Methods, systems and compositions for counting nucleic acid molecules

Abstract

Compositions, methods, systems and kits for detecting and quantifying changes in molecular number, specifically changes in gene dosage, such as due to gene duplication or changes in the normally euploid complement of chromosomes, such as trisomy of one or more chromosomes normally found in a diploid pair, in the absence of digital sequencing.

Description

[0001] This application claims priority to U.S. Provisional Application Serial No. 62 / 651,676, filed on April 2, 2018, and U.S. Provisional Application Serial No. 62 / 660,699, filed on April 20, 2018, each of which is incorporated herein by reference. Technical Field

[0002] The present invention relates to compositions and methods for determining the copy number of individual molecules such as nucleic acid molecules without digital sequencing. The technology of the present invention can be used, for example, to analyze the changes in the copy number of specific nucleic acid sequences that may be caused by changes in, for example, chromosome number, gene copy number, expression level, etc. The technology of the present invention can be specifically applied to genetic screening, such as prenatal testing, and specifically for non-invasive prenatal testing (NIPT). NIPT involves analyzing the free DNA (cfDNA) from the fetus circulating in the blood of women carrying fetuses in the uterus. Analysis of free DNA in maternal blood can be used to assess the health of the fetus. The technology of the present invention herein relates to methods, systems and kits for detecting and quantifying changes in gene dosage caused by changes in the number of molecules, specifically, for example, due to normal euploid complements of genes or chromosomes, such as changes in trisomy of one or more chromosomes usually found in diploid pairs. Background Art

[0003] The presence of the molecule in the detection sample or the change of the number of molecules is a useful way to characterize the source of the sample and the sample. For example, the change of gene dosage is a clinically significant indicator of the disease state of the experimenter from which the sample is collected. The change of gene dosage is due to the error in the DNA replication and can occur in germline cells to cause congenital defects and even embryonic death or occur in somatic cells to usually cause cancer. These replication abnormalities can cause the deletion or repetition of each part of the gene, the full-length gene and the regulatory region around it, the megabase long part of the chromosome or the whole chromosome. The analysis of other biomolecules is also very important clinically. For example, the change of the amount of RNA or protein can indicate the change of the expression of the gene associated with the disease state. Although embodiments of the present technology provided herein are discussed with respect to specific applications, such as measuring DNA, it should be understood that the present technology is not limited to these applications and that it is readily applicable to the analysis of many different types of molecules or moieties that can bind to a partner molecule in a specific manner, such as antigens with antibodies, nucleic acids with complementary nucleic acids, nucleic acid structures with proteins that bind to such structures (e.g., stem-loops, protruding nucleotides, flaps, promoter sequences), lectins with carbohydrates, proteins with protein binding partners, proteins with lipids (e.g., SH2 domains with lipids), etc.

[0004] Chromosomal abnormality can affect the number or structure of chromosomes. The situation that cells, tissues or individuals do not exist or also have one or more complete chromosomes or chromosome fragments in addition to the normal euploid complement of chromosomes can be referred to as aneuploidy. Germline replication errors caused by chromosome nondisjunction can lead to monosomy (autosomes instead of usually two or only one copy of sex chromosomes) or trisomy (three copies). When such events do not lead to complete embryonic death, they usually lead to various diseases that are generally considered to be syndromes, such as 21 trisomy and Down syndrome (Down's syndrome), 18 trisomy and Edward syndrome (Edward's syndrome) and 13 trisomy and Patau syndrome (Patau's syndrome). The structural chromosomal abnormality that affects the parts of chromosomes is caused by chromosome breakage and causes the disappearance, inversion, translocation or duplication of bulk genetic material. These events are often as devastating as the gain or loss of an entire chromosome and can lead to conditions such as Prader-Willi syndrome (del15q11-13), retinoblastoma (del 13q14), Cry-a-cat syndrome (del 5p), and others listed in U.S. Pat. No. 5,888,740, which is incorporated herein by reference in its entirety.

[0005] Major chromosomal abnormalities are detected in nearly 1 in 140 live births and in a higher proportion of preterm or non-live births. Hsu (1998) Prenatal diagnosis of chromosomal abnormalities through amniocentesis In: Milunsky A, ed., Genetic Disorders and the Fetus, 4th ed. Baltimore: The Johns Hopkins University Press. 179-180; Staebler et al. (2005) "Should determination of the karyotype be systematic for all malformations detected by obstetrical ultrasound?" Prenat Diagn 25: 567-573. The most common aneuploidy is trisomy 21 (Down syndrome) which currently occurs in 1 in 730 births. Hsu; Staebler et al. Although less common than trisomy 21, trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome) occur in 1 in 5,500 and 1 in 17,200 live births, respectively. Hsu. A variety of congenital defects, growth defects, and intellectual disabilities have been found in children with chromosomal aneuploidies, and these pose a lifelong challenge to families and society. Jones (2006) Smith's recognizable patterns of human malformation. Philadelphia: Elsevier Saunders. There are various prenatal tests that can indicate an increased risk of fetal aneuploidy, including invasive diagnostic tests such as amniocentesis or chorionic villus sampling), which are the current gold standard but are associated with a non-negligible risk of fetal loss. American College of Obstetrics and Gynecology (2007) ACOG Practice Bulletin 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 are based on the detection of fetal DNA in maternal plasma. It has been demonstrated that massively parallel sequencing of libraries generated from maternal plasma can reliably detect chromosome 21 abnormalities. See, e.g., Chiu et al., Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma, Proc Natl Acad Sci 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. Pat. No. 7,888,017.

[0006] Current methods that rely on next generation sequencing (NGS) for quantifying changes in molecular numbers, for example to screen for aneuploidy, are often time consuming, expensive, and require extensive bioinformatics analysis. Summary of the invention

[0007] The present invention provides compositions, methods and systems for detecting and characterizing samples by counting specific molecules (e.g., small molecules, haptens, proteins, antibodies, lipids, carbohydrates and nucleic acids, such as genes or other DNA molecules or fragments and / or RNA (e.g., messenger RNA, microRNA and other non-coding RNA)) that can be represented in the sample. The technology of the present invention can be applied, for example, to monitoring gene expression, measuring non-coding RNA abundance, and analyzing genetic variation, including but not limited to changes in gene dosage, such as aneuploidy. In a preferred embodiment, the technology of the present invention provides a method for detecting a target molecule and thereby counting a single copy of the target molecule without using "next generation" sequencing (NGS) technology (such as those described by Chiu et al. and Fan et al. (supra)) or single molecule amplification technology that relies on separate amplification reactions of individual target molecules (including nucleic acids) in different physical discrete elements (e.g., microcontainers or emulsion droplets).

[0008] Typically, these compositions, methods and systems provide improved ways to detect genome deletions and duplications of various sizes, including complete chromosomes, chromosome arms, microdeletions and duplications, submicrodeletions and deletions, and single nucleotide features (including single nucleotide polymorphisms, deletions, and insertions). In certain embodiments, the disclosed method can be used to detect subchromosomal gene damage, such as microdeletions. Exemplary applications of the method include children and prenatal diagnosis of aneuploidy, testing of the risk of pregnancy products or premature abortion, non-invasive prenatal testing (qualitative gene testing and quantitative gene testing, such as detecting Mendelian diseases, insertions / deletions, and chromosome imbalances), testing preimplantation genetics, tumor characterization, postnatal testing (including cytogenetics), and mutagenic effect monitoring.

[0009] In some embodiments, the technology of the present invention herein provides a method for characterizing nucleic acid, preferably DNA, more preferably circulating free DNA from blood or plasma in a sequence-specific and quantitative manner. In a preferred embodiment, a single copy of DNA is detected and counted in the absence of polymerase chain reaction or DNA sequencing. Embodiments of the technology of the present invention provide methods, compositions and systems for detecting target DNA using a method for amplifying a signal indicating the presence of a target DNA in a sample. In a preferred embodiment, the detectable signal from a single target molecule is amplified to such an extent and in such a manner that the signal derived from a single target molecule is detectable and identifiable, isolated from signals from other targets and from other copies of the target molecule.

[0010] In some embodiments, the technology of the present invention provides a method for counting target molecules on a solid support, the method comprising: forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a solid support; and detecting the formation of the at least one complex in a process comprising: i) extending the primer in the complex in a rolling circle amplification (RCA) reaction to form 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 silanized surface, preferably a surface comprising glass.

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

[0012] The surface is not limited to any particular format. For example, in any of the embodiments described above, the solid support may comprise a surface in an assay plate, preferably a glass bottom assay plate. In some embodiments, the assay plate is a multi-well assay plate, preferably a microtiter plate.

[0013] In some embodiments of the present technology, the primer of any embodiment described above is directly bound to the solid phase carrier, preferably covalently linked to the solid phase carrier. For example, in some embodiments, the primer includes a biotin moiety and the solid phase carrier includes avidin, preferably streptavidin. In certain embodiments, one or more complexes include an antibody bound to an antigen or hapten, and in some embodiments, the complex includes an antigen or hapten directly bound to the solid phase carrier. In certain embodiments, the antigen or hapten is covalently linked to the solid phase carrier.

[0014] In any of the embodiments described herein, forming the complex or complexes may comprise exposing the solid support to a solution comprising a crowding agent. In some embodiments, the crowding agent comprises polyethylene glycol (PEG), preferably at least 2% to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG. In certain preferred embodiments, the average molecular weight of the PEG is between 200 and 8000, preferably between 200 and 1000, preferably between 400 and 800, preferably 600.

[0015] In any of the embodiments described above, forming a complex or a plurality of complexes may include a step of exposing the solid support to a solution comprising graphene oxide. In a preferred embodiment, the solid support is exposed to graphene oxide before the step of detecting the hybridized labeled probe. In a particularly preferred embodiment, the solid support is exposed to a solution comprising a mixture of labeled probes and graphene oxide. In some embodiments, the solid support or the glass surface exposed to a solution comprising graphene oxide is washed with a solution comprising a detergent before the detection or counting. In certain preferred embodiments, the detergent comprises Tween 20.

[0016] The technology of the present invention can be used to detect many different types of molecules, including e.g. Fig.38 In some embodiments, the target molecule comprises a nucleic acid, preferably a sample from a subject, preferably DNA from a blood or blood product sample. In certain preferred embodiments, the DNA is free DNA from a blood or blood product sample. In some embodiments, the free DNA comprises maternal and / or fetal DNA from a maternal blood sample.

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

[0018] In certain embodiments of the technology of the present invention provided herein, a plurality of RCA products hybridized to a labeled probe are immobilized on the solid support in a dispersed manner, wherein at least a portion of the plurality of RCA products can be individually detected by detecting the label. In some embodiments, the dispersion of RCA products is irregular, and in some embodiments, the dispersion of RCA products is in an addressable array.

[0019] In any of the embodiments described herein, the complex immobilized on the surface can include at least one polypeptide (eg, an antibody) and / or the complex can include at least one specifically binding molecule selected from a hapten, a lectin, and a lipid.

[0020] In some embodiments, at least one labeled probe of the inventive technology described herein comprises a fluorescent label, and in some embodiments, at least one labeled probe comprises a quencher moiety. In certain preferred embodiments, at least one labeled probe comprises a fluorophore and a quencher moiety. In preferred embodiments, the at least one labeled probe is a molecular beacon probe.

[0021] In some embodiments of the present technology, multiple RCA products are hybridized to labelled probes all comprising the same label, while in some embodiments, multiple RCA products are hybridized to labelled probes comprising two or more different labels, preferably two or more different fluorescent dyes.

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

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

[0024] The present technology also provides compositions related to practicing the methods. In some embodiments, the present technology provides a composition comprising a silanized surface and a reaction mixture, wherein the silanized surface is bound to a plurality of complexes each comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a solid support, and the reaction mixture comprises: at least 0.2 units per μL of Phi29 DNA polymerase, preferably at least 0.8 units per μL; buffer; at least 400 μM, preferably at least 600 μM, more preferably at least 800 μM total dNTPs; and PEG, preferably at least 2% to 10% (w: v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG. In some embodiments, the average molecular weight of the PEG is between 200 and 8000, preferably between 200 and 1000, preferably between 400 and 800, preferably 600. In some embodiments, the reaction mixture further comprises at least 100 nM molecular beacon probe, preferably at least 1000 nM molecular beacon probe.

[0025] In some embodiments of the composition, the primers are bound to the solid support in an irregularly dispersed manner, and in some embodiments, the primers are bound to the solid support in an addressable array. In certain embodiments, the primers are covalently linked to the solid support, and in some embodiments, wherein the primers include a biotin moiety and the solid support includes avidin, preferably streptavidin. In some embodiments, the complex includes an antibody bound to an antigen or hapten, and in some embodiments, the complex includes an antigen or hapten directly bound to the solid support. In some embodiments, the antigen or hapten is covalently linked to the solid support.

[0026] In some embodiments of the compositions herein, 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 binding molecule selected from a hapten, a lectin, and a lipid.

[0027] Embodiments of the compositions described above may include a silanized surface and a solution, the silanized surface being combined with a plurality of complexes each comprising an RCA product, the RCA product comprising a plurality of hybridized labeled probes, the solution comprising graphene oxide. In some embodiments, the silanized surface is glass. In some preferred embodiments, the silanized surface comprises a surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate, preferably a glass surface.

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

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

[0030] The technology of the present invention provided herein is not limited to any specific use or application. In some embodiments, the technology of the present invention can be used to analyze chromosome aberrations (e.g., aneuploidy), preferably in the case of non-invasive prenatal testing. For example, some embodiments of the application of the technology of the present invention include obtaining a maternal sample including both maternal genetic material and fetal genetic material and measuring multiple target nucleic acids, wherein the target nucleic acid includes a specific sequence associated with a first chromosome, wherein the first chromosome is suspected to be a variant of the fetal material (e.g., in terms of gene dosage or chromosome count), and wherein the target nucleic acid further includes a specific sequence associated with a second chromosome, and the second chromosome is not suspected to be a variant of the fetal material. The method includes analyzing the amount of the target nucleic acid associated with the first chromosome in the sample and the amount of the target nucleic acid associated with the second chromosome to determine whether the amount of the target nucleic acid associated with the first chromosome is significantly different from the amount of the target nucleic acid associated with the second chromosome to indicate a chromosome or gene dosage variant in the fetus. In a preferred embodiment, the target nucleic acid associated with the first chromosome and the second chromosome is present in both maternal genetic material and fetal genetic material and is maternal nucleic acid and fetal nucleic acid, and the determination is not specific for the target nucleic acid. In a preferred embodiment, the maternal sample is free DNA from maternal blood. Statistical methods for analyzing chromosomal aberrations based on measuring the amount of DNA in a sample (including determining aberrations in fetal DNA when the fetal DNA is a small fraction of the total DNA in the maternal sample) are known in the art. See, for example, U.S. Patent No. 6,100,029, which is incorporated herein by reference.

[0031] definition

[0032] To facilitate understanding of the present invention, a number of terms and phrases are defined below:

[0033] Throughout this specification and claims, unless the context clearly indicates otherwise, the following terms adopt the meanings clearly associated with this document. Although the phrase "in one embodiment" used herein may, but does not necessarily refer to the same embodiment. In addition, the phrase "in another embodiment" used herein may, but does not necessarily refer to a different embodiment. Therefore, as described below, the various embodiments of the present invention can be easily combined without departing from the scope or spirit of the present invention.

[0034] In addition, unless the context clearly indicates otherwise, as used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or". Unless the context clearly indicates otherwise, the term "based on" is not exclusive and allows for being based on other factors not described. In addition, throughout the specification, the meanings of "a", "an" and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0035] The transitional phrase "consisting essentially of" as used in the claims of this application limits the scope of the claim to the specified materials or steps "and those materials or steps which do not materially affect one or more of the basic and novel characteristics 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 a level of unrecited contaminants such that the presence of the contaminants does not alter the function of the recited composition as compared to the pure composition (i.e., a composition "consisting of the recited components").

[0036] As used herein, the terms "subject" and "patient" refer to any organism, including plants, microorganisms, and animals (eg, mammals such as dogs, cats, livestock, and humans).

[0037] The term "sample" in this specification and claims is used in its broadest sense. On the one hand, it is meant to include specimens or cultures (e.g., microbial cultures). On the other hand, it is meant to include both biological samples and environmental samples. Samples may include specimens of synthetic origin. Biological samples may be animals, including humans, fluids, solids (e.g., feces) or tissues, as well as liquid and solid food and feed products and ingredients, such as dairy products, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from domestic animals of all different families as well as untamed or wild animals (including but not limited to ungulates, bears, fish, lagomorphs, rodents, etc.).

[0038] Environmental samples include environmental materials, such as surface materials, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, instruments, equipment, utensils, disposable and non-disposable items. These examples should not be construed as limiting the sample types applicable to the present invention.

[0039] As used herein, the term "target" refers to a molecule that is sought to be sorted out from other molecules for evaluation, measurement or other characterization. For example, a target nucleic acid can be sorted from other nucleic acids in a sample, for example, by probe binding, amplification, separation, capture, etc. When used with respect to hybridization-based detection (e.g., polymerase chain reaction), "target" refers to a region of nucleic acid defined by primers for polymerase chain reaction, and when used in an assay without amplifying target DNA (e.g., when captured by molecular inversion probes (MIPS)), the target includes a site defined by hybridization of the target-specific arms of the MIP, so that the MIP can be connected and the presence of the target nucleic acid can be detected.

[0040] 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, saliva and semen.

[0041] As used herein, the term "gene dosage" refers to the number of copies of a gene, gene region, chromosome, or fragment or portion thereof. Normal individuals carry two copies of most genes or gene regions, one copy on each of the two chromosomes. However, there are certain exceptions, for example, when a gene or gene region is located on chromosome X or Y, or when a gene sequence is present in a pseudogene.

[0042] As used herein, the term "aneuploidy" refers to a condition in which a cell, tissue, or individual has one or more entire chromosomes or chromosome segments absent or in addition to the normal euploid complement of chromosomes.

[0043] As used herein, the "sensitivity" of a given assay (or a set of assays used together) refers to reporting a specific form or variant of a threshold value that is higher than that distinguished between samples (e.g., cancer cells, aneuploidy) showing a variant phenotype and samples (e.g., non-cancerous cells, euploidy) showing a normal or wild-type phenotype, such as mutations, gene duplications, and percentages of samples of chromosome duplications. In some embodiments, "positive" is defined as a clinically confirmed variant of an assay result associated with the presence of a disease or condition to be detected, and a false negative is defined as a clinically confirmed variant of an assay result associated with the absence of a disease or condition. Therefore, the value of sensitivity reflects the probability that a given diagnostic assay performed on a known variant or a diseased sample will produce a result indicating the presence of a variation or disease. As defined herein, the clinical relevance of the calculated sensitivity value represents an estimate of the probability that a given assay will detect the presence of the condition when applied to a subject with a clinical condition. Using the technology of the present invention described herein, a certain level of accuracy can be achieved without the need to generate sequence reads. Accuracy can refer to sensitivity, accuracy can refer to specificity, or accuracy can refer to a certain combination thereof. The desired level of accuracy may be between 90% and 95%; the desired level of accuracy may be between 95% and 98%; the desired level of accuracy may be between 98% and 99%; the desired level of accuracy may be between 99% and 99.5%; the desired level of accuracy may be between 99.5% and 99.9%; the desired level of accuracy may be between 99.9% and 99.99%; the desired level of accuracy may be between 99.99% and 99.999%; the desired level of accuracy may be between 99.999% and 100%. An accuracy level above 95% may be referred to as high accuracy.

[0044] As used herein, the "specificity" of a given assay (or a set of assays used together) refers to the percentage of normal samples that report assay results associated with the presence of the disease or condition to be detected, and a false positive is defined as a clinically confirmed normal sample that reports an assay result associated with the presence of a disease or condition. Thus, the value of specificity reflects the probability that a given diagnostic assay performed on a known normal sample will produce a result indicating the presence of a variation or disease. As defined herein, the clinical relevance of a calculated specificity value represents an estimate of the probability that a given marker will detect the absence of a clinical condition when applied to a subject who does not suffer from the condition.

[0045] The term "gene" refers to a DNA sequence including control and coding sequences necessary to produce RNA (e.g., ribosomal RNA or transfer RNA), polypeptides or precursors having non-coding functions. RNA or polypeptides can be encoded by the full-length coding sequence or by any portion of the coding sequence, as long as the desired activity or function is retained.

[0046] As used herein, the term "gene region" refers to a gene, its exons, its introns, and the regions flanking it upstream and downstream, e.g., 5 to 10 kilobases 5' and 3' to the transcription start site and transcription stop site, respectively.

[0047] As used herein, the term "gene sequence" refers to a gene, its introns and its upstream and downstream regions flanking it, such as 5 to 10 kilobases of sequence 5' and 3' to the transcription start site and transcription stop site, respectively.

[0048] As used herein, the term "chromosome-specific" refers to a sequence that is only found in that particular type of chromosome.

[0049] As used herein, the term "hybridization" is used to refer to the pairing of complementary nucleic acids. Hybridization and hybridization strength (i.e., the strength of the association between nucleic acids) are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the T of the hybrid formed. m The process of "hybridization" involves annealing one nucleic acid to another complementary nucleic acid, i.e., a nucleic acid having a complementary nucleotide sequence. The ability of two nucleic acid polymers containing complementary sequences to find each other and anneal through base pairing interactions is a well-recognized phenomenon. Following the initial observation of the "hybridization" process by Marmur and Lane, PNAS 46:453 (1960) and Doty et al., PNAS 46:461 (1960), this process was refined into an important tool in modern biology.

[0050] 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 more preferably at least about 15 to 30 nucleotides. Exact size will depend on many factors, which in turn depend on the final function or purpose of the oligonucleotide. Oligonucleotides can be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR or a combination thereof.

[0051] Because the mononucleotides are reacted to form oligonucleotides in such a way that the 5' phosphate of one mononucleotide pentose ring is linked to the 3' oxygen of its adjacent mononucleotide pentose ring in one direction through a phosphodiester bond, the end of the oligonucleotide is referred to as the "5' end" if the 5' phosphate of the oligonucleotide is not linked to the 3' oxygen of the mononucleotide pentose ring, and the end of the oligonucleotide is referred to as the "3' end" if the 3' oxygen of the oligonucleotide is not linked to the 5' phosphate of the subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence may be referred to as having a 5' end and a 3' end even if it is inside a larger oligonucleotide. If the 3' end of the first region is ahead of the 5' end of the second region when moving along the nucleic acid chain in the 5' to 3' direction, the first region along the nucleic acid chain is referred to as upstream of another region.

[0052] When two different non-overlapping oligonucleotides anneal to different regions of the same straight-chain complementary nucleic acid sequence and the 3' end of one oligonucleotide points to the 5' end of the other oligonucleotide, the former can be referred to as an "upstream" oligonucleotide, while the latter is referred to as a "downstream" oligonucleotide. Similarly, when two overlapping oligonucleotides hybridize to the same straight-chain complementary nucleic acid sequence and the first oligonucleotide is positioned so that its 5' end is upstream of the 5' end of the second oligonucleotide and the 3' end of the first oligonucleotide is upstream of the 3' end of the second oligonucleotide, the first oligonucleotide can be referred to as an "upstream" oligonucleotide and the second oligonucleotide can be referred to as a "downstream" oligonucleotide.

[0053] The term "primer" refers to an oligonucleotide that can serve as a starting point for synthesis when placed under conditions such as in the presence of nucleotides and a suitable nucleic acid polymerase to initiate primer extension. Oligonucleotide "primers" can occur naturally, can be made using molecular biology methods (e.g., purification by restriction digestion), or can be produced synthetically. In a preferred embodiment, the primer is made of or includes DNA.

[0054] Primers are selected to be "substantially" complementary to a specific sequence strand of a template. Primers must have sufficient complementarity to hybridize with the template strand to allow primer extension to occur. The primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, and the remainder of the primer sequence may be substantially complementary to the strand. Non-complementary bases or longer sequences may be interspersed in the primer, provided that the primer sequence has sufficient complementarity to the sequence of the template to hybridize and thereby form a template-primer complex for the synthesis of an extension product of the primer.

[0055] As used herein, the term "sequence variation" refers to the difference in the nucleotide sequence between two nucleic acids. For example, a wild-type structural gene and a mutant form of the wild-type structural gene may differ in sequence by the presence of a single base substitution and / or the deletion or insertion of one or more nucleotides. These two forms of the structural gene are referred to as being different from each other in sequence. A second mutant form of the structural gene may exist. This second mutant form may be referred to as being different in sequence from both the wild-type gene and the first mutant form of the gene.

[0056] As used herein, the term "nucleotide analog" refers to modified or non-naturally occurring nucleotides, including but not limited to analogs with altered stacking interactions, such as 7-deazapurines (i.e., 7-deaza-dATP and 7-deaza-dGTP); base analogs with alternative hydrogen bonding configurations (e.g., Iso-C and Iso-G and other non-standard base pairs described in U.S. Pat. No. 6,001,983 to S. Benner); non-hydrogen bonding analogs (e.g., non-polar aromatic nucleoside analogs such as 2,4-difluorotoluene, derived from BA Kool, J. Org. Chem., 1994, 59, 7238-7242, BAS 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 (such as "K" nucleotides and "P" nucleotides, respectively; P. Kong et al., Nucleic Acids Res., 1989, 17, 10373-10383, P. Kong et al., Nucleic Acids Res., 1992, 20, 5149-5152). Nucleotide analogs include base analogs and include modified forms of deoxyribonucleotides and ribonucleotides and include, but are not limited to, the modified bases and nucleotides described in the following U.S. Patents: 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.

[0057] As used herein, the term "continuous nucleic acid chain" means a nucleic acid chain having a continuous, covalently linked backbone structure without nicks or other disruptions. The disposal of the base portion of each nucleotide (whether base-paired, single-stranded or mismatched) is not an element in the definition of a continuous chain. The backbone of a continuous chain is not limited to ribose-phosphate compositions or deoxyribose-phosphate compositions found in naturally occurring, unmodified nucleic acids. The nucleic acid of the present invention may include modifications in the structure of the backbone, including but not limited to phosphorothioate residues, phosphonate residues, 2' substituted ribose residues (e.g., 2'-O-methyl ribose) and alternative sugars (e.g., arabinose) containing residues.

[0058] As used herein, the term "continuous duplex" refers to a region of a double-stranded nucleic acid in which there is no disruption to the progression of base pairs within the duplex (i.e., base pairs along the duplex will not be twisted to accommodate a vacancy, protrusion or mismatch with the range of the region of the continuous duplex). As used herein, the term refers only to the arrangement of base pairs within the duplex, without implying continuity in the backbone portion of the nucleic acid chain. Double-stranded nucleic acids with uninterrupted base pairing but with nicks in one or both chains are within the definition of a continuous duplex.

[0059] The term "duplex" refers to the state of nucleic acid in which the base moieties of the nucleotides on one strand are bound by hydrogen bonding to their complementary bases arranged on the second strand. The condition of being in duplex form is reflected in the state of the bases of the nucleic acid. By means of base pairing, nucleic acid chains also generally adopt the tertiary structure of a double helix, with a major groove and a minor groove. Adopting a helical form is implicit in the act of becoming a duplex.

[0060] The term "template" refers to the nucleic acid strand on which a complementary copy is constructed from nucleoside triphosphates by the activity of a template-dependent nucleic acid polymerase. By convention, within a duplex, the template strand is depicted and described as the "bottom" strand. Similarly, the non-template strand is typically depicted and described as the "top" strand.

[0061] As applied to polynucleotides, the term "substantially identical" refers to the property of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 85% sequence identity, preferably at least 90% to 95% sequence identity, more usually at least 99% sequence identity compared to a reference sequence over a comparison window of at least 20 nucleotide positions (usually over a window of at least 25-50 nucleotides), wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence, the polynucleotide sequence may contain deletions or additions that total 20% or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence, for example, as a splice variant of the full-length sequence.

[0062] As applied to polypeptides, the term "substantially identical" means that two polypeptide sequences share at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or more (e.g., 99% sequence identity) when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic hydroxyl side chains are serine and threonine; a group of amino acids with amide-containing side chains are asparagine and glutamine; a group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains are lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine.

[0063] 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: radioactive labels, such as 32P; a binding moiety, such as biotin; a hapten, such as digoxgenin; a luminescent, phosphorescent or fluorescent moiety; a mass tag; and a fluorescent dye alone or in combination with a moiety that can suppress or shift the emission spectrum by fluorescence resonance energy transfer (FRET). FRET is a distance-dependent interaction between electronic excited states of two molecules (e.g., two dye molecules or a dye molecule and a non-fluorescent quencher molecule) in which excitation is transferred from a donor molecule to an acceptor molecule without emitting 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 part of the donor whose absorption spectrum overlaps with the emission spectrum of the donor and is capable of absorbing some or most of the emitted energy from the donor when near the donor group (usually between 1-100nm), such as a fluorophore, a chromophore or a quencher. If the acceptor is a fluorophore, it is usually re-emitted at a third still longer wavelength; if it is a chromophore or a quencher, it releases the energy absorbed from the donor without emitting photons. In some embodiments, a change in the detectable emission from the donor dye is detected (for example, when the acceptor part is close or away). In some embodiments, a change in the detectable emission from the acceptor dye is detected. In a preferred embodiment, the emission spectrum of the acceptor dye is different from the emission spectrum of the donor dye, so that the emission from the dye can be distinguished from each other (for example, spectrally resolved).

[0064] In some embodiments, the donor dye is used in combination with multiple acceptor moieties. In a preferred embodiment, the donor dye is used in combination with a non-fluorescent quencher and with an acceptor dye, so that when the donor dye is close to the quencher, its excitation is transferred to the quencher instead of 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 a particularly preferred embodiment, emission from the acceptor dye is detected. See, e.g., Tyagi et al., Nature Biotechnology 18: 1191 (2000), which is incorporated herein by reference.

[0065] The label can provide a signal detectable by fluorescence (e.g., simple fluorescence, FRET, time-resolved fluorescence, fluorescence polarization, etc.), radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, quality or a property of behavior affected by quality (e.g., MALDI time-of-flight mass spectrometer), etc. The label can be a charged moiety (positive or negative charge) or alternatively can be neutral. The label can comprise or consist of a nucleic acid or protein sequence, as long as the sequence comprising the label is detectable.

[0066] In some embodiments, the label includes particles for detection. In a preferred embodiment, the particles are phosphor particles. In a particularly preferred embodiment, the phosphor particles are up-conversion phosphor particles (see, for example, Ostermayer, FW 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 phosphor particles. Phosphor particles can be detected by any suitable method, including but not limited to up-conversion phosphor technology (UPT), in which up-conversion phosphors convert low-energy infrared (IR) radiation into high-energy visible light. Although the present invention is not limited to any particular mechanism, in some embodiments, UPT converts infrared light into visible light by multiphoton absorption and subsequent emission of dopant-dependent phosphorescence. See, e.g., U.S. Pat. No. 6,399,397, issued June 4, 2002 to Zarling et al.; van De Rijke et al., Nature Biotechnol. 19(3):273-6

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

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

[0067] As used herein, the term "solid phase carrier" or "carrier" refers to any material that provides another material to which a solid or semi-solid structure can be connected. Such materials include smooth carriers (e.g., smooth metals, glass, quartz, plastics, silicon, wafers, carbon (e.g., diamond) and ceramic surfaces, etc.) and textured and porous materials. Such materials also include but are not limited to gels, rubbers, polymers and other non-rigid materials. Solid phase carriers do not have to be flat. Carriers include any type of shape, including spherical shapes (e.g., beads).

[0068] As used herein, the term "bead" refers to a small solid support that can move around when in solution (e.g., having a size smaller than the size of the housing or container in which the solution is located). In some embodiments, the beads can settle out of the solution when the solution is not mixed (e.g., by shaking, hot mixing, vortexing), while in other embodiments, the beads can be suspended in the solution in a colloid form. In some embodiments, the beads are completely or partially spherical or cylindrical. However, the beads are not limited to any particular three-dimensional shape.

[0069] The material connected to the solid phase carrier can be connected to any part of the solid phase carrier (for example, it can be connected to the internal part of the porous solid phase carrier material or to the flat part on the carrier that is not flat in other ways, or vice versa). In a preferred embodiment of the present technology, biological molecules such as nucleic acids or protein molecules are connected to the solid phase carrier. The biological material is "attached" to the solid phase carrier when it is attached to the solid phase carrier by chemical or physical interactions. In some embodiments, the attachment is through a covalent bond. However, the attachment does not have to be covalent and does not have to be permanent. In some embodiments, the attachment can be revoked or dissociated by changes in conditions (for example, by temperature changes, ion changes, addition or removal of chelating agents or other changes in the solution conditions to which the surface and the bound molecules are exposed).

[0070] In some embodiments, the target molecule (e.g., biomaterial) is connected to the solid support via a "spacer molecule" or "linker group". Such spacer molecules are molecules having a first portion connected to the biomaterial and a second portion connected to the solid support. The spacer molecule typically comprises a chain of atoms (e.g., carbon atoms) that provide additional distance between the first portion and the second portion. Therefore, when connected to the solid support, the spacer molecule allows separation between the solid support and the biomaterial, but is connected to both.

[0071] As used herein, the terms "array" and "microarray" refer to a surface or container including addressable multiple predefined loci for analyzing loci (e.g., to determine the result of an assay). The analysis at the loci in the array is not limited to the analysis of any particular type and includes, for example, an analysis of a change (e.g., intensity or wavelength) for detecting atoms, molecules, chemical reactions, light or fluorescence emission, suppressing or indicating the result at the loci. The example of a predefined locus includes a grid or any other pattern, wherein the loci to be analyzed are determined by their known positions in the array pattern. For example, in Schena, "Microarray Biochip Technology", Eaton Publishing, Natick, MA, 2000, microarrays are generally described. The example of an array includes, but is not limited to, a carrier with multiple molecules (e.g., with a grid or other regular pattern) non-randomly combined with a surface and a container including a plurality of defined reaction loci (e.g., holes) in which molecules or signal generation reactions can be detected. In some embodiments, the array comprises a patterned distribution of wells that receive beads, e.g., as described above for SIMOA technology. See also U.S. Pat. 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.

[0072] As used herein, the term "irregular distribution" used about a solid phase carrier or a site on the surface refers to the distribution of loci on the surface or in a non-array mode. For example, molecules can be irregularly distributed on the surface by: a solution of a specific concentration is applied, the application provides the approximate average distance of expectation between the molecules on the surface, but not by any pattern on the surface or by means of an application solution (inkjet printing) and predefined or addressable sites. In such embodiments, the analysis on the surface can include finding the loci of molecules (for example, scanning the entire surface to detect fluorescence anywhere on the surface) by detecting any place where a signal may occur. This is opposite to locating signals by analyzing surfaces or containers only at predetermined loci (for example, points in a grid array) to determine how many (or which type) signals occur at each locus in the grid.

[0073] As used herein, the term "distinguishing" 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 properties, charge, or by the ability to interact with another moiety (such as with a chemical reagent, enzyme, antibody, etc.).

[0074] As used herein, the term "nucleic acid detection assay" refers to any method of determining the nucleotide composition of a nucleic acid of interest.Nucleic acid detection assays include, but are not limited to, DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., the INVADER assay (Hologic, Inc.) and, for example, U.S. Pat. Nos. 5,846,717; 5,985,557; 5,994,069; 6,001,567; 6,090,543; and 6,872,816; Lyamichev et al., Nature Biotechnology 17:292 (1999); Hall et al., National Academy of Sciences 17:294 (1999); No. 9,096,893, each of which is incorporated herein by reference in its entirety for all purposes); enzymatic mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, incorporated herein by reference in their entirety); polymerase chain reaction (PCR), described above; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, Nos. 5,710,264, 5,124,246, 5,624,802, each of which is incorporated herein by reference in its entirety); rolling circle amplification (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960, 6,235,502, each of which is incorporated herein by reference in its entirety); a variation of rolling circle amplification, known as "RAM amplification" (see, e.g., U.S. Pat. No. 5,942,391, incorporated herein by reference in its entirety); NASBA (e.g., U.S. Pat. No. 5,409,818, incorporated herein by reference in its entirety); Incorporated herein); Molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, incorporated herein by reference in its entirety); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, incorporated herein by reference in their entirety); Cyclic probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, incorporated herein by reference in their entirety); Dade Behring signal amplification method (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, each of which is incorporated herein by reference in its entirety); ligase chain reaction (e.g., Baranay, Proc. Natl. Acad. Sci. USA 88, 189-93 (1991)); and sandwich hybridization method (e.g., U.S. Pat. No. 5,288,609, each of which is incorporated herein by reference in its entirety).

[0075] In some embodiments, the target nucleic acid is amplified (e.g., by PCR) and the amplified nucleic acid is detected simultaneously using an invasive cleavage assay. The assay configured to be combined with an amplification assay for detection assays (e.g., invasive cleavage assays) is described in U.S. Patent No. 9,096,893, which is incorporated herein by reference in its entirety for all purposes. Additional amplification plus invasive cleavage detection configuration (referred to as QuARTS method) is described in, for example, U.S. Patent No. 8,361,720; No. 8,715,937; No. 8,916,344; and No. 9,212,392, each of which is incorporated herein by reference for all purposes. As used herein, the term "invasive cleavage structure" refers to a cleavage structure comprising: i) a target nucleic acid; ii) an upstream nucleic acid (e.g., an invasive or "INVADER" oligonucleotide); and iii) a downstream nucleic acid (e.g., a probe), wherein the upstream and downstream nucleic acids are annealed to a continuous region of the target nucleic acid, and wherein an overlap is formed between the 3' portion of the upstream nucleic acid and the duplex formed between the downstream nucleic acid and the target nucleic acid. When one or more bases of the upstream and downstream nucleic acids occupy the same position relative to the target nucleic acid base, an overlap occurs regardless of whether the one or more overlapping bases of the upstream nucleic acid are complementary to the target nucleic acid, and regardless of whether the bases are natural bases or non-natural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps the downstream duplex is a non-basic chemical portion, such as an aromatic ring structure, such as disclosed in U.S. Pat. No. 6,090,543, which is incorporated herein by reference in its entirety. In some embodiments, one or more nucleic acids in the nucleic acid may be attached to each other, for example, by a covalent bond (e.g., a nucleic acid stem loop) or by a non-nucleic acid chemical bond (e.g., a polycarbon chain). As used herein, the term "flap endonuclease assay" encompasses the "INVADER" invasive cleavage assay and the QuARTS assay as described above.

[0076] 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 an amplification product or signal from a single starting molecule. Typically, the sample is divided, for example, by serial dilution or by partitioning into sufficiently small portions (e.g., in a microchamber or in an emulsion) so that each portion or dilution has no more than a single copy of the target nucleic acid on average as assessed according to a Poisson distribution. The method of single molecule PCR is described, for example, in the following patents: U.S. 6,143,496 (which relates to a method comprising: dividing a sample into a plurality of chambers such that at least one chamber has at least one target; and amplifying the target to determine how many chambers have target molecules); U.S. 6,391,559; (which relates to an assembly for containing and distributing fluids); and U.S. US 7,459,315 (which relates to a method of dividing a sample into an assembly having a sample chamber (wherein the sample is partitioned into the chamber by surface affinity), and then sealing the chamber with a curable "displacing fluid"). See also US 6,440,706 and US 6,753,147 and Vogelstein et al., Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 9236-9241, August 1999. See also US 20080254474, which describes the combination of digital PCR coupled with methylation detection.

[0077] As used herein, the term "sequencing" is used in a broad sense and can refer to any technique known in the art that allows identification of the order of at least some consecutive nucleotides in at least a portion of a nucleic acid (including but not limited to at least a portion of an extension product or a vector insert). In some embodiments, sequencing allows differentiation of sequence differences between different target sequences. Exemplary sequencing technologies include targeted sequencing, single molecule real-time sequencing, electron microscopy-based sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy terminator sequencing, targeted 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, massively parallel signature tag sequencing, emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired end sequencing, recent sequencing, exonuclease sequencing, sequencing by ligation, short read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reverse terminator sequencing, ion semiconductor sequencing, nanoball sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, miSeq (Illumina), HiSeq 2000 (Illumina), HiSeq 2500 (Illumina), Illumina Genome Analyzer (Illumina), Ion Torrent PGM TM (Life Technologies), MinION TM (Oxford Nanopore Technologies), Real-time SMRT TM technology (Pacific Biosciences), probe-anchor ligation (cPAL TM )(Complete Genomics / BGI), Sequencing, MS-PET sequencing, mass spectrometry and combinations thereof. In some embodiments, sequencing includes detecting sequencing products using an instrument such as, but not limited to, ABI 377 DNA sequencer, 310, 3100, 3100-Avant, 3730 or 373OxI Genetic Analyzer, ABI 3700 DNA Analyzer, or Applied Biosystems SOLiD TMSystem (all from Applied Biosystems), Genome Sequencer 20 system (Roche Applied Science), or mass spectrometer. In certain embodiments, sequencing comprises emulsion PCR. In certain embodiments, sequencing comprises high-throughput sequencing technology, such as but not limited to massively parallel signature tag sequencing (MPSS).

[0078] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably with respect to a chain of two or more amino acids linked together by peptide bonds. Polypeptides can be synthetic or naturally occurring, and can be short (e.g., between two about 30 amino acid residues) or their length can be hundreds or thousands of amino acid residues. Polypeptides can be composed of the 20 main naturally occurring amino acids, or can include one or more non-natural amino acids (e.g., peptide nucleic acid residues including pyrimidine or purine bases on the peptide chain backbone) or modified versions of natural amino acids (e.g., modified in the structure of the side groups).

[0079] As used herein, the term "antibody" (Ab) refers to an antigen-binding immunoglobulin and includes monoclonal antibodies (mAb) and polyclonal Abs. The term further includes all modified forms of antibodies that have the ability to bind to an antigen, such as fragment antibodies (fAb) that include a portion of the immunoglobulin structure.

[0080] As used herein, the terms "crowding agent" and "volume exclusion agent" used with respect to components involving fluid reaction mixtures are used interchangeably and refer to compounds (typically, polymeric compounds) that reduce the available fluid volume in the reaction mixture, thereby increasing the effective concentration of reactant macromolecules (e.g., nucleic acids, enzymes, etc.). Crowding agents include, for example, glycerol, ethylene glycol, polyethylene glycol, ficoll, serum albumin, casein, and dextran.

[0081] 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 an individual nucleic acid molecule. Systems for individual molecule sequencing include but are not limited to the 454FLX TM or 454TITANIUM TM (Roche), SOLEXA TM / Illumina Genome Analyzer (Illumina), HELISCOPE TM Single-molecule sequencer (Helicos Biosciences) and SOLID TMDNA sequencers (Life Technologies / Applied Biosystems instruments) and other platforms still under development by companies such as Intelligent Biosystems and Pacific Biosciences. See also U.S. Patent No. 7,888,017, entitled "Non-Invasive Fetal Genetic Screening by Digital Analysis," which relates to digital analysis of maternal and fetal DNA (e.g., cfDNA).

[0082] As used herein, the term "probe" or "hybridization probe" refers to an oligonucleotide (i.e., a nucleotide sequence) that is capable of hybridizing at least partially with another oligonucleotide of interest, whether naturally present in a purified restriction digest or produced synthetically, recombinantly, or by PCR amplification. The probe may be single-stranded or double-stranded. The probe may be used to detect, identify, and separate specific sequences. In some preferred embodiments, the probe used in the present invention will be labeled with a "reporter molecule" so that it can be detected in any detection system (including but not limited to enzyme systems (e.g., ELISA and enzyme-based histochemical assays), fluorescent systems, radioactive systems, and luminescent systems). It is not intended that the present invention be limited to any specific detection system or labeling.

[0083] As used herein, the term "MIP" refers to a molecular inversion probe (or circular capture probe). A molecular inversion probe (or circular capture probe) is a nucleic acid molecule comprising a pair of unique polynucleotide arms, one or more unique molecular tags (or unique molecular identifiers), and a polynucleotide linker (e.g., a universal backbone linker). See, e.g., Figure 1 . In some embodiments, the MIP may include more than one unique molecular tag, such as two unique molecular tags, three unique molecular tags or more unique molecular tags. In some embodiments, the unique polynucleotide arms in each MIP are located at the 5' and 3' ends of the MIP, and one or more unique molecular tags and polynucleotide linkers are located inside the 5' and 3' ends of the MIP. For example, the MIP used in some embodiments of the present disclosure includes the following components in sequence: 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, WO 2017 / 020023 filed on July 29, 2016 and WO 2017 / 020024 filed on July 29, 2016, each of which is incorporated herein by reference for all purposes.

[0084] Unique molecular tags can be any detectable tags and can be incorporated into nucleic acid (e.g., polynucleotide) or connected to the nucleic acid and allow detection and / or identification of nucleic acids including tags. In certain embodiments, tags are incorporated into nucleic acid or attached to the nucleic acid during sequencing (e.g., by polymerase). Non-limiting examples of tags include nucleic acid tags, nucleic acid indexes or barcodes, radioactive labels (e.g., isotopes), metal labels, fluorescent labels, chemiluminescent labels, phosphorescent labels, fluorophore quenchers, dyes, proteins (e.g., enzymes, antibodies or parts thereof, joints, members of binding pairs), etc. or combinations thereof. In certain embodiments, particularly in sequencing embodiments, tags (e.g., molecular tags) are unique, known and / or identifiable sequences of nucleotides or nucleotide analogs (e.g., including nucleic acid analogs, sugars and one to three phosphate groups). In certain embodiments, tags are six or more continuous nucleotides. Various excitation and emission spectra can obtain many tags based on fluorophores. Any suitable type and / or quantity of fluorophores can be used as tags. In certain embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 500 or more, 1000 or more, 10,000 or more, 100,000 or more different labels are used in the methods described herein (e.g., nucleic acid detection and / or sequencing methods). In certain embodiments, one or two types of labels (e.g., different fluorescent markers) are connected to each nucleic acid in the library. In certain embodiments, chromosome-specific labels are used to make chromosome counting faster or more effective. Detection and / or quantification of the marker can be performed by a suitable method, machine or equipment, non-limiting examples of which include flow cytometry, quantitative polymerase chain reaction (qPCR), gel electrophoresis, luminometer, fluorometer, spectrophotometer, suitable gene chip or microarray analysis, protein blotting, mass spectrometry, chromatography, cell fluorescence analysis, fluorescence microscopy, suitable fluorescence or digital imaging methods, confocal laser scanning microscopy, laser scanning cytometer, affinity chromatography, manual batch mode separation, electric field suspension, suitable nucleic acid sequencing methods and / or nucleic acid sequencing equipment, the like and combinations thereof.

[0085] In MIP, unique polynucleotide arms are designed to hybridize immediately upstream and downstream with specific target sequences (or sites) in genomic nucleic acid samples. In certain embodiments, MIP includes unique molecular tags, which are short nucleotide sequences generated randomly. In certain embodiments, unique molecular tags do not hybridize with any sequence or site located on the genomic nucleic acid fragment or in the genomic nucleic acid sample. In certain embodiments, the polynucleotide linkers (or main chain linkers) in MIP are universal in all MIPs used in the embodiments of the present disclosure.

[0086] In some embodiments, MIPs are introduced into nucleic acid fragments derived from a test subject (or a reference subject) to perform capture of a target sequence or site (or a control sequence or site) located on a nucleic acid sample (e.g., genomic DNA). In some embodiments, fragmentation helps capture target nucleic acids by molecular inversion probes. In some embodiments, for example, when a nucleic acid sample contains free nucleic acids, fragmentation may not be required to improve capture of target nucleic acids by molecular inversion probes. As described in more detail herein, after capturing a target sequence of interest (e.g., a locus), an enzymatic gap filling and ligation step may be performed on the captured target so that a copy of the target sequence is incorporated into a circular structure. In some embodiments, for example, nucleic acid analogs containing markers, haptens, etc. may be incorporated into the filled segments for use in, for example, downstream detection, purification, or other processing steps. In some embodiments, the capture efficiency of MIPs for target sequences on nucleic acid fragments can be improved by extending the hybridization and gap filling incubation period. (See, for example, Turner EH et al., Nat Methods. 2009 April 6: 1-2).

[0087] In some embodiments, the MIP for capturing target sites or target sequences according to the present disclosure comprises the following components in sequence: a first targeting polynucleotide arm-a first unique targeting molecular tag-a polynucleotide linker-a second unique targeting molecular tag-a second targeting polynucleotide arm.

[0088] In some embodiments, the MIP for capturing control sites or control sequences in the present disclosure comprises the following components in sequence: a first control polynucleotide arm-a first unique control molecule tag-a polynucleotide linker-a second unique control molecule tag-a second control polynucleotide arm.

[0089] MIP technology can be used to detect or amplify specific nucleic acid sequences in complex mixtures. One of the advantages of using MIP technology is its ability to be highly multiplexed, which allows thousands of target sequences to be captured in a single reaction containing thousands of MIPs. Various aspects of MIP technology are described, for example, in the following references: 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, 15: 269-275 (2005); Biology, 632:99-124 (2010); Sissung et al., “Clinic alpharmacology 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. , "Allele quantification using molecular inversion probes (MIP)", Nucleic Acids Research, 33(21): e183 (2005), each of which is incorporated herein by reference in its entirety for all purposes. See also U.S. Pat. Nos. 6,858,412; 5,817,921; 6,558,928; 7,320,860; 7,351,528; 5,866,337; 6,027,889 and 6,852,487, each of which is incorporated herein by reference in its entirety for all purposes. ;

[0090] MIP technology has previously been successfully applied to other research areas, including the novel identification and subclassification of biomarkers in cancer. See, e.g., Brewster et al., “Copy number imbalances between screen-and symptom-detected breast cancers and impact on disease-free survival,” Cancer Prevention Research, 4(10): 1609-1616 (2011); Geiersbach et al., “Unknown partner for USP6 and unusual SS18 rearrangement detected by fluorescence in situ hybridization in a solid aneurysmal bone cyst,” Cancer Genetics, 204(4): 195-202 (2011); Schiffman et al., “Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas.” CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas” Cancer Research, 70(2): 512-519 (2010); Schiffman et al., “Molecular inversion probes reveal patterns of 9p21 deletion and copy number aberrations in childhood leukemia” Cancer Genetics and Cytogenetics, 193(1): 9-18 (2009);Press et al., "Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities," BMC Cancer, 8:17 (2008); and Deeken et al., "A pharmacogenetic study of docetaxel and thalidomide in patients with castration-resistant prostate cancer using the DMET genotyping platform," Pharmacogenomics, 10(3):191-199 (2009), each of which is incorporated herein by reference in its entirety for all purposes.;

[0091] MIP technology has also been applied to identify new drug-related biomarkers. See, for example, 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. See, e.g., Mega et al., "Cytochrome P-450 Polymorphisms and Response to Clopidogrel," New England Journal of Medicine, 360(4):354-362 (2009); Dumaual et al., "Comprehensive assessment of metabolic enzyme and transporter genes using the Affymetrix Targeted Genotyping System," Pharmacogenomics, 8(3):293-305 (2007); and Daly et al., "Multiplex assay for comprehensive genotyping of genes involved in drug metabolism, excretion, and transport," Clinical Chemistry, 10(5):154-163 (2008). Chemistry, 53(7): 1222-1230 (2007), each of which is incorporated herein by reference in its entirety for all purposes.Other applications of MIP technology include genotypic and phenotype databases. See, for example, Man et al., "Genetic Variation in Metabolizing Enzyme and Transporter Genes: Comprehensive Assessment in 3 Major East Asian Subpopulations with Comparison to Caucasians and Africans," Journal of Clinical Pharmacology, 50(8): 929-940 (2010), which is incorporated herein by reference in its entirety for all purposes.

[0092] As used herein, the term "capture" or "capturing" refers to the binding or hybridization reaction between a molecular inversion probe and its corresponding targeting site. In some embodiments, when captured, a circular replicon or MIP replicon is generated or formed. In some embodiments, the targeting 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 with the naturally occurring (e.g., wild-type) genomic region of interest where the target deletion is expected to be located. The target MIP is designed not to bind to the genomic region showing a deletion. In these embodiments, it is expected that the binding or hybridization between the target MIP and the target deletion site does not occur. The absence of such binding or hybridization indicates the presence of a target deletion. In these embodiments, the phrase "capture target site" or the phrase "capture target sequence" refers to detecting the target deletion by detecting the absence of such binding or hybridization.

[0093] As used herein, the term "MIP replicon" or "circular replicon" refers to a circular nucleic acid molecule generated by a capture reaction (e.g., a binding or hybridization reaction between a MIP and its targeted sequence). In some embodiments, the MIP replicon is a single-stranded circular nucleic acid molecule. In some embodiments, the targeted MIP captures a target sequence or site or hybridizes with a target sequence or site. After the capture reaction or hybridization, a connection / extension mixture is introduced to extend and connect the empty region between the two targeted polynucleotide arms to form a single-stranded circular nucleotide molecule, i.e., the targeted MIP replicon. In some embodiments, the control MIP captures a control sequence or site or hybridizes with a control sequence or site. After the capture reaction or hybridization, a connection / extension mixture is introduced to extend and connect the empty region between the two control polynucleotide arms to form a single-stranded circular nucleotide molecule, i.e., the control MIP replicon. The MIP replicon can be amplified by polymerase chain reaction (PCR) to produce a plurality of targeted MIP amplicons, which are double-stranded nucleic acid molecules. The MIP replicon can be particularly applied to rolling circle amplification or RCA. RCA is an isothermal nucleic acid amplification technique in which a DNA polymerase continuously adds single nucleotides to a primer annealed to a circular template, which produces long concatemers of single-stranded DNA containing tens to hundreds of tandem repeats (complementary to the circular template). See, e.g., M. Ali et al. "Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine," Chemical Society Reviews. 43(10): 3324-3341, which is incorporated herein by reference in its entirety for all purposes. See also WO 2015 / 083002, which is incorporated herein by reference in its entirety for all purposes.

[0094] The polymerases commonly used for DNA amplification in RCA are Phi29, Bst and Vent exo-DNA polymerases, with Phi29 DNA polymerase being preferred due to its superior processivity and strand displacement ability.

[0095] As used herein, the term "amplicon" refers to a nucleic acid generated by an amplification reaction (e.g., a PCR reaction). In certain embodiments, the amplicon is a single-stranded nucleic acid molecule. In certain embodiments, the amplicon is a double-stranded nucleic acid molecule. In certain embodiments, the targeted MIP replicon is amplified using conventional techniques to produce a plurality of targeted MIP amplicons, and the plurality of targeted MIP amplicons are double-stranded nucleotide molecules. In certain embodiments, the control MIP replicon is amplified using conventional techniques to produce a plurality of control MIP amplicons, and the plurality of control MIP amplicons are double-stranded nucleotide molecules.

[0096] The term "probe oligonucleotide" or "flap oligonucleotide" when used with respect to a flap assay (eg, the INVADER invasive cleavage assay) refers to an oligonucleotide that interacts with a target nucleic acid in the presence of an invasive oligonucleotide to form a cleavage structure.

[0097] The term "invasive oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid at a position adjacent to the hybridization region between the probe and the target nucleic acid, wherein the 3' end of the invasive oligonucleotide includes a portion (e.g., a chemical moiety or one or more nucleotides) that overlaps the hybridization region between the probe and the target. The 3' terminal nucleotide of the invasive oligonucleotide may or may not base pair with a nucleotide in the target. In some embodiments, the invasive oligonucleotide contains a sequence at its 3' end that is substantially identical to the sequence at the 5' end of the portion of the probe oligonucleotide that anneals to the target strand.

[0098] As used herein, "flap endonuclease" or "FEN" refers to a class of nucleolytic enzymes that act as structure-specific endonucleases on DNA structures, typically 5' end nucleases, where the duplex contains a single-stranded 5' overhang or flap on one strand of the strand that is displaced by the other strand of the nucleic acid (e.g., such that there are overlapping nucleotides at the junction between the single-stranded DNA and the double-stranded DNA). FEN catalyzes the hydrolytic cleavage of the phosphodiester bond at the junction of the single-stranded DNA and the double-stranded DNA, thereby releasing the overhang or flap. Flap endonucleases are reviewed by Ceska and Savers (Trends Biochem. Sci. 1998 23:331-336) and Liu et al. (Ann. Rev. Biochem. 2004 73:589-615; incorporated herein by reference in their entirety). FEN can be a separate enzyme, a multi-subunit enzyme, or can exist as an activity of another enzyme or protein complex (e.g., DNA polymerase).

[0099] The flap endonuclease can be thermostable. For example, the FEN-1 flap endonuclease from the archival thermophilic organism is a typical thermostable. As used herein, the term "FEN-1" refers to a non-polymerase flap endonuclease from a eukaryotic cell or an archaeal organism. See, for example, WO 02 / 070755 and Kaiser MW et al., (1999) J. Biol. Chem., 274: 21387, which are incorporated herein by reference in their entirety for all purposes.

[0100] As used herein, the term "cleaved flap" refers to a single stranded oligonucleotide that is the cleavage product of a flap assay.

[0101] The term "cassette" when used in relation to a flap cleavage reaction refers to an oligonucleotide or combination of oligonucleotides that is configured to generate a detectable signal in response to cleavage of a flap or probe oligonucleotide in a primary or first cleavage structure formed, for example, in a flap cleavage assay. In a preferred embodiment, the cassette hybridizes with a non-target cleavage product produced 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 (e.g., FEN-1 endonuclease).

[0102] In some embodiments, the box is a single oligonucleotide comprising a hairpin portion (i.e., a region in which one portion of the box oligonucleotide hybridizes with the second portion of the same oligonucleotide under reaction conditions to form a duplex). In other embodiments, the box comprises at least two oligonucleotides, and the at least two oligonucleotides comprise complementary portions that can form a duplex under reaction conditions. In a preferred embodiment, the box comprises a label, such as a fluorophore. In a particularly preferred embodiment, the box comprises a labeled portion that produces a FRET effect. In such embodiments, the box may be referred to as a "FRET box". See, for example, U.S. 9,096,893, issued on 08 / 04 / 15, which is incorporated herein by reference in its entirety for all purposes.

[0103] As used herein, the phrase "substantially non-complementary" when used with respect to a probe flap or arm means that the flap portion has sufficient non-complementarity to selectively not hybridize to a nucleic acid sequence (e.g., a target nucleic acid or amplified DNA) under specified annealing conditions or stringent conditions, which encompasses the terms "substantially non-complementary" and "completely non-complementary."

[0104] As used herein, the term "signal" refers to any detectable effect such as would be caused or provided by a label or by the action or accumulation of a component or product in an assay reaction.

[0105] As used herein, the term "detector" refers to a system or component of a system that can communicate the presence of a signal or effect to a user or another component of the system (e.g., a computer or controller), such as an instrument (e.g., a camera, a fluorometer, a charge-coupled device, a scintillation counter, a solid-state nanopore device, etc.) or a reactive medium (x-ray or camera film, a pH indicator, etc.). The detector is not limited to the specific type of signal detected and can be: a photometric system or a spectrophotometric system (which can detect ultraviolet, visible, or infrared light, including fluorescence or chemiluminescence); a radiation detection system; a charge detection system; a system for detecting electronic signals (e.g., current or charge disturbances); a spectroscopic system, such as nuclear magnetic resonance spectroscopy, mass spectrometry, or surface enhanced Raman spectroscopy; a system such as gel or capillary electrophoresis or gel exclusion chromatography; or other detection systems or combinations thereof known in the art.

[0106] 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. Detection assays are assays that produce a detectable signal or effect when performed in the presence of a target analyte and include, but are not limited to, assays combined with processes of hybridization, nucleic acid cleavage (e.g., exonucleases or endonucleases), nucleic acid amplification, nucleotide sequencing, primer extension, nucleic acid ligation, antigen-antibody binding, interaction of a primary antibody with a secondary antibody, and / or conformational changes in nucleic acids (e.g., oligonucleotides) or polypeptides (e.g., proteins or small peptides).

[0107] 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 associated with pregnancy or directly or indirectly caused by pregnancy. These diseases or conditions may include any and all birth defects, congenital conditions or hereditary diseases or conditions. Examples of prenatal or pregnancy-related diseases include, but are not limited to, rhesus disease, hemolytic disease of the newborn, β-thalassemia, sex determination, pregnancy determination, hereditary Mendelian genetic disorders, chromosomal aberrations, fetal chromosomal aneuploidy, fetal chromosomal trisomy, fetal chromosomal monosomy, 8 trisomy, 13 trisomy (Patau syndrome), 16 trisomy, 18 trisomy (Edwards syndrome), 21 trisomy (Down syndrome), X chromosome linkage disorders, X trisomy (XXX syndrome), X monosomy (Turner syndrome (Turner syndrome) 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, cry-a-cat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, vulnerable compression neuropathy, Smith-Magenis syndrome 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, Pelmer-Merzheimer disease, testicular determining factor on the Y, azoospermia (factor a), azoospermia (factor b), azoospermia (factor c), 1p36 deletion, phenylketonuria, Tay-Sachs diseasedisease), adrenal hyperplasia, Fanconi anemia, spinal muscular atrophy, Duchenne muscular dystrophy, Huntington's disease, myotonic dystrophy, Robertsonian translocation, Angelman syndrome, tuberous sclerosis, ataxia-telangiectasia, open spina bifida, neural tube defects, abdominal wall defects, small for gestational age, congenital cytomegalovirus, achondroplasia, Marfan's syndrome syndrome), congenital hypothyroidism, congenital toxoplasmosis, biotinidase deficiency, galactosemia, maple syrup urine disease, homocystinuria, medium-chain acyl Co-A dehydrogenase deficiency, structural birth defects, heart defects, limb anomalies, ectopod, anencephaly, arrhythroencephaly / holoprosencephaly, hydrocephalus, anophthalmia / microphthalmia, athymia / 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 / stenosis with or without fistula, intestinal atresia / stenosis, anorectal atresia / stenosis, hypospadias, indeterminate sex, renal dysgenesis, cystic kidney, preaxial polydactyly, short limb defects, diaphragmatic hernia, blindness, cataracts, visual problems, hearing loss, deafness, X-linked adrenoleukodystrophy, Rett syndrome syndrome), lysosomal disorders, cerebral palsy, autism, anglotonsia, albinism, ocular albinism, oculocutaneous albinism, gestational diabetes, Arnold-Chiari malformation, CHARGE syndrome, congenital diaphragmatic hernia, brachydactyly, aniridia, cleft hands and feet, heterochromia, Dwarnian ear, Ehlers Danlossyndrome, epidermolysis bullosa, Gorham's disease, Hashimoto's syndrome, hydrops fetalis, hypotonia, Klippel-Feil syndrome, muscular dystrophy, osteogenesis imperfecta, progeria, Smith-Lemli-Opitz syndrome symdrom), color blindness, X-linked lymphoproliferative disorder, omphalocele, gastroschisis, preeclampsia, eclampsia, preterm birth, preterm birth, miscarriage, intrauterine growth retardation, ectopic pregnancy, hyperemesis gravidarum, morning sickness, or possible successful induction of labor.

[0108] In some NIPT embodiments, the present technology described herein further comprises estimating a fetal fraction of a sample, wherein the fetal fraction is used to help determine whether genetic data from a test subject indicates aneuploidy. Methods for determining or calculating fetal fraction are known in the art.

[0109] As used herein, the term "valid detection assay" refers to a detection assay that has been shown to accurately predict the association between detection of a target and a phenotype (e.g., a medical condition). Examples of valid detection assays include, but are not limited to, detection assays that accurately predict a medical phenotype 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% of the time when the target is detected. Other examples of valid detection assays include, but are not limited to, detection assays that are suitable and / or marketed as analyte-specific reagents (i.e., as defined by FDA regulations) or in vitro diagnostics (i.e., approved by the FDA).

[0110] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of reaction determination, such delivery systems include systems that allow reaction reagents (e.g., oligonucleotides, enzymes, etc. in suitable containers) and / or support materials (e.g., buffers, written instructions for determination, etc.) to be stored, transported, or delivered from one location to another. For example, the kit includes one or more housings (e.g., boxes) containing relevant reaction reagents and / or support materials. As used herein, the term "fragmentation kit" refers to a delivery system comprising two or more separate containers of sub-parts each containing all kit components. The container can be delivered to the intended recipient together or individually. For example, the first container can contain an enzyme for use in determination, and the second container contains oligonucleotides. The term "fragmentation kit" is intended to encompass a kit containing an analyte-specific reagent (ASR) regulated by Article 520 (e) of the Federal Food, Drug, and Cosmetic Act, but not limited thereto. In fact, any delivery system comprising two or more separate containers of sub-parts each containing all kit components is included in the term "fragmentation kit". In contrast, a "combination kit" refers to a delivery system that contains all components of a reaction assay in a single container (eg, in a single box containing each of the desired components). The term "kit" encompasses both fragmentation kits and combination kits.

[0111] As used herein, the term "information" refers to any collection of facts or data. With respect to information stored or processed using one or more computer systems (including but not limited to the Internet), the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term "information related to a subject" refers to facts or data related to a subject (e.g., people, plants, or animals). 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 allele identity, the statistical correlation between the presence of alleles and the characteristics of a subject (e.g., human subjects), the presence or absence of alleles in individuals or groups, the percentage probability of alleles being present in individuals with one or more specific characteristics, etc.

[0112] As used herein, the term "assay validation information" refers to genomic information and / or allele frequency information generated by processing (e.g., processing by means of a computer) 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

[0113] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0114] Figure 1 A schematic diagram of a molecular inversion probe (MIP) for chromosome-specific recognition suitable for use in large-scale multiplex capture assays is provided.

[0115] Figure 2 A schematic diagram of an example of multiplex chromosome-specific rolling circle amplification is provided.

[0116] Figure 3 A schematic diagram of an example of multiplex chromosome-specific rolling circle amplification using molecular beacon probes for detection is provided.

[0117] Figure 4 Provided are methods including the use of a single-stranded ligase (e.g., CircLigase TM Schematic diagram of an embodiment of the present technology in which a thermostable RNA ligase) circularizes cfDNA to form a "native circle" for detection.

[0118] Figure 5Schematic diagrams of embodiments of the present technology including circularizing cfDNA and adding segments for detection using "Golden Gate Assembly" are provided (see, e.g., Engler, C., Kandzia, R. and Marillonnet, S. (2008) PLoS One 3, e3647).

[0119] Figure 6 Schematic diagrams are provided of embodiments of the present technology including circularization of cfDNA using extended ligation on a unique molecular inversion inducing template, wherein detection is performed using embodiments of RCA.

[0120] Figure 7 Schematic diagrams of embodiments of the present technology comprising unique molecular inversion induced templates that are extended and ligated to generate circular DNA molecules, wherein detection is performed using embodiments of RCA.

[0121] Figure 8 Schematic diagrams of embodiments of the present technology including synthetic circular DNA used as a template, for example, for rolling circle amplification, including a binding site for probe binding and a primer binding site for replication are provided.

[0122] Fig. 9 Schematic diagrams are provided of embodiments of the present technology involving the use of probe pairs configured for collisional quenching when hybridizing to strands of DNA for detecting products from RCA.

[0123] Fig.10 Schematic diagrams of embodiments of the present technology involving the use of probe pairs configured for fluorescence resonance energy transfer (FRET) when hybridized to strands of DNA for detecting products from RCA are provided.

[0124] Fig.11 Schematic diagrams are provided of embodiments of the present technology comprising the use of a probe configured to cleave upon hybridisation to a strand of DNA, for example using a duplex-specific nuclease such as a restriction enzyme, comprising a dye and a quencher for detecting products from RCA.

[0125] Fig.12 A schematic diagram of an embodiment of the present technology comprising RCA using CID followed by CID-specific digestion and CID-specific labeling is provided.

[0126] Fig.13An example is shown in which a MIP hybridizes to a target nucleic acid, such as cfDNA, leaving a single nucleotide vacancy. The vacancy is filled by extension to incorporate a biotinylated nucleotide and closed by ligation. The circularized MIP can then be bound to a streptavidin-coated surface.

[0127] Fig.14 A schematic diagram showing the initiator oligonucleotide hybridized to a MIP immobilized on a surface.

[0128] Fig.15 Schematic diagram showing hairpin oligonucleotides working together in the presence of an initiator oligonucleotide to form a self-assembling scaffold.

[0129] Fig.16 Self-assembled scaffolds including multiple labels such as fluorescent dyes are demonstrated.

[0130] Fig.17 Schematic diagrams of invasive cutting structures according to embodiments of the present technology are provided.

[0131] Fig.18 Provided are methods for forming a flap endonuclease assay (e.g., Schematic representation of the hairpin probe used in the invasive cleavage structure of the assay).

[0132] Fig.19 A graphical representation of the accumulation of cleaved flap fragments in a flap endonuclease assay is provided.

[0133] Fig. 20 Examples are shown in which the cleaved biotinylated flap is captured using an immobilized complementary probe and the biotin is reacted with streptavidin linked to an enzyme (eg, β-galactosidase).

[0134] Fig.21 Embodiments of the present technology are demonstrated in which MIPs designed to target different chromosomes each require a different nucleotide for extension and ligation and in which the MIPs are extended and ligated in a chromosome-specific manner using nucleotides carrying a different dye or hapten for each different dNTP.

[0135] Fig. 22 Figures A, B and C of illustrate embodiments of the technology of the present invention in which a MIP contains or is modified to contain an immobilization moiety or is hybridized to an oligonucleotide containing an immobilization moiety and immobilized on a surface.

[0136] Fig.23 A schematic diagram of the rolling circle amplification reaction is provided.

[0137] Fig.24A-24D provides a graph showing the results from examining the effect of including biotin residues in the MIP complex on the RCA signal.

[0138] Figures 25A-25C Graphs showing the results of varying the amounts of components in a standard RCA reaction in solution are provided.

[0139] Fig.26 Graphs comparing the effects on signal accumulation using different molecular weights of PEG at the indicated percentages (w:v) are provided.

[0140] Figures 27A-27B and Fig.28 A-28B shows the results obtained in an RCA reaction using primers bound to a glass surface in a randomly dispersed manner, wherein detection was performed using a molecular beacon probe including a quencher and a fluorophore.

[0141] Fig.27A Microscope images of the surface of an APTES silanized plate as described in Example 1 are shown and the RCA signal with and without PEG is compared.

[0142] Fig.27B Provided is a diagram showing the effect of PEG on Fig.27A The effects of the number of spots and fluorescence intensity are shown in the figure.

[0143] Fig.28 Graphs showing the effect of different molecular weights of PEG in 20% solution on the number and fluorescence intensity of spots on APTES silanized plates as described in Example 1 are provided.

[0144] Fig.29A Microscope images of the surface of an APTES silanized plate as described in Example 1 are shown and the RCA signal is compared for reactions hybridized for either 18 hours or 1 hour prior to initiating the RCA reaction.

[0145] Fig.29B Provides comparison of hybridization time and buffer pairs Fig.29A The effect of the number of spots and fluorescence intensity (area) is shown in the graph.

[0146] Fig.30 Graphs comparing the effect of PEG 200 with or without a 2 hour hybridization time for standard RCA reaction conditions and the effect of PEG 2000 with a 2 hour hybridization on the number and fluorescence intensity (area) of spots are provided.

[0147] Fig.31Graphs comparing the effect of PEG 200 with or without a 2 hour hybridization time on standard RCA reaction conditions at 25°C and the effect of PEG 2000 on the number and fluorescence intensity (area) of spots with 2 hours of hybridization are provided.

[0148] Fig.32 Graphs comparing the effect of PEG 200 with or without a 2 hour hybridization time on standard RCA reaction conditions at 37°C and the effect of PEG 2000 on the number and fluorescence intensity (area) of spots with a 2 hour hybridization are provided.

[0149] Fig.33 Microscope images of the surface of an APTES silanized plate as described in Example 1 are shown and RCA signals are compared for reactions including the indicated concentrations of PEG 600 performed at 37°C or 45°C.

[0150] Fig.34 Schematic representation of RCA molecular beacon production on a surface with or without graphene oxide is provided, where graphene oxide quenches the fluorescence background from beacons nonspecifically bound to the surface.

[0151] Fig.35 A schematic diagram of a two-step RCA reaction as described in Example 1 is provided, in which a rolling circle reaction is initiated, a molecular beacon and graphene oxide are added, and the RCA reaction is further incubated.

[0152] Fig.36 A microscope image of the surface of an APTES silanized plate as described in Example 1 is shown and the RCA signal of the two-step reaction graphene oxide is shown.

[0153] Fig.37 Graphs comparing spot counts for RCA reactions performed in one step (without GO) or two steps (with or without GO) comparing reactions with 100 fmol of target to reactions without target are provided.

[0154] Fig.38 Schematic diagrams of different capture complexes for applying embodiments of the present technology to detect different types of target molecules are provided.

[0155] Fig.39 A schematic diagram of applying the technology of the present invention to the detection of immobilized antigens is provided.

[0156] Fig.40 A schematic diagram of applying the technology of the present invention to the detection of immobilized antigen-antibody complexes is provided. DETAILED DESCRIPTION

[0157] The goal of molecular diagnosis is to realize accurate and sensitive detection of analyte with the least possible workload and step in the least possible time.A way to realize this is to carry out multiple detection to the analyte in the sample, thereby allowing multiple detection events to be carried out in a single reaction vessel or solution.However, many existing diagnostic methods (comprising multiple reactions) still require many steps, comprising sample preparation steps that increase the time, complexity and cost of reacting.In certain embodiments, the present invention provides solutions to these problems by providing a determination that can be carried out directly in an unpurified or untreated biological sample (for example, blood or blood plasma).

[0158] In some embodiments, the technology of the present invention provided herein provides an economical method for testing samples in a manner that the number of copies of a specific nucleic acid or protein in a sample or sample portion is counted digitally (i.e., by detecting a separate copy of the molecule) without using a sequencing step (e.g., a digital or "next generation" sequencing step). The technology of the present invention can be used to measure target molecules (such as nucleic acid molecules) in any type of sample (including but not limited to, for example, samples collected from subjects for diagnostic screening). The embodiments of the technology of the present invention provided herein can be used, for example, for non-invasive prenatal testing (NIPT) and other genetic analysis. The embodiments of the technology of the present invention implement one or more steps of nucleic acid extraction, MIP probe design, MIP amplification / replication and / or a method for measuring signals from circularized MIPs. In a preferred embodiment, the technology of the present invention provides a method for fixing MIPs on a surface and detecting immobilized MIPs. In a preferred embodiment, rolling circle amplification is used to detect immobilized MIPs.

[0159] In preferred embodiments, the methods of the present technology include target recognition events, typically including hybridization of a target nucleic acid (e.g., a sample of patient DNA) with another nucleic acid molecule (e.g., a synthetic probe). In preferred embodiments, the target recognition event creates conditions that produce unique products (e.g., probe oligonucleotides that have been extended, connected and / or cut), which then indicate the presence of the target in the reaction and the hybridization of the probe thereto.

[0160] Many different "front-end" methods for identifying target nucleic acids and generating new products are described herein. For example, as shown in the exemplary embodiments in the figure, the present technology provides a variety of ways to generate circularized molecules for "back-end" detection / readout steps (see, e.g., Figure 1-3 , Figure 13-18 , Fig.34 , Fig.35 and Figure 38-40The present technology also provides methods for using other probe types (such as probes that can be cleaved by flap endonucleases in the presence of target nucleic acids) to signal the presence of target nucleic acids (see, e.g., Figure 17-19 ). Each of these front end embodiments can be used to generate unique molecules, such as circular or cleaved oligonucleotides.

[0161] These unique molecules can be configured to have one or more features useful for capture and / or identification in downstream back-end detection steps. Examples of molecules and features generated in the front-end reaction include circularized MIPs with attached sequence (e.g., the complete target-specific sequence formed by ligation of the 3' and 5' ends of the probe), with added sequence (e.g., a copied portion of the target template), and / or labeled nucleotides (e.g., nucleotides attached to biotin, dyes, quenchers, haptens, and / or other moieties), or products released from flap cleavage reactions (e.g., single-stranded arms) (see, e.g., Figure 17-19 ). In some embodiments, the MIP comprises a feature in a portion of the probe (eg, in the backbone of the probe).

[0162] For example, in Figure 2-3 , Figure 6-7 , Figure 9-12 , Figure 15-16 , Figure 20-21 , Fig.34 , Fig.35 and Figure 38-40 Examples of back-end analysis methods for amplifying and / or detecting unique products of the front-end are provided in.

[0163] Although the present technology has been discussed with reference to specific embodiments (e.g., combinations of certain front-end target-dependent reactions with specific back-end signal amplification methods and detection platforms (e.g., Figure 13-16 The biotin-incorporated MIP is coupled to the back end of the non-enzymatic hybridization chain reaction); the cleaved flap with a biotin tag (such as Fig.19 ) is coupled to the capture surface and then catalytically hybridized with an enzyme-linked probe that generates a fluorescent signal (e.g. Fig. 20 ), but the invention is not limited to the specific combination of front-end methods and configurations and back-end methods and configurations disclosed herein or any specific method of detecting signals from assay products. It should be understood that a skilled person can easily adapt a front end to work with an alternative back end. For example, Figure 2-3 , Figure 8-7 , Figure 9-12 , Fig.21 , Fig.34 , Fig.35 and Figure 38-40 as exemplified in Fig.14 The cyclized MIP can be used Fig. 20 The PCR products can be captured and detected using enzyme-linked probes or can alternatively be amplified in a rolling circle amplification assay. Figure 19-20 As described in Fig.19 The cleaved flap shown in can be detected using hybridization chain reaction; and the circularized MIP or RCA amplicons can be detected using Fig.17 The invasive cleavage reaction shown in the figure is used for detection, etc.

[0164] Further, although the present technology is discussed with reference to specific target nucleic acids (e.g., free DNA in plasma), the present invention is not limited to any specific form of DNA or any specific type of nucleic acid or any specific type of nucleic acid variation. It should be understood that a skilled person can easily configure embodiments of the present technology to detect and count epigenetic variations of mutations, insertions, deletions, single nucleotide polymorphisms (SNPs), and methylation (e.g., by analyzing methylation variations of specific CpG dinucleotides produced by treating DNA with an agent that converts unmethylated cytosine to uracil, thereby producing detectable sequence variations that reflect cytosine methylation variations in the target DNA).

[0165] In some embodiments, the assays are performed in a multiplexed fashion. In some embodiments, the multiplexed assays can be performed under conditions that allow different loci to reach more similar levels of amplification.

[0166] Figure 1 A schematic diagram of a molecular inversion probe (MIP) is provided. The molecular inversion probe contains first and second targeting polynucleotide arms complementary to adjacent or proximal regions on a target nucleic acid to be detected, wherein a polynucleotide linker or "backbone" connects the two arms (see Figure 1 ).

[0167] In the presence of a complementary target nucleic acid, the MIP can be circularized to form a MIP replicon suitable for detection. In some embodiments, the MIP is simply ligated using a nick repair enzyme (e.g., T4 DNA ligase), while in some embodiments, sealing the probe to form a circle includes: additionally modifying the probe to produce a ligatable nick (e.g., an overlapping cut between the ends), filling the gap between the ends using a nucleic acid polymerase, etc.

[0168] As used herein, target site or sequence refers to the part or region of the nucleic acid sequence sorted out from other nucleic acids in the sample with other sequences, which is important for determining the presence or absence of a gene disorder or condition (e.g., mutation, polymorphism, deletion, insertion, aneuploidy, etc.). As used herein, control site or sequence refers to a site with a known or normal copy number of a specific control gene. In certain embodiments, the targeting MIP includes the following components in sequence: the first targeting polynucleotide arm-the first unique targeting molecule tag-polynucleotide linker-the second unique targeting molecule tag-the second targeting polynucleotide arm. In certain embodiments, the target population of the targeting MIP is used in the method disclosed herein. In the target population, several pairs of the first and second targeting polynucleotide arms in each targeting MIP in the targeting MIP are identical and substantially complementary to the first and second regions of the respective flanking target sites in the nucleic acid. See, for example, WO2017 / 020023 and WO 2017 / 020024, each of which is incorporated herein by reference in its entirety.

[0169] In some embodiments, each of the targeting polynucleotide arms is between 18 and 35 base pairs in length. In some embodiments, each of the targeting polynucleotide arms is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 base pairs in length or any size ranging between 18 and 35 base pairs. In some embodiments, each of the control polynucleotide arms is between 18 and 35 base pairs in length. In some embodiments, each of the control polynucleotide arms is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 base pairs in length or any size ranging between 18 and 35 base pairs. In some embodiments, each of the targeting polynucleotide arms has a melting temperature between 57° C. and 63° C. In some embodiments, each of the targeting polynucleotide arms has a melting temperature between 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., or 63° C. or any size ranging between 57° C. and 63° C. In some embodiments, each of the control polynucleotide arms has a melting temperature between 57° C. and 63° C. In some embodiments, the melting temperature of each of the control polynucleotide arms is 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, or 63°C or any size ranging between 57°C and 63°C. In some embodiments, the GC content of each of the targeting polynucleotide arms is between 30% and 70%. In some embodiments, the GC content of each of the targeting polynucleotide arms is between 30% and 70%. In some embodiments, the GC content of each of the targeting polynucleotide arms is between 30% and 40%, or 30-50%, or 30-60%, or 40-50%, or 40-60%, or 40-70%, or 50-60%, or 50-70%, or any size ranging between 30% and 70%, or any specific percentage between 30% and 70%. In some embodiments, the GC content of each of the control polynucleotide arms is between 30% and 70%. In some embodiments, the GC content of each of the control polynucleotide arms is 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 range between 30% and 70% or any specific percentage between 30% and 70%.

[0170] In some embodiments, the polynucleotide joint is substantially non-complementary to any genomic region of a sample or subject. In some embodiments, the length of the polynucleotide joint is between 30 and 40 base pairs. In some embodiments, the length of the polynucleotide joint is 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 base pairs or any interval between 30 and 40 base pairs. In some embodiments, the melting temperature of the polynucleotide joint is between 60°C and 80°C. In some embodiments, the melting temperature of the polynucleotide joint is 60°C, 65°C, 70°C, 75°C or 80°C or any interval between 60°C and 80°C or any specific temperature between 60°C and 80°C. In some embodiments, the GC content of the polynucleotide joint is between 40% and 60%. In some embodiments, the GC content of the polynucleotide adaptor is 40%, 45%, 50%, 55%, or 60%, or any interval between 40% and 60%, or any specific percentage between 40% and 60%.

[0171] In some embodiments, the targeted MIP replicon is generated by: i) first and second targeting polynucleotide arms hybridizing to first and second regions in a nucleic acid that flank the target site, respectively; and ii) after hybridization, extending and ligating the gap region between the two targeting polynucleotide arms using a ligation / extension mixture to form a single-stranded circular nucleic acid molecule.

[0172] In certain embodiments, the methods described herein are used to detect the deletion or insertion or duplication of an exon. In certain embodiments, the target site (or sequence) is a deletion or insertion or duplication in a gene of interest or a genomic region of interest. In certain embodiments, the target site is a deletion or insertion or duplication in one or more exons of a gene of interest. In certain embodiments, a plurality of exons of targeting are continuous. In certain embodiments, a plurality of exons of targeting are non-continuous. In certain embodiments, the first and second targeting polynucleotide arms of MIP are designed to hybridize upstream and downstream with a deletion (or insertion or duplication) in a gene or a genomic region (e.g., one or more exons) of a deletion or duplication or a genomic region of interest. In certain embodiments, the first or second targeting polynucleotide arms of MIP include sequences that are substantially complementary to the genomic region of a gene of interest that encompass a target deletion or duplication site (e.g., an exon or a portion of an exon).

[0173] 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 is hybridized to a circular nucleic acid molecule (e.g., ligated MIPs) or circularized cfDNA. A strand-displacing DNA polymerase (e.g., E. coli Pol I DNA polymerase) is used to amplify the cfDNA. Extension primers (Phi29, Bst large fragment, and Klenow fragment) will generate long single-stranded DNA molecules containing repeat sequences of nucleic acid sequences complementary to the MIP circular molecules.

[0174] In some embodiments, a ligation-mediated rolling circle amplification (LM-RCA) involving a ligation operation before replication is used. In the ligation operation, the probe is hybridized with its complementary target nucleic acid sequence (if present) and the end of the hybridized probe is connected by connection to form a covalently closed single-stranded nucleic acid. After connection, the rolling circle replication primer hybridizes with the probe molecule to start rolling circle replication as described above. Generally, LM-RCA includes: mixing a hollow probe with a target sample to produce a probe-target sample mixture, and incubating the probe-target sample mixture under conditions that promote hybridization between the hollow probe and the target sequence; mixing a ligase with the probe-target sample mixture to produce a connection mixture, and incubating the connection mixture under conditions that promote hollow probe connection to form an amplification target ring (ATC, which is also referred to as RCA replicator). A rolling circle replication primer (RCRP) is mixed with the connection mixture to produce a primer-ATC mixture, and the primer-ATC mixture is incubated under conditions that promote hybridization between the amplification target ring and the rolling circle replication primer. A DNA polymerase is mixed with the primer-ATC mixture to produce a polymerase-ATC mixture, and the polymerase-ATC mixture is incubated under conditions that promote replication of the amplified target circle, wherein replication of the amplified target circle results in the formation of tandem sequence DNA (TS-DNA) (i.e., a long chain of single-stranded DNA containing concatemers of sequences complementary to the amplified target circle).

[0175] exist Figure 2In the embodiment shown in , the circularized molecules A, B, C and D are composed of MIPs specific to chromosomes 13, 8, 21 or a reference chromosome (such as Chr. 1). The sequence of the MIP surrounds the vacant complementary region of the targeted chromosome, and the backbone of the MIP contains a unique sequence for hybridizing a probe containing a specific fluorescent dye (FITC, ALEXA, Dylight, Cyan, Rhodamine dye, quantum dots, etc.). Step 1 includes hybridizing the MIP with cfDNA, extending a single base pair (or longer extension) and connecting to circularize the extended MIP. Step 2 includes rolling circle amplification of the circularized MIP so that the sequence required for hybridization with the fluorescently labeled oligonucleotide is amplified. A*, B*, C*, D* are complements of the MIP sequence. Step 3 includes hybridizing the fluorescently labeled probe with the rolling circle product. In Figure 3 In the examples presented in , the detection of the RCA products is facilitated by molecular probes rather than fluorescent dye-labeled oligonucleotides.

[0176] There are a variety of ways to immobilize the MIP to a surface (e.g., a bead or glass surface). For example, this can be accomplished by priming a rolling circle amplification with a modified oligonucleotide that includes a bindable moiety. Groups used to modify the priming oligonucleotide include, but are not limited to, thiols, amino groups, azides, alkynes, and biotin, such that the modified oligonucleotide can be immobilized using appropriate reactions as outlined in, for example, Meyer et al., "Advances in DNA-mediated immobilization," Current Opinions in Chemical Biology 18:8:8-15 (2014), which is incorporated herein by reference in its entirety for all purposes.

[0177] Imaging of fluorescent dye-incorporated MIPs can be accomplished using methods that include immobilizing the MIP to a surface (slide or bead) (e.g., using a method that modifies the MIP backbone to contain modified bases that can be immobilized using appropriate reactions as outlined above and in Meyer et al. (supra) and detected using antibodies). Once immobilized to the surface, antibodies directed against the incorporated tag can be used to form antibody-MIP complexes that can be imaged using a microscope. In some embodiments, the antibody can be conjugated to enhance or amplify the detectable signal from the complex. For example, conjugation of β-galactosidase to an antibody allows detection in a single molecule array ("SIMOA") using a process described by Quanterix, wherein each complex is immobilized on a bead so that any bead has no more than one labeled immune complex, and the beads are distributed to an array of femtoliter-sized wells so that each well contains at most one bead. The β-galactosidase on the immobilized immune complex catalyzes the production of fluorescent resorufin by the addition of resorufin β-galactopyranoside. After visualization, the fluorescence emitted in the wells with the immobilized individual immune complexes can be detected and counted. See, e.g., Quanterix Whitepaper 1.0, Scientific Principle of Simoa (Single Molecule Array) Technology, 1-2 (2013); and Quanterix Whitepaper 6.0, Simoa for Ultrasensitive Multiplex Immunoassays of Protein Biomarkers. TM Practical Application of Simoa TM 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 can be directly detected using, for example, a solid-state nanopore with antibodies labeled with poly(ethylene glycol) of various molecular weights as described in Morin et al., "Nanopore-Based Target Sequence Detection," Public Library of Science: Comprehensive, DOI: 10.1371 / journal.pone.0154426 (2016), which is incorporated herein by reference.

[0178] Figure 4Provided are methods including the use of a single-stranded ligase (e.g., CircLigase TM Schematic diagram of an embodiment of the present technology in which a thermostable RNA ligase (RT-RNA) cyclizes circulating cfDNA (ccfDNA) to form a "natural circle" for detection. Once created, the "circular ccfDNA" can be detected using a variety of different methods (including a variety of RCA methods). For example, Figure 5 As illustrated in , one embodiment of the technology of the present invention includes circularizing cfDNA and adding segments for detection using "Golden Gate Assembly" (see, e.g., Engler, C., Kandzia, R. and Marillonnet, S. (2008) PLoS ONE 3, e3647).

[0179] Figure 6 Another method for detecting ccfDNA is shown. In this embodiment, as previously described, plasma samples are treated to purify ccfDNA (see, e.g., M. Fleischhacker et al., Methods for isolation of cell-free plasma DNA strongly affect DNA yield, Clin ChimActa, November 20, 2011; 412(23-24): 2085-8). In step 1, ccfDNA is heat denatured and treated with T4 polynucleotide kinase to produce 5' phosphorylated and 3' hydroxyl-terminated DNA fragments. Before heat denaturation and T4 polynucleotide kinase treatment, additional DNA repair (such as with T4 DNA polymerase) can be used to repair DNA. A complementary oligonucleotide with a 3' protected end (so it will not be extended by the polymerase) hybridizes with ccfDNA. This complementary oligonucleotide consists of chromosome-specific regions A and C and a universal sequence B. ccfDNA is extended and connected to complete a circular DNA molecule. The circularized ccfDNA was purified from the oligonucleotides and RCA was used by annealing the oligonucleotides to the universal sequence B. After RCA, a fluorescently labeled probe was hybridized to the rolling circle product.

[0180] Figure 7Another method for detecting ccfDNA is shown. As previously described, plasma samples are processed to purify ccfDNA. Step 1, ccfDNA is heat denatured. A complementary oligonucleotide with phosphorylated 5 primer protection ends is hybridized with ccfDNA. This complementary oligonucleotide consists of chromosome-specific regions A and C and universal sequence B. Both 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 universal sequence B. After rolling circle amplification, a fluorescently labeled probe hybridizes with the rolling circle product.

[0181] Figure 8 A schematic diagram of a synthetic circular DNA used as a template for rolling circle amplification and comprising a rolling circle primer binding site and two probe binding sites and an optional binding moiety (eg, biotin) is shown.

[0182] Fig. 9 Provided are methods comprising using a method for detecting a product from RCA (e.g., Figure 8 Schematic diagram of an embodiment of the technology of the present invention of a probe pair configured for collision quenching when hybridizing with a strand of DNA (circular DNA similar to the circular DNA shown in ). In this embodiment, the dye-labeled probe in solution is not quenched and generates a signal. The probe hybridized to the target close to the probe labeled with a quencher will be quenched, thereby reducing the fluorescent signal. As the amount of RCA product increases, the fluorescence decreases.

[0183] Fig.10 Schematic diagrams are provided of embodiments of the present technology involving the use of probe pairs configured for fluorescence resonance energy transfer (FRET) as described above when hybridised to a strand of DNA for detecting products from RCA.

[0184] Fig.11 Schematic diagrams are provided of embodiments of the present technology comprising the use of a probe configured to cleave upon hybridisation to a strand of DNA, for example using a duplex-specific nuclease such as a restriction enzyme, comprising a dye and a quencher for detecting products from RCA.

[0185] like Fig.12 As illustrated in the figure, an embodiment of the technology of the present invention includes RCA using chromosome-specific identifier sequences (CIDs), followed by CID-specific digestion of non-targeted chromosomes and CID-specific labeling of targeted CIDs. CIDs are amplified by RCA, but their individual single-molecule identities are maintained. CID amplification increases the fluorescent signal from individual target molecules. Sequences from chromosomes that have not been analyzed are double suppressed by enzymatic digestion and using labels that are specific only to the analyzed chromosomes.

[0186] In some embodiments, the MIP can be detected using non-enzymatic methods of signal amplification. For example, in some embodiments, the MIP is immobilized on a surface and detected using methods such as "hybridization chain reaction" (HCR), such as described by RM Dirks et al., Proc. Natl. Acad. Sci. USA 101(43): 15275-15278 (2004) and U.S. Pat. No. 8,105,778, each of which is incorporated herein by reference. Figure 13-16 An exemplary configuration for signal amplification using HCR is shown.

[0187] Fig.13 An example is shown in which a MIP is hybridized to a target nucleic acid, such as cfDNA, leaving a single nucleotide vacancy. The vacancy is filled by extension to incorporate a biotinylated nucleotide and closed by ligation. The circularized MIP can then be hybridized to a target nucleic acid, such as cfDNA. Fig.14 The MIP is bound to a streptavidin-coated surface as shown in FIG. 1 , and after washing away any unbound MIP, the backbone of the bound MIP is hybridized to the initiator oligonucleotide. In a preferred embodiment, a spacer is included between the initiator sequence and the backbone binding sequence, such as an 18-atom hexaethylene glycol spacer. Preferably, the footprint of the MIP binding region is selected to have a high T m (eg, about 79° C.) for stable binding. As discussed above, binding tags other than biotin (such as amines, thiols, azides, or haptens) can be used to label and immobilize the MIPs to an appropriately reactive surface.

[0188] Fig.15 Examples of hairpin oligonucleotides used to form self-assembling scaffolds in HCR are shown. One or both oligonucleotides include at least one label, such as a fluorophore. In preferred embodiments, the dye is positioned to provide a sufficiently large spacing in the assembly scaffold to prevent quenching effects. For example, in some embodiments, such as Fig.14 As shown in , the dyes are located at opposite ends of the hairpin. Fig.16 As shown in , once the reaction is initiated by hybridization of an initiator oligonucleotide bound to the MIP backbone, the HCR hairpin unfolds and hybridizes in long chains, resulting in a scaffold comprising a large number of labels.

[0189] The flap endonuclease reaction (e.g., Invader assay) can be used for specific quantitative detection of chromosomes. Figure 17-20 An exemplary embodiment is shown in FIG. Fig.17An intruder oligonucleotide and a probe oligonucleotide are shown hybridizing to a target region of a chromosome. The 3′ end of the intruder oligonucleotide overlaps the 5′ end of the probe oligonucleotide in a region complementary to the target region. In this embodiment, the probe oligonucleotide includes a 5′ flap that includes a biotin moiety and a 3′ tail that includes a label (e.g., a fluorophore). A flap endonuclease (e.g., FEN-1 nuclease) recognizes the overlapping intruder cleavage structure and cleaves the probe in a highly specific, structure-dependent manner, thereby releasing the 5′ flap. In a preferred embodiment, as Fig.19 As schematically shown in FIG. 1 , the reaction is run isothermally and produces linear signal amplification, providing 10 Å per target in one to three hours. 3 to 10 4 A cleaved probe.

[0190] In a preferred embodiment, if Fig.18 As shown in , the probe oligonucleotide used includes a hairpin structure in which the 5' flap and 3' tail of the probe are hybridized to each other. A fluorophore or another moiety (e.g., 2,4-dinitrophenyl) can be used as a hapten so that the uncleaved probe and / or the 3' portion of the cleaved probe can be removed from the reaction using an antibody against the hapten used for capture.

[0191] The cleaved flap from the flap endonuclease reaction can be detected in a variety of ways. In a preferred embodiment, Fig. 20 As shown in , the cleaved flap is captured using an immobilized complementary probe and the biotin is reacted with streptavidin linked to a detectable moiety. In the illustrated embodiment, the streptavidin is coupled to β-galactosidase and the fluorescent signal is generated by providing non-fluorescent resorufin-β-galactopyranoside catalyzed by β-galactosidase to produce D-galactose and the fluorescent dye resorufin. Using femtoliter arrays and Poisson statistics to generate a digital readout variant, a single hybridization event can be detected using this enzymatic signal amplification. See, e.g., DM Rissin and DR Walt, Digital Concentration Readout of Single Enzyme Molecules Using Femtoliter Arrays and Poisson Statistics. Nano letters 6(3):520-523 (2006); Quanterix White Paper 1.0, Scientific Principles of Simoa (Single Molecule Array) Technology, 1-2 (2013); Quanterix White Paper 6.0, Simoa for Ultrasensitive Multiplex Immunoassays of Protein Biomarkers.TM Practical Applications of the HD-1 Analyzer, 1-3 (2015), each of which is incorporated herein by reference for all purposes. In certain preferred embodiments, a kinetic readout is used, ie, signals are collected from the array at two time points.

[0192] Fig.21 In the embodiment shown in, A, B, C and D are composed of MIPs that are specific to chromosomes 13, 8, 21 or reference chromosomes (such as 1). The sequence of the MIP surrounding the vacancy is complementary to the region of the targeted chromosome and is designed to contain a single nucleotide vacancy. Step 1: fill this vacancy with dNTPs conjugated with haptens (such as fluorescent dyes, biotin, etc.). Filling the vacancy introduces different haptens into the MIPs of each specific chromosome in the targeted different specific chromosomes. For example, adding A only completes the MIP targeting chromosome 21, T completes the MIP targeting chromosome 18, G completes the MIP targeting chromosome 13, and C completes the MIP targeting the reference chromosome (such as chromosome 1). This method uses four unique hapten labels for these four different MIPs. Targeting requires specific dNTPs to complete the pool of MIPs of each chromosome of a single extension and connection for increasing the number of capture events. Step 2 includes incubating the MIP containing haptens with labeled antibodies that are specific to each hapten. Labeling can include, for example, fluorescent dyes, quantum dots or other fluorescent particles. Step 3 includes the optional step of exposing the immune complex including the hapten-targeted primary antibody to a labeled secondary antibody directed against the primary antibody, thereby amplifying the fluorescent signal.

[0193] like Fig.21 As shown in , in this embodiment of the present technology, the MIPs designed to target different chromosomes each require different nucleotides to extend and connect and the MIPs are extended and connected in a chromosome-specific manner using nucleotides carrying different dyes for each different dNTP. For example, in a preferred embodiment, CY2, CY3, CY5 and CY7 are used. The MIPs with dye labels can be detected using antibodies specific to each different dye (and by extension, each different chromosome to be detected). The signal can be amplified by using a secondary antibody. For example, the CY2 primary rabbit antibody binds to the target MIP and the secondary goat anti-rabbit antibody binds to the primary antibody to amplify the signal, etc.

[0194] As discussed above, many different fluorescent labeling systems can be applied in embodiments of the present technology. In certain embodiments, fluorescent dyes (e.g., fluorescein, Texas Red, TAMRA, Cy3, Cy5) can be used, for example, to connect them to nucleotide analogs incorporated into oligonucleotides or extension products. In certain embodiments, fluorescent particles, such as nanoparticles, nanocrystals, quantum dots, silicon dioxide (e.g., mesoporous silicon dioxide nanoparticles) polymer beads (e.g., latex) can be used.

[0195] There are many options for detecting and quantifying the fluorescent signal from the embodiments of the present invention technology described above herein.Detection can be based on measuring characteristics such as physicochemical properties, electromagnetic properties, electrical properties, photoelectric properties or electrochemical properties or immobilized molecules and / or target molecules.Two factors related to the single molecule detection of molecules on the surface are to achieve sufficient spatial resolution to distinguish separate molecules and to distinguish the desired single molecule from background signals (for example, with probes non-specifically bound to the surface).For example, WO2016 / 134191 is found to be an exemplary method for detecting single molecule related signals, and the patent is incorporated herein by reference in its entirety for all purposes.In certain embodiments, determination is configured to, for example, perform standard SBS microplate detection in SpectraMax microplate reader or other plate reader.Although this method generally requires low-variation fluorescence (multiple holes, multiple measurements), this format can be multiplexed and read on multiple different fluorescence channels.In addition, the throughput of the format is very high.

[0196] Embodiments can also be configured for detection on a surface (e.g., a glass surface, a gold surface, or a carbon (e.g., diamond) surface). In some embodiments, signal detection is accomplished by any method for detecting electromagnetic radiation (e.g., light) (e.g., selected from far-field optical methods, near-field optical methods, epifluorescence spectroscopy, confocal microscopy, two-photon microscopy, optical microscopy, and total internal reflection microscopy), wherein the target molecule is labeled with an electromagnetic radiation emitter. Other microscopic methods (e.g., atomic force microscopy (AFM) or other scanning probe microscopy (SPM)) are also applicable. In some embodiments, it may not be necessary to label the target. Alternatively, a label that can be detected by SPM can be used. In some embodiments, signal detection and / or measurement includes surface reading by counting fluorescent clusters using imaging systems such as ImageXpress imaging systems (Molecular Devices, San Jose, CA, San Jose, CA) and similar systems.

[0197] Embodiments of the present technology can be configured for detection using many other systems and instrument platforms (e.g., bead assays (e.g., Luminex), array hybridization, NanoString nCounter single molecule counting device). See, for example, GK Geiss et al., Direct multiplexed measurement of gene expression with color-coded probe pairs; Nature Biotechnology 26(3): 317-25 (2008), U.S. Patent Application 2018 / 0066309 Al (PN Hengen et al., inventors of Nanostring Technologies, Inc.) published on August 3, 2018, etc.

[0198] In the Luminex bead assay, color-coded beads pre-coated with analyte-specific capture antibodies for the molecule of interest are added to the sample. Multiple analytes can be detected simultaneously in the same sample. The analyte-specific antibodies capture the analyte of interest. A biotinylated detection antibody also 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. On a dual laser flow-based detection instrument such as the Luminex 200 TM or The beads are read on a PCR analyzer. One laser sorts the beads and identifies the analyte to be detected. A second laser determines the magnitude of the PE-derived signal, which is proportional to the amount of bound analyte.

[0199] NanoString nCounter is a single molecule counting device for digital quantification of hundreds of different genes in a single multiplex reaction. The present technology uses molecular "barcodes" (each of which is color-coded to correspond to a single probe of a gene (or other nucleic acid) of interest and is connected to it) in combination with solid phase hybridization and automatic imaging and detection. See, for example, Geiss et al. (supra), which describes the use of several unique pairs of capture probes and reporter probes constructed to detect each nucleic acid of interest. In the described embodiment, the probe is mixed with nucleic acid (e.g., unpartitioned cfDNA) or total RNA from a sample in a single solution phase hybridization reaction. Hybridization results in the formation of a three-part structure consisting of a target nucleic acid bound to its specific reporter probe and capture probe, and unhybridized reporter probes and capture probes are removed, for example, by affinity purification. When a biotin immobilized label is used, the hybrid complex is exposed to an appropriate capture surface (e.g., a streptavidin-coated surface). After capture on the surface, the applied electric field extends and directs each complex in the same direction in the solution. Then, the complex is fixed in an elongated state and imaged. Thus, each target molecule of interest can be identified and counted by the color code generated by the ordered fluorescent segments present on the reporter probes to enumerate the target molecules.

[0200] Fig. 22 Figures A, B and C of the present invention illustrate embodiments of the present technology in which a MIP including an immobilization moiety or attached thereto is immobilized on a surface. While not limited to any particular embodiment for incorporating unique features indicative of target recognition into a cyclized MIP molecule, Fig. 22 The embodiments are illustrated using an embodiment that includes using a polymerase to replicate one or more nucleotides of a target nucleic acid to extend a linear MIP, followed by ligation to circularize the extended probe.

[0201] exist Fig. 22In the embodiment shown in Figure A of , in step 1, the MIP is hybridized to the target DNA, and then extended by a DNA polymerase in the presence of modified dNTPs, so that the immobilized portion is incorporated into each MIP during extension. Then, the MIP is connected to itself to complete the circularized probe. The modified dNTPs may include, but are not limited to, dNTPs including reactive chemicals (such as amine or thiol groups) or other binding 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 immobilized features of the MIP to bind to the MIP. Such surfaces include, but are not limited to, derivatized or underivatized glass, silica, diamond, gold, agarose, plastics, ferromagnetic materials, alloys, etc., and may be in any form, such as a slide, a sample well, a channel, a bead, a particle, and / or a nanoparticle, any of which may be porous or non-porous.

[0202] exist Fig. 22 In the embodiment shown in FIG. B of , in step 1, the MIP is hybridized to the target DNA and ligated for circularization. In the embodiment shown, the MIP is extended by a DNA polymerase to fill in sequence gaps prior to ligation, while in other embodiments, the MIP can be designed to simply hybridize to the target nucleic acid and ligated for circularization without using a polymerization step, such as in the manner of a padlock probe. See, e.g., 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 an immobilization moiety as described above (e.g., a reactive amine, a reactive thiol group, biotin, a hapten, etc.). In step 3, the hybridized MIP complex of the MIP and the oligonucleotide including the immobilized moiety is exposed to the surface under conditions where the surface interacts with the immobilized features of the MIP complex to bind the MIP complex. As described above, the surface includes but is not limited to derivatized or underivatized glass, silica, diamond, gold, agarose, plastic, ferromagnetic material, alloy, etc., and can be in any form, such as a slide, a sample well, a channel, a bead, a particle and / or a nanoparticle, any of which can be porous or non-porous.

[0203] exist Fig. 22In the embodiment shown in Figure C of , in step 1, the MIP containing the immobilization part built into the probe backbone is hybridized with DNA, extended by DNA polymerase and connected to cyclize the probe. As in the embodiment of Figure B described above, the MIP can be designed to hybridize with the target nucleic acid only and connect to cyclize without using a polymerization step. In step 2, the cyclized MIP containing the immobilization part is exposed to the surface under the condition that the surface interacts with the immobilization feature of the MIP to bind the MIP. As described above, the surface includes but is not limited to derivatized or underivatized glass, silica, diamond, gold, agarose, plastic, ferromagnetic material, alloy, etc., and can be in any form, such as a slide, a sample well, a channel, a bead, a particle and / or a nanoparticle, any of which can be porous or non-porous.

[0204] exist Fig. 22 In each of the embodiments presented in, once the MIP has been immobilized to the surface, labeling and / or signal amplification (e.g., fluorescent labeling and / or fluorescent signal amplification) and detection can be accomplished using any of the various back-end analytical methods discussed herein. Suitable methods for amplifying and / or detecting unique immobilized MIP products include, but are not limited to, the NanoString nCounter technology described above and Figure 2-3 , Figure 6-7 , Figure 9-12 , Figure 15-16 and Figure 20-21 In some embodiments, labeling and / or signal amplification (eg, fluorescent labeling and / or fluorescent signal amplification) is accomplished before the MIP has been immobilized on the surface.

[0205] In preferred embodiments, back-end processes configured for single-molecule visualization are used. For example, as described above, the Quanterix platform uses an array of femtoliter-sized wells that capture beads with no more than one labeled complex, where the signal from the captured complex is visualized using a resorufin-β-galactopyranoside / β-galactosidase reaction to produce fluorescent resorufin. Visualization of the array allows detection of the signal from each individual complex. In certain preferred embodiments, a solid-state nanopore device is used, such as described by Morin et al. (see "Nanopore-Based Target Sequence Detection" PLoS ONE 11(5): e0154426 (2016)). A solid-state nanopore is a nanoscale opening formed in a solid film that separates two aqueous volumes

[23] . A voltage clamp amplifier applies a voltage across the membrane while measuring the ionic current passing through the pore ( Figure 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). Although DNA alone can be detected using this system, unique tags (e.g., polyethylene glycols (PEGs) of varying sizes) can be attached to highly sequence-specific probes (e.g., peptide nucleic acid probes, PNA) to give any particular DNA-PNA-PEG complex a unique signature tag representing the target nucleic acid detected in the front end of the assay.

[0206] exist Fig.23 In the embodiment shown in , a complex comprising an oligonucleotide primer and a circular probe (such as a MIP or a connected padlock probe) is formed. The primer is extended in a rolling circle amplification reaction to produce a long chain of single-stranded DNA containing a concatemer of a sequence complementary to the circular probe. The RCA product is combined with a plurality of molecular beacon probes having a fluorophore and a quencher. Hybridization of the beacon separates the quencher from the fluorophore, thereby allowing the fluorescence from the beacon to be detected. The accumulation of the RCA product can be monitored in real time by measuring the increase in fluorescence intensity indicating that the beacon binds to an increased amount of product during the time course of the reaction.

[0207] Real-time quantification of the accumulated fluorescence in the reaction is used to examine the effect of the attached biotin moiety on the MIP or on the primer. Fig.24 A-24D shows the results obtained from examining the effect of including a biotin residue in only the circularized MIP (A), in only the RCA primer (B), in both (C), and in neither (D) on the RCA signal. In this experiment, the MIP contained the following sequence:

[0208]

[0209] ATGCTGCTGCTGTACTACGAGGCTAAGGCATTCTGCAAACAT-3' (circularized).

[0210] In the above biotinylated MIPs, the boxed "T" indicates the attachment site of biotin in the biotin-containing MIP (Integrated DNA Technologies, "Internal Biotin dT"). The biotinylated primer includes biotin attached at the terminal '5' phosphate (Integrated DNA Technologies, "5' Biotin-TEG"). The rolling circle reaction was performed at 37°C for one hour according to the "Standard Rolling Circle Reaction" procedure described in Example 1 below. These data show that the presence of biotin in the circularized MIP inhibits RCA, while the presence of biotin on the primer does not inhibit the reaction.

[0211] Figures 25A-25C Shown are the results of varying the amounts of components in a standard RCA reaction in solution. Fig.25A A comparison was made between the use of 5 units and 25 units of Phi29 polymerase in each reaction and it was shown that the higher concentration of polymerase consistently produced higher signals under the conditions tested. Fig.25B The effect of using different concentrations of molecular beacon probes ("Beacons") is shown; Fig.25C The effects of using different concentrations of Phi29 polymerase and molecular beacon probes were compared to the effects of using 200 μM or 800 μM total dNTPs on standard reactions. Based on these data, reactions adjusted to include 1000 nM beacon, 800 μM dNTPs, and 2000 nM phi 29 polymerase (80 units) were further tested.

[0212] The effects of adding different concentrations of PEG and using different sizes of PEG on enhanced RCA conditions (E-RCA, see Example 1 below) were examined. Fig.26 The effects of using different sizes of PEG (200 and 8000) under E-RCA conditions at the percentages (w:v) shown were compared. Under the conditions tested for this example, PEG 200 provided superior results at all concentrations tested, with 20% PEG 200 providing the best results. In contrast, PEG 8000 significantly reduced the efficiency of the RCA. Based on these data, RCA reactions including at least 20% w:v of PEG 200 were further tested.

[0213] As discussed above, when performing single molecule detection on a surface, it is preferred that the spot size of the signal from any individual bound molecule be minimized so that separation between spots is ensured. The effect of using PEG 200 on spot size and the number of spots detected was examined. The assay was performed using the E-RCA conditions described below with or without 20% w:vPEG 200 and incubated for 140 minutes. Figures 27A-27B and Fig.28 The results are shown in A-28B. Fig.27A It was shown that the presence of PEG reduced the spot size, thereby enhancing the measurement of the fluorescent signal from individual spots. Fig.27B The PEG Fig.27A The effects of PEG on the number of spots and fluorescence intensity are shown in Figure 3 and show that the addition of PEG increases the number of detectable spots while decreasing the size of the detected spots.

[0214] The effect of using different molecular weights of PEG in 20% solution on spot counts and spot size in reactions performed on APTES silanized plates was examined. Reactions were performed on APTES treated surfaces as described in "One-step rolling circle amplification on surfaces" in Example 1, where Fig.28 The PEG component was modified as indicated in . Fig.28 It is shown that when the PEG used has an average molecular weight of less than 1000, preferably between 200 and 800, more preferably 600, the number of spots is maximized and the size of the spots is minimized.

[0215] The length of hybridization time was checked before initiating the RCA reaction. Fig.29A Microscope images of the surface of an APTES silanized plate as described in Example 1 are shown and the RCA signal is compared for hybridizations in TBS buffer or RCA buffer for 18 hours or 1 hour before initiating the RCA reaction. Enhanced RCA was performed as described above with 20% PEG600 for 140 minutes. Fig.29B Provides comparison of hybridization time and buffer pairs Fig.29A The effect of the number of spots and the fluorescence intensity (area) is shown in FIG. These data show that the number of spots increases significantly when the hybridization time is longer.

[0216] Fig.30 , 31 32 provide graphs comparing the effects of PEG 200 with or without a 2 hour hybridization time and replacing PEG 200 with PEG 2000 with a 2 hour hybridization on standard RCA reaction conditions, enhanced RCA (E-RCA) conditions, and E-RCA conditions with other variations. The reactions in each graph were performed at the same temperature, with Fig.30 , 31 In and 32, the reactions were carried out at 30°C, 25°C and 37°C, respectively.

[0217] The number and fluorescence intensity (area) of spots were evaluated for each condition. These data show that in the presence of PEG 200, a 37°C reaction temperature produces the best combination of high spot counts and small spot sizes. The effect of varying the concentration of beacon probes using a higher RCA reaction temperature was also examined. Reactions containing 1000nM, 2000nM, 4000nM or 8000nM molecular beacon probes were performed at 37°C or 42°C, and the reactions showed that at higher temperatures, the number of spots counted increased significantly (data not shown). Although the technology of the present invention is not limited to any particular mechanism of action, these data indicate that reactions performed at higher temperatures (e.g., 42°C or higher) produce more RCA products and more bound beacon probes.

[0218] The effect of elevated temperature in the presence of varying concentrations of PEG 600 was further examined. Fig.33 Microscope images of the surface of an APTES silanized plate as described in Example 1 are shown and the RCA signals are compared for reactions including the indicated concentrations of PEG600 performed at 37° C. or 45° C. These data show that the 45° C. reactions produced significantly higher spot counts and that 10% to 15% w:v PEG600 produced the best combination of spot count and spot size at 45° C.

[0219] The effect of adding graphene oxide to the RCA surface binding reaction was examined. Fig.35 The two-step RCA procedure is schematically shown in FIG. Fig.36 Shown is a microscope image of the surface of an APTES silanized plate as described in Example 1 and shows the RCA signal of the two-step reaction graphene oxide. The negative control contains no input target and shows the background from the molecular beacon probe. Fig.37 Graphs comparing spot counts for RCA reactions performed in one step (no GO) or two steps (with or without GO) comparing reactions with 100 fmol of target to reactions without target are provided. These data show that the use of GO significantly reduced the number of background spots in the no-target control reaction, thereby improving the signal: background results in the assay.

[0220] experiment

[0221] Example 1

[0222] This example provides an example of a workflow for analyzing DNA, such as cfDNA, from a sample, such as a blood sample.

[0223] Sample collection

[0224] Blood was collected from the patient in a standard draw. 10 mL of blood was stored in a Streck blood collection tube or an alternative blood collection tube containing EDTA. The sample was transported to the laboratory at ambient temperature and processed as follows:

[0225] - Centrifuge the blood at 2000 x g for 20 minutes at room temperature to obtain a plasma fraction from the blood.

[0226] • Transfer plasma to new sterile nuclease-free polypropylene tubes and centrifuge at 3220 xg for 30 minutes.

[0227] Cell-free DNA (cfDNA) purification

[0228] Cell-free DNA is purified from plasma using standard methods, such as using the MagMAX Cell-Free DNA Isolation Kit (ThermoFisher Scientific, Catalog No: A29319).

[0229] Assay plate preparation

[0230] Glass bottom microtiter plates are treated to immobilize oligonucleotides that initiate rolling circle amplification of circularized MIPs. Several methods can be used (see, e.g., EJ Devor et al., "Strategies for Attaching Oligonucleotides to Solid Supports," Integrated DNA Technologies (2005), which is incorporated herein by reference in its entirety for all purposes).

[0231] 1) Acid pre-wash

[0232] For each method, the glass substrate was first pickled as follows:

[0233] (a) Add 100 μL of 0.5 N sulfuric acid to each well.

[0234] (b) Add foil seal to the plate.

[0235] (c) The plate was incubated at 37°C for 2 hours with rotation at 300 RPM.

[0236] (d) Removal of pore contents.

[0237] (e) Wash the wells twice with 100 μL of molecular grade water.

[0238] (f) Wash the wells twice with 100 μL of 95% ethanol.

[0239] 2) 3-Aminopropyltriethoxysilane (APTES) silanization and streptavidin-biotin primer immobilization:

[0240] (a) Prepare 2% APTES by adding 200 μL 99% APTES (Sigma-Aldrich, catalog number: 440140), 500 μL molecular grade water, and 9.3 ml 95% ethanol.

[0241] (b) Vortex the solution and pipette 100 μL into each well.

[0242] (c) Incubate at room temperature for 15 minutes.

[0243] (d) Removal of pore contents.

[0244] (e) Wash the wells twice with 100 μL of 95% ethanol.

[0245] (f) Remove the last wash.

[0246] (g) The plates were incubated at 37°C for 24 hours.

[0247] Primer immobilization

[0248] (h) 1 ng of streptavidin in 100 μL of Tris-buffered saline was added to the amine-functionalized glass plate.

[0249] (i) Incubate at room temperature for 1 hour.

[0250] (j) Wash each well three times with 100 μL of TBS.

[0251] (k) Add 100 μL of 1 μM biotinylated oligonucleotide solution.

[0252] (1) Incubate at room temperature for 1 hour.

[0253] (m) Each well was washed three times with 100 μL of TBS.

[0254] 3) Acrylic silanization and acrydite primer immobilization

[0255] (a) Prepare 4% acrylated silane by adding 400 μL of 99% acrylated silane (3-(trimethoxysilyl)propyl methacrylate; Sigma-Aldrich, Catalog No. 440159), 1 mL of molecular grade water, and 18.6 mL of 100% ethanol.

[0256] (b) Add 100 μL of 4% acrylic silane solution to each well.

[0257] (c) Incubate at room temperature for 15 minutes.

[0258] (d) Removal of 4% acrylic silane solution.

[0259] (e) Wash each well four times with 100 μL of 100% ethanol each time.

[0260] (f) The plates were incubated at 37°C for 24 hours.

[0261] (g) Prepare an acrylate primer solution by adding:

[0262] (i) 250 μL 5x TRIS boron EDTA (TBE) buffer,

[0263] (ii) 500 μL 40% acrylamide,

[0264] (iii) 17.5 μL 10% ammonium persulfate,

[0265] (iv) 5 μL tetramethylethylenediamine (TEMED),

[0266] (v) 25 μL of 100 μM oligonucleotide primer including a '5' acrylate (or acrylate-phosphoramidite)

[0267] (vi) 1.7 mL of molecular grade water.

[0268] (h) Add 25 μL of the acrylate primer solution to each well and gently agitate the plate to cover the bottom of the wells.

[0269] (i) Incubate at room temperature for 30 minutes.

[0270] (j) Wash the wells four times with 100 μL 0.5 x TBE, discarding the first three washes and leaving the final wash in the wells before proceeding with the RCA assay.

[0271] Primers can be immobilized by other methods (eg, as described by Devor et al., supra).

[0272] Molecular Inversion Probe Pool

[0273] The probe pool is used to capture specific loci in a DNA sample, such as a cfDNA sample, and to generate circularized MIPs for rolling circle amplification. NIPT assays typically include a pool of molecular inversion probes. In a preferred embodiment, the NIPT assay includes about 5,000-10,000 molecular inversion probes.

[0274] • Generate targeted MIPs to target the features to be studied by the assay (eg, chromosomes 13, 18, 21, X, Y, and CHR22q11.2).

[0275] • Approximately 10,000 unique MIPs were generated for each feature.

[0276] • MIPs are mixed together to create a pool of probes, where each probe has a customized concentration.

[0277] MIP capture and ligation of cfDNA

[0278] ●In the following reaction, add the MIP pool to the purified cfDNA.

[0279] o 2 μL AMP ligase buffer (10x), 1 μL MIP probe library, 16 μL cfDNA preparation, and 1 μL AMP ligase (80 units).

[0280] o Incubate the reaction at 98°C for 2 minutes and cool at 1 degree per minute until the reaction reaches 45°C, then hold at 45°C for 2 hours.

[0281] Molecular beacon probes

[0282] Examples of molecular beacon probes that can be used in the present technology are as follows:

[0283] 1)5′Alexa 405-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-dabcyl 3′

[0284] 2)5′ Alexa 488-CC TCA ATG CTG CTG CTG TAC TAC mGmAmG mG-dabcyl 3′

[0285] 3)5′Alexa 594-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-BHQ2 3′

[0286] 4)5′ Alexa 647-CCTCAGCGCTGCCTATTCGAACTmGmAmGmG-BHQ2 3′

[0287] 5) 5′ Alexa 750-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-BHQ3 3′

[0288] Standard rolling circle amplification assay conditions

[0289] ○ For 100 μL RCA solution, combine on ice

[0290] ■MIP probe-target DNA preparation (e.g., the entire MIP trap / cfDNA preparation described above, approximately 20 μL)

[0291] ■10 μL of 10X Phi29 buffer, final concentration is 1X

[0292] 1X Phi29 DNA polymerase reaction buffer

[0293] -50mM Tris-HCl

[0294] -10mM MgCl2

[0295] -10mM(NH4)2SO4

[0296] -4mM DTT

[0297] -(pH 7.5, at 25°C)

[0298] 200 μM dNTP

[0299] ■5 units of Phi29 DNA polymerase

[0300] ■100nM beacon probe

[0301] ■Molecular grade water, up to 100μL

[0302] o Incubate at 30°C to 37°C for the reaction time (eg, 90 to 120 minutes).

[0303] Enhanced RCA (E-RCA) conditions:

[0304] ○ For 100 μL Enhanced RCA Solution, combine on ice

[0305] ■ MIP probe-target DNA preparation (e.g., the entire MIP trap / cfDNA preparation described above, approximately 20 μL);

[0306] ■10 μL of 10X Phi29 buffer, final concentration is 1X

[0307] 800 μM dNTP

[0308] ■80 units of Phi29 DNA polymerase

[0309] ■1000nM beacon probe

[0310] ■Molecular grade water, up to 100μL

[0311] o Incubate at 30°C to 37°C for the reaction time (eg, 90 to 120 minutes).

[0312] One-step enhanced rolling circle amplification on surfaces

[0313] ●Preparation of rolling circle amplification (RCA) solution

[0314] ○ For 100 μL RCA solution, combine on ice

[0315] ■ MIP probe-target DNA preparation (e.g., the entire MIP trap / cfDNA preparation described above, approximately 20 μL);

[0316] ■10 μL of 10X Phi29 buffer, final concentration is 1X

[0317] 1X Phi29 DNA polymerase reaction buffer

[0318] -50mM Tris-HCl

[0319] -10mM MgCl2

[0320] -10mM(NH4)2SO4

[0321] -4mM DTT

[0322] -(pH 7.5, at 25°C)

[0323] ■4 μL of 10 mM dNTPs, the final total dNTP concentration is 0.4 mM;

[0324] 50 μL of filtered 30% PEG 600;

[0325] ■0.5 μL of 100 μM molecular beacon, final concentration is 0.5 μM

[0326] 8 μL of Phi29 polymerase (10 units / μL); and

[0327] ■22.5μL of molecular grade water

[0328] o The solution is mixed, for example by vortexing, and pipetted onto the treated glass surface including the bound primers, and the plate is then sealed;

[0329] o Incubate the plate at 45°C for 90 minutes on a flat bottom heating block on a Thermomixer with heated lid;

[0330] ○ Remove well contents and wash wells twice with 100 μL of 1X TBS; discard wash solution;

[0331] o Add 100 μL of IX TBS and image in a microscope as described below.

[0332] Samples were imaged using an IXM4 microscope (Megumi Molecular Instruments, San Jose, CA).

[0333] Images are typically captured using a 20x, 40x, or 60x objective.

[0334] The plate was placed in the IXM4 microscope and imaged as follows:

[0335] o Plates were automatically exposed to ensure a wide dynamic range of fluorescence intensity values ​​(maximum range of the camera used, eg 16-bit images).

[0336] ○ Each well of the plate was subdivided into approximately 100 images.

[0337] For high-throughput assays, automated microscopy can be used.

[0338] Image analysis

[0339] ●The image is analyzed as follows:

[0340] ○ Relative fluorescence intensity was determined in images without sample (negative control).

[0341] o The threshold was determined by multiplying the mean relative fluorescence intensity from the negative control by three.

[0342] o In each channel, spots above the threshold were counted.

[0343] One-step variant

[0344] Crowding agent (eg, PEG) addition: Prepare 30% solution in molecular grade water; filter with a 0.2 μm pore size filter. Add PEG to the RCA reaction, adjusting the water added to the RCA to maintain a consistent volume.

[0345] Beacon: Add desired concentration, thereby adjusting water added to the RCA to maintain a consistent volume.

[0346] dNTPs: Add desired concentration, adjusting water added to RCA to maintain consistent volume.

[0347] Graphene oxide: A 2-step reaction was performed as described in Example 2 to add graphene oxide and labeled probe.

[0348] Example 2

[0349] Detection using two-step rolling circle amplification on surfaces with graphene oxide

[0350] Prepare the rolling circle amplification (RCA) solution on ice:

[0351] o For 100 μL RCA solution (without molecular beacon), combine:

[0352] ■ MIP probe-target DNA preparation (e.g., the entire MIP trap / cfDNA preparation described above, approximately 20 μL);

[0353] ■10 μL of 10X Phi29 buffer, final concentration is 1X

[0354] 1X Phi29 DNA polymerase reaction buffer

[0355] -50mM Tris-HCl

[0356] -10mM MgCl2

[0357] -10mM(NH4)28O4

[0358] -4mM DTT

[0359] -(pH 7.5, at 25°C)

[0360] ■4 μL of 10 mM dNTPs, the final total dNTP concentration is 0.4 mM;

[0361] 50 μL of filtered 30% PEG 600;

[0362] 8 μL of Phi29 polymerase (10 units / μL); and

[0363] ■23μL of molecular grade water

[0364] o The solution was mixed by vortexing and pipetted onto the treated glass surface and the plate was then sealed;

[0365] o Incubate the plate at 45°C for 90 minutes on a flat bottom heating block on a Thermomixer with heated lid;

[0366] ○ Remove well contents and wash wells three times with 100 μL of 1X TBS; discard wash solution;

[0367] ○ Add 50 μL of graphene oxide-molecular beacon solution consisting of:

[0368] ■5 μL of 10X Phi 29 buffer, final concentration is 1X

[0369] ■0.5 μL of 100 μM molecular beacon, final concentration is 0.5 μM

[0370] ■5 μL of 2 mg / mL graphene oxide solution, final concentration 0.2 mg / mL;

[0371] ■Molecular grade water, up to 50 μL

[0372] ○ Incubate the reaction at 37°C for 60 minutes

[0373] ○ Wash three times with 100 μL 1x TBS;

[0374] ○ Wash once with 100 μL 1x TBS containing 5% w:v Tween 20:

[0375] ○ Wash twice with 100 μL of 1X TBS; discard wash solution;

[0376] o Add 100 μL of IX TBS and image in microscope as described above.

[0377] It is readily apparent that each of the disclosed front-end target identification systems provided with respect to the disclosure herein may be configured to generate a detectable signal for use with any of the back-end instruments and systems described above.

[0378] Additional references

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[0401] 23. EJ Devor et al., “Strategies for Attaching Oligonucleotides to Solid Supports”, Integrated DNA Technologies (2005)

[0402] 24. WO 2015 / 083002 “Multiplex Detection of Nucleic Acids”

[0403] All documents and similar materials cited in this application, including publications described in the above bibliography and including but not limited to patents, patent applications, papers, books, monographs and Internet web pages, are expressly incorporated by reference in their entirety for any purpose. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which the various embodiments described herein belong. When the definition of a term in an incorporated reference appears different from the definition provided in this teaching, the definition provided in this teaching shall prevail.

[0404] Various modifications and variations of the compositions, methods and uses of the described technology of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the described technology of the present invention. Although the present technology has been described in conjunction with specific exemplary embodiments, it should be understood that the claimed invention cannot be unduly limited to such specific embodiments. In fact, various modifications of the described modes for carrying out the present invention that are apparent to those skilled in the art of molecular biology, molecular diagnostics, nucleic acid structure, biochemistry, medical science or related fields are intended to fall within the scope of the claims. Sequence Listing <110> PROGENITY, INC. <120> Methods, systems and compositions for counting nucleic acid molecules <130> PRGNY-36313 / WO-1 / ORD <150> US 62 / 660,699 <151> 2018-04-20 <150> US 62 / 651,676 <151> 2018-04-02 <160> 7 <170> PatentIn Version 3.5 <210> 1 <211> 56 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 1 agtctaggat tcggcgtggg ttaaggtggc ttccttggcc gaagtgcggg gaccgc 56 <210> 2 <211> 100 <212> DNA <213> Artificial sequence <220> <221> misc_feature <222> (1) <223> The sequence is circular, with position 1 connected to position 100 <400> 2 ccttcgactt caagagacca tgagttgtgc ggtccccgca cttcggccaa ggaagccacc 60 atcatcagta gtgtgatggc agcctagcac gggcattagc 100 <210> 3 <400> 3 000 <210> 4 <211> 48 <212> DNA <213> Artificial sequence <400> 4 gtttcatcag atcctaagcc gcacaattgg gtgcggctta ggatctga 48 <210> 5 <211> 103 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 5 agtctaggat tcggcgtggg ttaacacgcc gaatcctaga ctactttgat ctaggattcg 60 gcgtgggtta aggtggcttc cttggccgaa gtgcggggac cgc 103 <210> 6 <211> 96 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 6 ttaacccacg ccgaatccta gactcaaagt agtctaggat tcggcgtgtt aacccacgcc 60 gaatcctaga ctcaaagtag tctaggattc ggcgtg 96 <210> 7 <211> 104 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 7 agtctaggat tcggcgtggg ttaacacgcc gaatcctaga ctactttgag tctaggattc 60 ggcgtgggtt aaggtggctt ccttggccga agtgcgggga cccc 104

Claims

1. A method for detecting a target molecule on a solid phase carrier, the method comprising: a) forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a surface on a solid support, and wherein forming the at least one complex comprises exposing the solid support to a solution comprising a crowding agent, wherein the crowding agent comprises at least 12% w:v polyethylene glycol (PEG), wherein the PEG has an average molecular weight between 400 and 800; b) detecting the formation of the at least one complex in a process comprising: i) extending the primer in the complex in a rolling circle amplification (RCA) reaction to form an RCA product; ii) hybridizing a plurality of labeled probes to the RCA product; as well as iii) detecting the hybridized labeled probe; wherein the hybridized labeled probe indicates the presence of the target molecule on the solid support; Wherein the method is for non-diagnostic purposes.

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

3. The method according to claim 1, wherein: i) the surface on the solid support comprises a silanized surface, wherein the silanized surface comprises at least one of the following: an acrylic group; and reactive amine groups; ii) the forming of at least one complex further comprises forming a plurality of complexes, the plurality of complexes comprising a double-stranded scaffold product, the double-stranded scaffold product comprising a plurality of concatenated labeled scaffold oligonucleotides; iii) wherein the formation of the complex indicates the presence of the target molecule on the silanized surface of the solid support; and iv) the method further comprises counting the plurality of complexes.

4. The method of claim 1, wherein the solid support comprises an assay plate.

5. The method of any one of claims 2 to 3, wherein the silanized surface comprises a glass surface treated with 3-aminopropyltriethoxysilane or 3-(trimethoxysilyl)propyl methacrylate, and / or wherein the solid support comprises an assay plate.

6. The method according to any one of claims 1 to 3, wherein: The primer is directly bound to the surface on the solid support; or wherein the primer comprises a biotin moiety and the surface on the solid support comprises avidin.

7. The method of claim 6, wherein the avidin is streptavidin.

8. The method of claim 6, wherein the primer is covalently attached to a surface on the solid support.

9. The method of any one of claims 1 to 3, wherein the crowding agent comprises at least 14% w:v PEG, and / or wherein the PEG has an average molecular weight of 600.

10. The method of claim 9, wherein the crowding agent comprises at least 16% w:v PEG.

11. The method of claim 9, wherein the crowding agent comprises at least 18% w:v PEG.

12. The method of claim 9, wherein the crowding agent comprises at least 20% w:v PEG.

13. The method of any one of claims 1 to 3, wherein the target molecule comprises a nucleic acid comprising DNA from a sample from a subject.

14. The method of any one of claims 1 to 3, wherein forming the RCA product comprises extending a primer on the circularised nucleic acid probe in a reaction mixture comprising Phi29 DNA polymerase.

15. The method of claim 14, wherein the reaction mixture comprises: - At least 0.2 units per μL of Phi29 DNA polymerase; - At least 400 μM total dNTPs.

16. The method of claim 15, wherein the reaction mixture comprises: At least 0.8 units of Phi29 DNA polymerase per μL.

17. The method of claim 15, wherein the reaction mixture comprises: 10 units of Phi29 polymerase per μL.

18. The method of claim 15, wherein the reaction mixture comprises: At least 600 μM total dNTPs.

19. The method of claim 15, wherein the reaction mixture comprises: At least 800 μM total dNTPs.

20. The method of claim 14, wherein hybridizing a labelled probe to the RCA product comprises forming the RCA product in a reaction mixture further comprising greater than 100 nM of the labelled probe.

21. The method of claim 20, wherein there is at least 1000 nM of labeled probe in the reaction mixture.

22. The method according to any one of claims 1 to 3, wherein a plurality of RCA products hybridized to the labeled probe are immobilized in a dispersed manner on the surface of the solid support, wherein at least a portion of the plurality of RCA products can be individually detected by detecting the label.

23. The method of any one of claims 1 to 3, wherein the plurality of labeled probes comprises a fluorescent label, and wherein the detecting comprises detecting fluorescence.

24. The method of claim 3, wherein said counting comprises detecting fluorescence.

25. A method according to any one of claims 1 to 3, wherein a plurality of RCA products are hybridised to labelled probes which all comprise the same label, or wherein a plurality of RCA products are hybridised to labelled probes which comprise two or more different labels.

26. A method for nucleic acid molecule analysis, the method comprising: a) contacting a sample comprising nucleic acid molecules with a plurality of chromosome-specific molecular inversion probes (MIPs), or with a plurality of MIPs specific for chromosomes 8, 13, 21, X, and Y; b) using ligation to circularize the MIPs in the plurality of MIPs hybridized to the nucleic acid molecule, thereby forming a plurality of circularized nucleic acid probes; c) providing a solid phase support, wherein the solid phase support comprises a plurality of oligonucleotide primers bound to the surface of the solid phase support; d) forming a plurality of complexes by hybridizing the circularized nucleic acid probe to a plurality of oligonucleotides bound to a surface of a solid support to form immobilized complexes, and wherein forming the plurality of complexes comprises exposing the solid support to a solution comprising a crowding agent, wherein the crowding agent comprises at least 12% w:v polyethylene glycol (PEG), wherein the average molecular weight of the PEG is between 400 and 800, e) detecting the formation of the plurality of complexes in a process comprising: i) extending the plurality of primers in the immobilized complex in a rolling circle amplification reaction to form a plurality of RCA products immobilized to the solid support; ii) hybridizing a plurality of labeled oligonucleotide probes to a plurality of RCA products immobilized on a solid support to produce a plurality of RCA products comprising hybridized labeled oligonucleotide probes immobilized on the solid support; iii) detecting the hybridized labeled oligonucleotide probe immobilized on the solid support; and f) counting a plurality of RCA products hybridized to a plurality of hybridized labeled oligonucleotide probes immobilized on a solid support based on detection, Wherein the method is for non-diagnostic purposes.

27. A composition comprising a silanized surface on a solid support and a reaction mixture, the silanized surface being bound to a plurality of complexes each comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primers are bound to the solid support, the reaction mixture comprising - At least 0.2 units per μL of Phi29 DNA polymerase; - buffer; - At least 400 μM total dNTPs; - at least 12% w:v PEG, wherein the average molecular weight of the PEG is between 400 and 800.

28. The composition of claim 27, wherein the reaction mixture comprises at least 0.8 units per μL of Phi29 DNA polymerase.

29. The composition of claim 27, wherein the reaction mixture comprises Phi29 DNA polymerase buffer.

30. The composition of claim 27, wherein the reaction mixture comprises at least 600 μM total dNTPs.

31. The composition of claim 27, wherein the reaction mixture comprises at least 800 μM total dNTPs.

32. The composition of claim 27, wherein the reaction mixture comprises at least 14% w:vPEG.

33. The composition of claim 27, wherein the reaction mixture comprises at least 16% w:vPEG.

34. The composition of claim 27, wherein the reaction mixture comprises at least 18% w:vPEG.

35. The composition of claim 27, wherein the reaction mixture comprises at least 20% w:vPEG.

36. The composition of claim 27, wherein the PEG has an average molecular weight of 600.

37. A composition according to claim 27, wherein the reaction mixture further comprises at least 100 nM molecular beacon probe, and / or wherein the plurality of complexes each comprise an RCA product, the RCA product comprising a plurality of hybridized molecular beacon probes, and the composition further comprises a solution comprising graphene oxide, and / or wherein the primers are bound to the solid support in an irregularly dispersed manner, and / or wherein the primers are covalently linked to a silanized surface on the solid support, and / or wherein the primers comprise a biotin portion and the silanized surface on the solid support comprises avidin.

38. The composition of claim 37, wherein the reaction mixture further comprises at least 1000 nM of a molecular beacon probe.

39. The composition of claim 37, wherein the avidin is streptavidin.

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