-specific capture antibody for the molecule of interest are added to the sample. Multiple analytes can be detected simultaneously in the same sample. The analyte-specific antibody captures the analyte of interest. A biotinylated detection antibody specific for the analyte of interest is added so that an antibody-antigen sandwich is formed. 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 such as a Luminex200 (trademark) or a Bio-Rad (registered trademark) Bio-Plex (registered trademark) analyzer. One laser classifies the beads and determines the analyte to be detected. The second laser determines the magnitude of the signal derived from the PE, which is directly proportional to the amount of the bound analyte.

[0154] The NanoString nCounter is a single molecule counting device for digitally quantifying hundreds of different genes in a single multiplex reaction. This technology uses molecular "barcodes" in combination with solid-phase hybridization and automated imaging and detection: each barcode is color-coded and attached to a single probe corresponding to the gene (or other nucleic acid) of interest. See, for example, Geiss et al., supra, which describes the use of unique pairs of capture and reporter probes constructed to detect each nucleic acid of interest. In the described embodiments, the probes are mixed together with nucleic acids, such as undivided cfDNA, or total RNA from a sample, in a single liquid-phase hybridization reaction. Hybridization results in the formation of a ternary structure consisting of the target nucleic acid bound to its specific reporter and capture probes, and the unhybridized reporter and capture probes are removed, for example, by affinity purification. The hybridization complex is exposed to a suitable capture surface, such as a surface coated with streptavidin, if a biotinylated tag is used. After capture on the surface, an applied electric field spreads and orients each complex in the same direction. The complexes are then immobilized in an elongated state and imaged. Thus, each target molecule of interest is identified by the color code generated by the regular fluorescent segments present in the reporter probe and aggregated to count the target molecules.

[0155] Figures 22, panels A, B, and C, show embodiments of the present technology where MIPs containing or attached to immobilization moieties are immobilized on a surface. While not limited to any particular embodiment for incorporating unique features indicative of target recognition into the cyclic MIP molecules, the embodiment of Figure 22 is shown using an embodiment that includes the extension of a linear MIP using a polymerase to copy one or more nucleotides of the target nucleic acid, followed by ligation to circularize the extended probe.

[0156] In the embodiment shown in Panel A of FIG. 22, in step 1, the MIP is hybridized to the target DNA and then extended by DNA polymerase in the presence of modified dNTPs, whereby the immobilization moiety is incorporated into each MIP during the extension. The MIP is then ligated to itself to complete the circular probe. Modified dNTPs can include, but are not limited to, dNTPs containing reactive chemical species such as amine groups or thiol groups, or other bindable features such as biotin or antibody haptens. In step 2, the circularized MIP is exposed to the surface under conditions where the surface interacts with the immobilization function of the MIP and binds to the MIP. Such surfaces can 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 such as slides, sample wells, channels, beads, particles, and / or nanoparticles, any of which can be porous or non-porous.

[0157] In the embodiment shown in Panel B of FIG. 22, in step 1, the MIP is hybridized to the target DNA and ligated to circularize. In the embodiment shown, the MIP is extended by DNA polymerase to fill in sequence gaps prior to ligation, but in other embodiments, the MIP may simply hybridize to the target nucleic acid without using a polymerization step and be designed to ligate and circularize, for example, in the manner of a padlock probe. 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 MIP is hybridized to a complementary oligonucleotide containing the above-described immobilization moiety, such as a reactive amine, reactive thiol group, biotin, hapten, etc. In step 3, the hybrid MIP complex of the MIP and the oligonucleotide containing the immobilization moiety is exposed to a surface under conditions where the surface interacts with the immobilization function of the MIP complex and binds to the MIP complex. As described above, the surface includes, but is not limited to, derivatized glass or non-derivatized glass, silica, diamond, gold, agarose, plastic, ferromagnetic material, alloy, etc., and can be in any form, such as slides, sample wells, channels, beads, particles, and / or nanoparticles, any of which can be porous or non-porous.

[0158] In the embodiment shown in panel C of FIG. 22, in step 1, a MIP containing an immobilized moiety incorporated into the backbone of the probe is hybridized with DNA, extended by DNA polymerase, and ligated to circularize the probe. Similar to the embodiment of panel B described above, the MIP can be designed to simply hybridize to the target nucleic acid and ligate to circularize without using a polymerization step. In step 2, the circularized MIP containing the immobilized moiety is exposed on the surface under conditions where the surface interacts with the immobilization function of the MIP and binds to the MIP. As described above, the surface includes, but is not limited to, derivatized glass or underivatized glass, silica, diamond, gold, agarose, plastic, ferromagnetic material, alloy, etc., and can be in any form such as, for example, slides, sample wells, channels, beads, particles, and / or nanoparticles, any of which can be porous or non-porous.

[0159] In each of the embodiments shown in FIG. 22, once the MIP is immobilized on the surface, labeling and / or signal amplification (e.g., fluorescent labeling and / or amplification of the fluorescent signal) as well as detection can be achieved using any of the various backend analysis methods discussed herein. Suitable methods for amplifying and / or detecting the native immobilized MIP product include, but are not limited to, the NanoString nCounter technology described above, as well as the methods shown in FIGS. 2-3, 6-7, 9-12, 15-16, and 20-21. In some embodiments, the labeling and / or signal amplification (e.g., fluorescent labeling and / or amplification of the fluorescent signal) is performed before the MIP is immobilized on the surface.

[0160] In a preferred embodiment, a backend process configured for visualization of single molecules is used. For example, as described above, the Quanterix platform uses an array of femtoliter-sized wells that capture beads having only one tagged complex, and the signal from the captured complex is generated using a resorufin-β-galactopyranoside / β-galactosidase reaction that produces fluorescent resorufin. Visualization of the array enables detection of the signal from each individual complex. In certain preferred embodiments, for example, a solid nanopore device such as that described by Morin et al. (see “Nanopore-Based Target Sequence Detection” PLoS ONE 11(5):e0154426 (2016)) is used. A solid nanopore is a nanoscale aperture formed in a thin solid membrane that separates two aqueous volumes

[23] . A voltage clamp amplifier measures the ionic current passing through the open pore while applying a voltage across the membrane (FIG. 1a). When a single charged molecule such as double-stranded DNA is captured and driven through the pore by electrophoresis, the measured current shift, as well as the shift depth (δI) and duration, are used to characterize the event. (Morin et al., supra). While this system can be used to detect only DNA, different tags (e.g., polyethylene glycol (PEG) of different sizes) can be attached to sequence-specific probes (e.g., peptide nucleic acid probes, PNAs) to obtain any particular DNA-PNA-PEG complex that is a unique signature representing the target nucleic acid detected at the front end of the assay.

[0161] In the embodiment shown in FIG. 23, a complex including an oligonucleotide primer and a circular probe such as an MIP or a ligated padlock probe is formed. The extension of the primer in the rolling circle amplification reaction generates a long 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 beacon separates the quencher from the fluorophore, enabling detection of fluorescence from the beacon. Accumulation of the RCA product can be monitored in real time by measuring the increase in fluorescence intensity indicating binding of the beacon to the product, the amount of which increases over time of the reaction.

[0162] Using real-time quantification of fluorescence accumulating in the reaction, the effect of the biotin moiety attached to the MIP or primer was investigated. FIGS. 24A-24D show the results of examining the effect of including the biotin residue in the circularized MIP only (A), the RCA primer only (B), both (C), and neither (D) on the RCA signal. In this experiment, the MIP included the following sequence: [Sequence Listing 1]

[0163] [Chemical Formula]

[0164] In the above biotinylated MIP, the "T" enclosed by a square indicates the attachment site of biotin (Integrated DNA Technologies, "Internal Biotin dT") within the MIP containing biotin. The biotinylated primer contained biotin attached to the terminal 5'-phosphate (Integrated DNA Technologies, "5'Biotin-TEG"). The rolling circle reaction was carried out at 37 °C according to the "standard rolling circle reaction" procedure described in Example 1 below. It was carried out for 1 hour. These data indicate that while the presence of biotin in the circularized MIP inhibits RCA, the presence of biotin on the primer does not inhibit the reaction.

[0165] Figures 25A - 25C show the results of various amounts of components in a standard RCA reaction in solution. Figure 25A compares the use of 5 units and 25 units of Phi29 polymerase in each reaction, showing that a higher concentration of polymerase consistently yields a higher signal under the tested conditions. Figure 25B shows the effect of using different concentrations of molecular beacon probes ("beacons"). Figure 25C compares the effect of using different concentrations of Phi29 polymerase and molecular beacon probes to the effect on a standard reaction when using 200 μM or 800 μM total dNTP. Based on these data, reactions adjusted to contain 1000 nM beacon, 800 μM dNTP, and 2000 nM phi29 polymerase (80 units) were further tested.

[0166] The effects of adding different concentrations of PEG and using different sizes of PEG were examined for enhanced RCA conditions (E - RCA, see Example 1 below). Figure 26 compares the effects of using different sizes of PEG (200 and 8000) at the indicated percentages (w:v) under E - RCA conditions. Under the conditions tested for this embodiment, PEG200 provided excellent results at all concentrations tested, with 20% PEG200 providing the best results. In contrast, PEG8000 significantly reduced the efficiency of RCA. Based on these data, RCA reactions containing at least 20% w:v of PEG200 were further tested.

[0167] As described above, for the detection of single molecules on a surface, it is preferable that the spot size of the signal from any individual binding molecule is also minimized so as to ensure separation between spots. The influence of using PEG200 on spot size and the number of detected spots was investigated. The assay was performed by incubating for 140 minutes with or without 20% w:v PEG200 using the E-RCA conditions described below. The results are shown in FIGS. 27A - 27B and FIGS. 28A - 28B. FIG. 27A shows that the presence of PEG decreased the spot size and improved the measured fluorescence signal from individual spots. FIG. 27B shows the effect of PEG on the number and fluorescence intensity of the spots shown in FIG. 27A, indicating that the addition of PEG decreased the size of the detected spots while increasing the number of detectable spots.

[0168] In the reaction carried out on an APTES-silanized plate, the influence of using PEGs with different molecular weights in a 20% solution on the number of spots and spot size was investigated. The reaction on the APTES-treated surface was carried out by modifying the PEG component as shown in FIG. 28 and as described in "One-step rolling circle amplification on the surface" of Example 1. FIG. 28 shows that when the PEG used has an average molecular weight of less than 1000, preferably 200 - 800, more preferably 600, the number of spots is maximized and the spot size is minimized.

[0169] The length of the hybridization time before starting the RCA reaction was investigated. FIG. 29A shows a microscopic image of the surface of an APTES-silanized plate as described in Example 1, comparing the RCA signals for reactions hybridized for 18 hours or 1 hour before starting the RCA reaction with either TBS buffer or RCA buffer. Enhanced RCA was performed as described above for 140 minutes using 20% PEG600. FIG. 29B provides a graph comparing the influence of hybridization time and buffer on the number and fluorescence intensity (area) of the spots shown in FIG. 29A. These data show that as the hybridization time increases, the number of spots increases significantly.

[0170] Figures 30, 31, and 32 provide graphs comparing the effect of PEG200 on standard RCA reaction conditions, enhanced RCA (E-RCA) conditions, and E-RCA conditions with further variations (with or without a 2-hour hybridization time, using PEG2000 instead of PEG200, 2-hour hybridization). All reactions in each figure were carried out at the same temperature, and in Figures 30, 31, and 32, the reactions were carried out at 30 °C, 25 °C, and 37 °C, respectively.

[0171] The number of spots and fluorescence intensity (area) were evaluated for each condition. These data indicate that in the presence of PEG200, a reaction temperature of 37 °C resulted in the best combination of a high number of spots and small spot size. The effect of varying the concentration of the beacon probe using a higher RCA reaction temperature was also investigated. Reactions containing 1000, 2000, 4000, or 8000 nM molecular beacon probe were carried out at 37 °C or 42 °C, and it was shown that at higher temperatures, the number of spots counted increased significantly (data not shown). Without limiting the technique to any particular mechanism of action, these data suggest that carrying out the reaction at a higher temperature, such as 42 °C or above, results in more RCA products and more bound beacon probes.

[0172] The effect of increasing temperature in the presence of various concentrations of PEG600 was further investigated. Figure 33 shows microscopic images of the surface of an APTES silanized plate as described in Example 1, comparing the RCA signals for reactions containing PEG600 at the specified concentrations carried out at 37 °C or 45 °C. These data indicate that the number of spots increased significantly for the 45 °C reaction, and that 10 - 15% w:v PEG600 at 45 °C resulted in the best combination of number of spots and spot size.

[0173] The effect of adding graphene oxide to the RCA surface binding reaction was investigated. A two-step RCA procedure as described in Example 2 and schematically shown in Figure 35 was developed. Figure 36 shows a microscopic image of the surface of an APTES-silanized plate as described in Example 1 and shows the RCA signal for graphene oxide in the two-step reaction. The negative control contains no input target and shows the background from the molecular beacon probe. Figure 37 provides a graph comparing the number of spots in the RCA reaction performed in one step (without GO) or two steps (with or without GO), comparing reactions containing 100 fmol of target with reactions without target. These data show that the use of GO significantly reduces the number of background spots in the control reaction without target and improves the signal:background result of the assay.

Example

[0174] Example 1 This example provides an example of a workflow for the analysis of DNA, such as cfDNA, from a sample such as a blood sample. Sample collection Blood is collected from the patient in a standard blood draw. 10 mL of blood is stored in a Streck blood collection tube or an alternative EDTA-containing blood collection tube. The sample is transported to the laboratory at ambient temperature and processed as follows: · Centrifuge the blood at 2000 x g for 20 minutes at room temperature to obtain the plasma fraction from the blood. · Transfer the plasma to a new sterile nuclease-free polypropylene tube and centrifuge at 3220 x g for 30 minutes. Purification of cell-free DNA (cfDNA) Cell-free DNA is purified from the plasma using standard methods, for example, using the MagMAX Cell-Free DNA Isolation Kit (Thermofisher Scientific, catalog number No. A29319). Preparation of assay plate Treat a glass-bottom microtiter plate and immobilize oligonucleotides that initiate rolling circle amplification of circularized MIP. Several approaches can be used (see, for example, E.J. Devor, et al., “Strategies for Attaching Oligonucleotides to Solid Supports,” Integrated DNA Technologies (2005), which is hereby incorporated by reference in its entirety for all purposes).

[0175] 1) Pre-washing with acid In either method, the glass-bottom plate is first acid-washed as follows: (a) Add 100 μL of 0.5 N sulfuric acid to each well.

[0176] (b) Add a foil seal to the plate.

[0177] (c) Incubate at 37 °C for 2 hours while rotating the plate at 300 RPM.

[0178] (d) Remove the well contents.

[0179] (e) Wash the wells twice with 100 μL of molecular grade water.

[0180] (f) Wash the wells twice with 100 μL of 95% ethanol.

[0181] 2) Silanization with 3-aminopropyltriethoxysilane (APTES) and streptavidin-biotin primer immobilization: (a) Prepare 2% APTES by adding 200 μL of 99% APTES (Sigma Aldrich, catalog number 440140), 500 μL of molecular grade water, and 9.3 ml of 95% ethanol.

[0182] (b) Vortex the solution and pipette 100 μL into each well.

[0183] (c) Incubate at room temperature for 15 minutes.

[0184] (d) Remove the contents of the well.

[0185] (e) Wash the well twice with 100 μL of 95% ethanol.

[0186] (f) Remove the last wash solution.

[0187] (g) Incubate the plate at 37 °C for 24 hours. Primer Immobilization (h) Add 1 nanogram of streptavidin in 100 μL of Tris-buffered saline to the amine-functionalized glass plate.

[0188] (i) Incubate at room temperature for 1 hour.

[0189] (j) Wash each well three times with 100 μL of TBS.

[0190] (k) Add 100 μL of a 1 μM solution of biotinylated oligonucleotide.

[0191] (l) Incubate at room temperature for 1 hour.

[0192] (m) Wash each well three times with 100 μL of TBS.

[0193] 3) Acrylsilanization and Acrydite Primer Immobilization (a) Prepare 4% acrylsilane by adding 400 μL of 99% acrylsilane (3-(trimethoxysilyl)propyl methacrylate, Sigma Aldrich, catalog number 440159), 1 mL of molecular grade water, and 18.6 mL of 100% ethanol.

[0194] (b) Add 100 μL of the 4% acrylsilane solution to each well.

[0195] (c) Incubate at room temperature for 15 minutes.

[0196] (d) Remove the 4% acrylsilane solution.

[0197] (e) Wash each well 4 times with 100 μL of 100% ethanol per wash.

[0198] (f) Incubate the plate at 37 °C for 24 hours.

[0199] (g) The Acrydite primer solution is (i) prepared by adding 250 μL of 5xTRIS Boron EDTA (TBE) buffer, (ii) 500 μL of 40% acrylamide, (iii) 17.5 μL of 10% ammonium persulfate, (iv) 5 μL of tetramethylethylenediamine (TEMED), (v) 25 μL of 100 μM oligonucleotide primer containing ’5’ Acrydite (or acrylyl-phosphoramidite), (vi) 1.7 mL of molecular grade water.

[0200] (h) Add 25 μL of the Acrydite primer solution to each well and gently stir the plate to cover the bottom of the well.

[0201] (i) Incubate at room temperature for 30 minutes.

[0202] (j) Before continuing the RCA assay, wash the wells 4 times with 100 μL of 0.5xTBE, discard the first 3 washings, and leave the last washing in the well.

[0203] The primer may be immobilized by other methods such as those described by Devor et al. above, for example. Molecular inversion probe pool Using a probe pool to capture specific loci in a DNA sample, such as a cfDNA sample, and create circular MIPs for rolling circle amplification. NIPT assays generally include a pool of molecular inversion probes. In a preferred embodiment, the NIPT assay includes from about 5,000 to 10,000 molecular inversion probes. · The target MIPs are created to target the features investigated by the assay (e.g., chromosome 13, chromosome 18, chromosome 21, chromosome X, chromosome Y, and chromosome CHR22q11.2). · Approximately 10,000 unique MIPs are created for each feature. · The MIPs are mixed together to create a probe pool with a custom concentration for each probe. Capture and Ligation of cfDNA by MIP · The MIP pool is added to the purified cfDNA in the following reaction.

[0204] ○ 2 μL of AMPligase buffer (10x), 1 μL of the MIP probe pool, 16 μL of the cfDNA prep, and 1 μL of AMPligase (80 units).

[0205] ○ Incubate the reaction at 98 °C for 2 minutes, cool it at 1 °C per minute until it reaches 45 °C, and then maintain it at 45 °C for 2 hours. Molecular Beacon Probes Examples of molecular beacon probes that can be used in this technique are as follows: 1) 5′Alexa 405-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-dabcyl 3′ 2) 5’Alexa 488-CC TCA ATG CTG CTG CTG TAC TAC mGmAmG mG-dabcyl 3’ 3) 5’Alexa 594-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-BHQ2 3’_ 4) 5’Alexa 647-CCTCAGCGCTGCCTATTCGAACTmGmAmGmG-BHQ2 3’ 5) 5′ Alexa 750-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-BHQ3 3′ Standard rolling circle amplification assay conditions ○ For 100 μL of RCA solution, combine on ice □ MIP probe - target DNA preparation (e.g., the above MIP capture whole / cfDNA preparation, ~20 μL) □ 10 μL of 10X Phi29 buffer for 1X final concentration 1X Phi29 DNA polymerase reaction buffer - 50 mM Tris - HCl - 10 mM MgCl 2 - 10 mM (NH 4 ) 2 SO 4 - 4 mM DTT - (pH 7.5 at 25 °C) □ 200 μM dNTP □ 5 units of Phi29 DNA polymerase □ 100 nM beacon probe □ Molecular grade water up to 100 μL ○ Incubate at 30 °C - 37 °C for reaction time, e.g., 90 - 120 minutes Enhanced RCA (E - RCA) conditions: ○ For 100 μL of enhanced RCA solution, combine on ice □ MIP probe - target DNA preparation (e.g., the above MIP capture whole / cfDNA preparation, ~20 μL) □ 10 μL of 10X Phi29 buffer for 1X final concentration □ 800 μM dNTP □ 80 units of Phi29 DNA polymerase □ 1000 nM beacon probe □ Molecular grade water up to 100 μL ○ Incubate at 30 °C - 37 °C for reaction time, e.g., 90 - 120 minutes

[0206] One - step enhanced rolling circle amplification on the surface □ Preparation of Rolling Circle Amplification (RCA) Solution ○ For 100 μL of RCA solution, combine on ice □ MIP Probe - Target DNA Preparation (e.g., the above MIP Capture Whole / cfDNA Preparation, ~20 μL) □ 10 μL of 10X Phi29 Buffer for 1X final concentration □ 1X Phi29 DNA Polymerase Reaction Buffer - 50 mM Tris - HCl - 10 mM MgCl 2 - 10 mM (NH 4 ) 2 SO 4 - 4 mM DTT - (pH 7.5 at 25 °C) □ 4 μL of 10 mM dNTP for 0.4 mM total dNTP final concentration □ 50 μL of filtered 30% PEG600 □ 0.5 μL of 100 μM Molecular Beacon at 0.5 μM final concentration □ 8 μL of Phi29 Polymerase (10 units / μL), and □ 22.5 μL of molecular grade water ○ Mix the solution, for example, by vortexing, pipette it onto the treated glass surface containing the annealed primers, and then seal the plate.

[0207] ○ Incubate the plate at 45 °C for 90 minutes on a flat - bottom heat block of a thermomixer equipped with Thermo - lid.

[0208] ○ Remove the well contents, wash the wells twice with 100 μL of 1×TBS, and discard the wash buffer.

[0209] ○ Add 100 μL of 1×TBS as described below and image with a microscope. Imaging of Samples with an IXM4 Microscope (Molecular Devices, San Jose, CA) Typically, 20x, 40x, or 60x objective lenses are used for imaging. · Place the plate on the IXM4 microscope and image as follows: ○ The plate is automatically exposed to ensure a wide dynamic range (such as a 16-bit image, the maximum range of the camera used) in the fluorescence intensity values.

[0210] ○ Each well of the plate is further divided into approximately 100 images.

[0211] For high-throughput assays, an automated microscope may be used. Image analysis · The images are analyzed as follows: ○ The relative fluorescence intensity is determined in an image without the sample (negative control).

[0212] ○ The threshold is determined by multiplying the average relative fluorescence intensity from the negative control by 3.

[0213] ○ Count the spots that exceed the threshold in each channel. Variation of the one-step protocol Addition of crowding reagent (e.g., PEG): Prepare a 30% solution in molecular grade water and filter through a 0.2 μm pore size filter. Add PEG to the RCA reaction and adjust so that the water added to the RCA maintains a constant volume.

[0214] Beacon: Add the desired concentration and adjust so that the water added to the RCA maintains a constant volume.

[0215] dNTP: Add the desired concentration and adjust so that the water added to the RCA maintains a constant volume.

[0216] Graphene oxide: Perform a two-step reaction as described in Example 2 and add graphene oxide with the labeled probe.

[0217] Example 2 Detection using two-step rolling circle amplification on a surface containing graphene oxide Prepare a rolling circle amplification (RCA) solution on ice.

[0218] ○ For 100 μL of RCA solution (without molecular beacon), combine the following: □ MIP probe - target DNA preparation (e.g., the entire MIP capture / cfDNA preparation as described above, approximately 20 μL) □ 10 μL of 10X Phi29 buffer for a final concentration of 1X □ 1X Phi29 DNA polymerase reaction buffer - 50 mM Tris - HCl - 10 mM MgCl 2 - 10 mM (NH 4 ) 2 SO 4 - 4 mM DTT - (pH 7.5 at 25 °C) □ 4 μL of 10 mM dNTP for a final total dNTP concentration of 0.4 mM □ 50 μL of filtered 30% PEG600 □ 8 μL of Phi29 polymerase (10 units / μL), and □ 23 μL of molecular grade water ○ Mix the solution by vortexing, pipette it onto the treated glass surface, and seal the plate.

[0219] ○ Incubate the plate at 45 °C for 90 minutes on a flat - bottom heat block of a thermomixer equipped with a thermowell.

[0220] ○ Remove the well contents, wash the wells 3 times with 100 μL of 1×TBS, and discard the wash solution.

[0221] ○ Add 50 μL of graphene oxide - molecular beacon solution containing the following: □ 5 μL of 10X Phi29 buffer for a final concentration of 1X □ 0.5 μL of 100 μM molecular beacon for a final concentration of 0.5 μM □ 5 μL of 2 mg / mL graphene oxide solution for a final concentration of 0.2 mg / mL Molecular grade water up to 50 μL ○ Incubate at 37 °C for 60 minutes ○ Wash three times with 100 μL of 1xTBS

[0222] ○ Wash once with 100 μL of 1×TBS containing 5% w:v Tween20

[0223] ○ Wash twice with 100 μL of 1×TBS and discard the wash buffer

[0224] ○ As described above, add 100 μL of 1×TBS and image using a microscope

[0225] It is readily apparent from the disclosure herein that each of the disclosed front - end target recognition systems can be configured to generate a detectable signal for use with any one of the above - mentioned back - end devices and systems

[0226] Other reference materials 1. F. Dahl, et al., Imaging single DNA molecules for high precision NIPT; Nature Scientific Reports 8:4549(2018) p1 - 8 2. R. M. Dirks, et al., Triggered amplification by hybridization chain reaction, Proc. Natl. Acad. Sci. USA101(43):15275 - 15278(2004) 3. T. J. Morin, et al., Nanopore - Based Target Sequence Detection, PLoS ONE11(5):e0154426(2016) 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) 5. J. R. Epstein, et al., High-Density Fiber-Optic Genosensor Microsphere Array Capable of Zeptomole Detection Limits; Anal.Chem. 74: 1836-1840(2002) 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) 7. R. Roy, et al., A Practical Guide to Single Molecule FRET Nat Methods. 5(6): 507-516(2008) 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) 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) 10.Quanterix Whitepaper 1.0,Scientific Principle of Simoa(Single Molecule Array)Technology,1-2(2013) 11.Quanterix Whitepaper 6.0,Practical Application of Simoa(Trademark)HD-1 Analyzer for Ultrasensitive Multiplex Immunodetection of Protein Biomarkers,1-3(2015) 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) 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) 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.) 15. G. K. Geiss, et al., Direct multiplexed measurement of gene expression with color - coded probe pairs; Nature Biotechnology 26(3): 317 - 25 (March 2008) and Corrigendum regarding authors’affiliations at 26(6): 1 (June 2008) 16. P. N. Hengen, et al., Inventors, U.S. Pat. Appl. Ser No. 15 / 729,421, published 03 / 08 / 2018 as U.S. Patent Pub. 2018 / 0066309 Al (Nanostring Technogies, Inc.) 17. M. Nilsson, et al. “Padlock probes: circularizing oligonucleotides for localized DNA detection”. Science. 265(5181): 2085 - 2088 (1994) 18. P. - J. J. Huang, and J. Liu, “Molecular Beacon Lighting up on Graphene Oxide,” Anal. Chem. 84: 4192 - 4198 (2012) 19. Y. Phillip, et al., “Common Crowding Agents Have Only a Small Effect on Protein-Protein Interactions,” Biophysical Journal 97:875-885(2009) 20. L. M. Dominak, et al., “Polymeric Crowding Agents Improve Passive Biomacromolecule Encapsulation in Lipid Vesicles,” Langmuir 26(16):13195-13200(2010) 21. B. Schweitzer, et al., “Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection,” Proc. Natl. Acad. Sci. USA97(18):10113-10119(2000) 22. C. Hong, et al., “Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide,” Anal. Chem. 88:2999-3003(2016) 23. E. J. Devor, et al., “Strategies for Attaching Oligonucleotides to Solid Supports,” Integrated DNA Technologies(2005) 24. WO 2015 / 083002”Multiplex Detection of Nucleic Acids” All documents and similar materials cited in this application, including but not limited to publications described in the above references, such as patents, patent applications, articles, books, papers, and Internet web pages, are hereby expressly incorporated by reference in their entirety for any purpose. 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 case the definitions of the terms in the incorporated references are considered to be different from the definitions provided in this teaching, the definitions provided in this teaching shall govern.

[0227] Various modifications and variations of the described compositions, methods, and uses of the described technology will be apparent to those of ordinary skill 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 invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described manner of practicing the invention that are apparent to those of ordinary skill in the fields of molecular biology, molecular diagnostics, nucleic acid structure, biochemistry, medicine, or related fields are intended to be within the scope of the claims.

Claims

1. 1. A method for enumerating target molecules on a solid support, comprising: a) forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, said primer being bound to a solid support; b) i) extending the primer in the complex in a rolling circle amplification (RCA) reaction to form an RCA product; ii) hybridizing a plurality of labeled probes to the RCA product; iii) detecting the formation of said at least one complex in a process comprising: A hybridized labeled probe indicates the presence of said target molecule on said solid support.

2. The method of claim 1 , wherein the solid support comprises a silanized surface.

3. The method of claim 2 , wherein the silane treated surface comprises glass.

4. 1. A method for enumerating target molecules on a solid support, comprising: a) - acrylic group, - reactive amine groups; b) forming a plurality of composites on a glass surface, said plurality of composites comprising: - an RCA product containing multiple hybridized labeled probes; - a double-stranded scaffold product comprising a plurality of concatemerized, labeled scaffold oligonucleotides; forming a plurality of complexes, the formation of which indicates the presence of a target molecule on the glass surface, and forming the plurality of complexes comprises exposing the glass surface to a solution comprising graphene oxide; and c) counting said plurality of complexes.

5. The method of claim 4 , wherein the silane treated surface is glass.

6. The method of claim 1 or claim 4, wherein the silane-treated surface comprises a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate.

7. 5. The method of claim 1 or claim 4, wherein the solid support comprises a surface within an assay plate, preferably a glass-bottom assay plate.

8. The method of claim 7, wherein the assay plate is a multi-well assay plate, preferably a microtiter plate.

9. The method of claim 1 , wherein the primer is directly attached to the solid support, preferably covalently attached to the solid support.

10. The method of claim 1 , wherein the primer comprises a biotin moiety and the solid support comprises avidin, preferably streptavidin.

11. 5. The method of claim 1 or claim 4, wherein the complex or complexes comprise an antibody bound to an antigen or hapten.

12. The method of claim 11 , wherein the complex comprises an antigen or hapten directly bound to the solid support.

13. The method of claim 12, wherein the antigen or hapten is covalently attached to the solid support.

14. 10. The method of claim 1 or claim 4, wherein forming the complex or complexes comprises exposing the solid support to a solution comprising a crowding agent.

15. 15. The method of claim 14, wherein the crowding agent comprises polyethylene glycol (PEG), preferably at least 2-10% (w:v) PEG, preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18%-20%.

16. The method according to claim 15, wherein the PEG has an average molecular weight of 200-8000, preferably 200-1000, preferably 400-800, preferably 600.

17. 10. The method of claim 1, comprising exposing the solid support to a solution comprising graphene oxide.

18. 20. The method of claim 17, wherein the solid support is exposed to graphene oxide prior to step b)iii).

19. 20. The method of claim 17, wherein the solid support is exposed to graphene oxide prior to step b)ii).

20. The method of claim 1 , wherein the target molecule comprises a nucleic acid.

21. The method of claim 4 , wherein the target molecule comprises a nucleic acid.

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

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

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

25. Forming the RCA product comprises extending a primer on the circularized nucleic acid probe in a reaction mixture, the reaction mixture comprising: at least 0.2 units / μL, preferably at least 0.8 units / μL of Phi29 DNA polymerase, - at least 400 μM, preferably at least 600 μM, more preferably at least 800 μM in total dNTPs.

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

27. 26. The method of claim 25, wherein forming an RCA product comprising a plurality of hybridized labeled probes comprises forming the RCA product further comprising greater than 100 nM of molecular beacon probe in the reaction mixture, preferably at least 1000 nM of molecular beacon probe in the reaction mixture.

28. 26. The method of claim 25, wherein the reaction mixture further comprises PEG, preferably at least 2-10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18%-20% PEG.

29. 29. The method according to claim 28, wherein the PEG has an average molecular weight of 200-8000, preferably 200-1000, preferably 400-800, preferably 600.

30. 26. The method of any one of claims 1, 4, and 25, wherein a plurality of RCA products hybridized to labeled probes are dispersed and immobilized on the solid support, and at least a portion of the plurality of RCA products are individually detectable by detection of the label.

31. 31. The method of claim 30, wherein the distribution of RCA products is irregular.

32. 31. The method of claim 30, wherein the distribution of RCA products is in an addressable array.

33. The method of claim 30 , wherein the complex comprises at least one polypeptide.

34. 34. The method of claim 33, wherein the at least one polypeptide comprises an antibody.

35. 31. The method of claim 30, wherein the complex comprises at least one specifically binding molecule selected from heptanes, lectins, and lipids.

36. 26. The method of any one of claims 1, 4, and 25, wherein at least one labeled probe comprises a fluorescent label.

37. 26. The method of any one of claims 1, 4, and 25, wherein the at least one labeled probe comprises a quencher moiety.

38. 26. The method of any one of claims 1, 4, and 25, wherein the at least one labeled probe comprises a fluorophore and a quencher moiety.

39. 39. The method of claim 38, wherein the at least one labeled probe is a molecular beacon probe.

40. 26. The method of any one of claims 1, 4, and 25, wherein the multiple RCA products are all hybridized to labeled probes containing the same label.

41. 26. The method of any one of claims 1, 4, and 25, wherein a plurality of RCA products are hybridized to a labeled probe comprising two or more different labels.

42. 42. The method of claim 41 , wherein the two or more different labels comprise two or more different fluorescent dyes.

43. 26. The method of any one of claims 1, 4, and 25, wherein the detecting or counting comprises detecting fluorescence.

44. 44. The method of claim 43, wherein detecting fluorescence comprises fluorescence microscopy.

45. 26. The method of any one of claims 1, 4, and 25, wherein forming an RCA product comprises incubating the reaction mixture at at least 37 degrees, preferably at least 42 degrees, preferably at least 45°C.

46. 46. ​​The method of claim 45, wherein the reaction mixture comprises PEG, preferably at least 2-10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18%-20% PEG.

47. 26. The method of any one of claims 4, 17-19, and 25, wherein the solid support or the glass surface exposed to a solution comprising graphene oxide is washed with a solution comprising a surfactant prior to the detecting or counting.

48. 48. The method of claim 47, wherein the surfactant comprises Tween 20.

49. 1. A composition comprising a silane-treated surface bound to a plurality of complexes, each of which comprises an oligonucleotide primer hybridized to a circularized nucleic acid probe, the primers being bound to a solid support, the reaction mixture comprising: at least 0.2 units / μL, preferably at least 0.8 units / μL, of Phi29 DNA polymerase; a buffer solution, at least 400 μM, preferably at least 600 μM, more preferably at least 800 μM in total dNTPs, - PEG, preferably at least 2-10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18%-20% PEG.

50. 50. The composition of claim 49, wherein the PEG has an average molecular weight of 200-8000, preferably 200-1000, preferably 400-800, preferably 600.

51. 50. The composition of claim 49, wherein the reaction mixture further comprises at least 100 nM of molecular beacon probe, preferably at least 1000 nM of molecular beacon probe.

52. The composition of any one of claims 49 to 51, wherein the primers are bound to the solid support in a randomly distributed manner.

53. The composition of any one of claims 49 to 51, wherein the primers are bound to the solid support in an addressable array.

54. 50. The composition of claim 49, wherein the primer is covalently attached to the solid support.

55. 50. The composition of claim 49, wherein the primer comprises a biotin moiety and the solid support comprises avidin, preferably streptavidin.

56. 50. The composition of claim 49, wherein the complex comprises an antibody bound to an antigen or hapten.

57. 50. The composition of claim 49, wherein the complex comprises an antigen or hapten directly bound to the solid support.

58. 58. The composition of claim 57, wherein the antigen or hapten is covalently attached to the solid support.

59. 50. The composition of claim 49, wherein the complex comprises at least one polypeptide.

60. 60. The composition of claim 59, wherein the at least one polypeptide comprises an antibody.

61. 50. The composition of claim 49, wherein the complex comprises at least one specifically binding molecule selected from heptanes, lectins, and lipids.

62. A composition comprising: a silane-treated surface bound to a plurality of complexes comprising RCA products, each complex comprising a plurality of hybridized labeled probes; and a solution comprising graphene oxide.

63. 63. The composition of claim 62, wherein the silane treated surface is glass.

64. 63. The composition of claim 62, wherein the silane-treated surface comprises a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate.

65. 63. The composition of claim 62, wherein the solution comprising graphene oxide further comprises a molecular beacon probe, preferably greater than 100 nM of a molecular beacon probe, preferably at least 1000 nM of a molecular beacon probe.

66. The solution containing graphene oxide contains MgCl ２ 63. The composition of claim 62, comprising a buffer comprising:

67. MgCl ２ 67. The composition of claim 66, wherein the buffer comprising is a Phi29 DNA polymerase buffer.

68. The method of any one of claims 1 to 4, wherein the solid support comprises a surface within an assay plate, preferably a glass-bottom assay plate.

69. 69. The method of claim 68, wherein the assay plate is a multi-well assay plate, preferably a microtiter plate.

70. The method of any one of claims 1 to 3 and 68 to 69, wherein the primer is directly attached to the solid support, preferably covalently attached to the solid support.

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

72. The method of any one of claims 1 to 4 and 68 to 71, wherein the conjugate or conjugates comprise an antibody bound to an antigen or hapten.

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

74. 74. The method of claim 73, wherein the antigen or hapten is covalently attached to the solid support.

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

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

77. 77. The method of claim 76, wherein the PEG has an average molecular weight of 200-8000, preferably 200-1000, preferably 400-800, preferably 600.

78. 80. The method of any one of claims 1-3 and 68-77, comprising exposing the solid support to a solution comprising graphene oxide.

79. 80. The method of claim 78, wherein the solid support is exposed to graphene oxide prior to step b)iii).

80. 80. The method of claim 78, wherein the solid support is exposed to graphene oxide prior to step b)ii).

81. The method of any one of claims 1 to 4 and 68 to 80, wherein the target molecule or molecules comprise a nucleic acid.

82. 82. The method of claim 81 , wherein the nucleic acid comprises DNA from a sample from a subject, preferably a blood sample or a blood product sample.

83. 83. The method of claim 82, wherein the DNA is cell-free DNA from a blood sample or a blood product sample.

84. 84. The method of claim 83, wherein the cell-free DNA comprises fetal DNA from a maternal blood sample.

85. Forming the RCA product comprises extending a primer on the circularized nucleic acid probe in a reaction mixture, the reaction mixture comprising: at least 0.2 units / μL, preferably at least 0.8 units / μL of Phi29 DNA polymerase, The method according to any one of claims 1 to 4 and 68 to 84, comprising at least 400 μM, preferably at least 600 μM, more preferably at least 800 μM in total dNTPs.

86. 86. The method of claim 85, wherein hybridizing a labeled probe to the RCA product comprises forming the RCA product further comprising greater than 100 nM of molecular beacon probe in the reaction mixture, preferably at least 1000 nM of molecular beacon probe in the reaction mixture.

87. 87. The method of claim 85 or 86, wherein forming an RCA product comprising a plurality of hybridized labeled probes comprises forming the RCA product further comprising greater than 100 nM of molecular beacon probe in the reaction mixture, preferably at least 1000 nM of molecular beacon probe in the reaction mixture.

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

89. 89. The method of claim 88, wherein the PEG has an average molecular weight of 200-8000, preferably 200-1000, preferably 400-800, preferably 600.

90. The method of any one of claims 1 to 4 and 68 to 89, wherein a plurality of RCA products hybridized to labeled probes are dispersed and immobilized on the solid support, and at least a portion of the plurality of RCA products are individually detectable by detection of the label.

91. 91. The method of claim 90, wherein the distribution of RCA products is irregular.

92. 91. The method of claim 90, wherein said distribution of RCA products is in an addressable array.

93. The method of any one of claims 90 to 92, wherein the complex comprises at least one polypeptide.

94. 94. The method of claim 93, wherein the at least one polypeptide comprises an antibody.

95. 95. The method of any one of claims 90 to 94, wherein the complex comprises at least one specifically binding molecule selected from heptanes, lectins, and lipids.

96. The method of any one of claims 1 to 4 and 68 to 95, wherein the at least one labeled probe comprises a fluorescent label.

97. The method of any one of claims 1-4 and 68-96, wherein the at least one labeled probe comprises a quencher moiety.

98. The method of any one of claims 1 to 4 and 68 to 97, wherein the at least one labeled probe comprises a fluorophore and a quencher moiety.

99. 99. The method of claim 98, wherein the at least one labeled probe is a molecular beacon probe.

100. The method of any one of claims 1-4 and 68-99, wherein the multiple RCA products are hybridized to labeled probes that all contain the same label.

101. The method of any one of claims 1-4 and 68-100, wherein the multiple RCA products are hybridized to a labeled probe comprising two or more different labels.

102. 102. The method of claim 101, wherein the two or more different labels comprise two or more different fluorescent dyes.

103. The method of any one of claims 1 to 4 and 68 to 102, wherein the detecting or counting comprises detecting fluorescence.

104. 104. The method of claim 103, wherein detecting fluorescence comprises fluorescence microscopy.

105. The method of any one of claims 1 to 4 and 68 to 104, wherein forming an RCA product comprises incubating the reaction mixture at at least 37 degrees, preferably at least 42 degrees, preferably at least 45°C.

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

107. 107. The method of any one of claims 78 to 106, wherein the solid support or glass surface exposed to a solution comprising graphene oxide is washed with a solution comprising a surfactant prior to said detecting or counting.

108. 108. The method of claim 107, wherein the surfactant comprises Tween 20.

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

110. 110. The composition of claims 49 and 109, wherein the primer is covalently attached to the solid support.

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

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

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

114. The composition of any one of claims 49 and 109-113, wherein the antigen or hapten is covalently attached to the solid support.

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

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

117. 117. The composition of any one of claims 49 and 109-116, wherein the complex comprises at least one specifically binding molecule selected from heptanes, lectins, and lipids.

118. The composition of claim 62 or 63, wherein the silane-treated surface comprises a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate.

119. 119. The composition of any one of claims 62, 63 and 118, wherein the solution comprising graphene oxide further comprises a molecular beacon probe, preferably greater than 100 nM of a molecular beacon probe, preferably at least 1000 nM of a molecular beacon probe.

120. The solution containing graphene oxide contains MgCl ２ The composition according to claims 62-63 and 118-119, comprising a buffer comprising:

121. MgCl ２ 121. The composition of claim 120, wherein the buffer comprising is a Phi29 DNA polymerase buffer.

122. A method according to any one of claims 1 to 48 and 68 to 108, comprising detecting or enumerating for diagnostic purposes.

123. 124. The method of claim 123, wherein the diagnostic objective comprises detecting aneuploidy.

124. 124. The method of claim 123, wherein the aneuploidy is a fetal aneuploidy detected in a maternal blood sample.

125. 125. The method of claim 123 or 124, wherein detecting aneuploidy comprises detecting or enumerating cfDNA molecules from a maternal blood sample.

126. 126. The method of claim 125, wherein the cfDNA from a maternal blood sample comprises maternal DNA and fetal DNA.

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

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