TW200907067A - Measurement of an insoluble analyte in a sample - Google Patents

Abstract

The present invention relates to compositions, apparatus and methods useful for concurrently performing singular, multiple, high throughput, biological or chemical assays, using nuclease protection molecules which specifically bind to a target of interest. The nuclease protection molecules are capable of detecting targets in complex biological samples, including, preserved, fixed, dried, and/or cross-linked specimen. The reagents and methods of the instant invention provide an effective means for analyzing a target of interest from a complex biological sample without solubilizing or disrupting the sample. Utilization of such methods in clinical and/or diagnostic applications is also described.

Description

200907067 IX. Inventor's Note: The application for the provisional application of the country's provisional application is claimed to be US 60/920,814, filed on March 30, 2007, and US 61 /01 8,71, 7 on January 3, 2008. The right to apply for the date. [Prior Art] A plurality of molecular probes arranged on a surface or "wafer" have been used in various biological and chemical assays. A verification is carried out to determine whether the positive molecules of interest should be the same as those of the probes (4). After exposing the probes to the silk molecules under the conditions of the conditions, the debt measuring device measures whether the molecules interact with the specific probes. These procedures are applicable to multiple (four) inspection procedures to obtain information about the probe filament molecules. For example, it has been used to screen peptides or potential drugs that bind to receptors of interest, in particular: for screening genetically-mutated, dual-gene variants in samples containing insoluble standards, for example, especially in populations Or the presence of a specific pathogen or pathogen strain; used to study gene expression, for example, for I to identify mRNAs that are associated with specific physiological conditions, developmental stages, or disease states. SUMMARY OF THE INVENTION Accurate measurement of genes from a fixed tissue and, in particular, gene expression or recruitment of nucleotides will have four gains. In the case of a clinical sample, the method allows the target oligonucleotide to be directly self-fixed to the tissue without the need to change clinical procedures, rather than having to prepare a frozen sample. There are a large number of stocks of preserved fixation materials that can be used in retrospective studies to identify and validate biomarkers 130267.doc 200907067

And target genes, either used to develop and validate surveillance, prognostic or diagnostic tests, or to combine safety and gene expression, or to understand disease processes. However, performing such measurements from a fixed tissue is still problematic. Measurements by the pcR or heterogeneous methods require extensive tissue and complex extraction and sample preparation methods. In addition, it is often observed that the quality of the measurement depends on how long the tissue has been stored and the sputum is reduced. In contrast, in situ measurements (where rna is labeled and visible in the tissue) can be performed on fresh fixed tissue or preserved tissue and produce similar quality data. Described herein is a self-immobilizing tissue for measuring oligonucleotides, which method comprises recovering a probe from the tissue, wherein the probe is the basis of the measurement' rather than the transfusion acid itself. The invention further relates to the use of nuclease protection as a method of measuring oligoacids from a self-fixing tissue. Methods such as PCR require the target RNA to be solubilized, extracted and purified, followed by reverse transcription and amplification of the resulting eDNA. bDNAf will dissolve the legs. Thus 'crosslinking needs to be reversed or only - part of it can be recovered in a soluble R N A form for measurement by these methods. The square points of the present invention are -ttr, -η.Ι θ 々 Ling Yun 4 to detect the parental oligonucleotide and the soluble oligonucleotide. Tissue sections obtained from clinical or animal experiments are typically fixed, embedded, and stored in a form suitable for subsequent microscopic examination. Conventionally, a method is generally used to immobilize a tissue by immobilizing a tissue between a tissue protein and a tissue protein. The cross-linking tends to maintain tissue morphology and integrity, harden tissue to facilitate sectioning, and inhibit microbial attack. After the tissue-like prints are fixed, they are typically embedded in an embedding medium so that the samples can be cut into thin sections. Stone is the most common embedding medium, but it can also be used for 130267.doc 200907067 with acrylamide and celloidin. Other non-formal in fixations such as ethanol or acid fixation are also known in the art. Aldehyde fixation tends to produce a solid bead edge of the structure of the tissue sample. Variations such as hai often tend to lose the reactivity of antibodies that may be present in tissue samples to antibodies that target such antigens. One effect of formalin fixation is to substantially lock the three-dimensional shape of the protein molecules in the tissue sample. Due to the recent development of novel histochemical reagents, it is now possible to perform histochemical analysis that was impossible when many tissues were previously stored. Thus, a variety of procedures have been developed that can reverse some of the changes produced by aldehyde immobilization and enhance the histochemical staining properties of tissue samples. - A routine laboratory protocol for improving the stainability of tissue samples that have been compared to formalin and embedded in acetamide, involves the encapsulation of acrylamide gel in 1% 2% mercaptoethanol The tissue was treated for 15 minutes and then rinsed with phosphate buffered saline. A method for restoring histochemical staining of tissue samples is described in U.S. Patent No. 5, to Key et al. Another method for restoring the histochemical staining properties of a tissue sample is described in U.S. Patent No. 5,578,452 to Shi et al. Fixing or conserving biological targets in a sample is a technically challenging adventure. It is known that proteins are denatured in the process and usually lose antigenicity, i.e., antibody recognition). Carbohydrates can be chemically altered, such as carbohydrates associated with peptides and protein f in the brewed protein fraction. The process of high-filament time at the expense of nucleic acid in the cell environment (iv) and between other molecules (including proteins, lipids, and carbohydrates). ...recycling and analysis of the child 130267.doc 200907067 The study of nucleic acids in fixed tissues is particularly difficult for a variety of reasons. Formalin immobilization means that the addition of a layer or coating to the embedded sample towel presents a considerable challenge to the probe and the reagent. In addition, because most probes rely on direct connection for detection, the secondary junction is self-identifying and detecting.

The structure and/or the structure of the tertiary structure is like the miscellaneous σ Q. Another aspect is the specificity of identification, 1±' which may be lost due to non-natural exposure to chemicals and other factors during fixation. There is a relatively lack of technical knowledge about the analysis of nucleic acids in fixed biological samples. Daniel DOrazio {American Journal 〇f Pathology 2〇〇2·16〇: 383·384)_Measurement of fixed sample towels, but self-matching frozen tissue quantitative measurements are not comparable to fixed tissue measurements. The authors further indicate that the large variability between the fixed, tissue samples (e.g., Her-2/Neu mRNA) seen can be an anefaet caused by the effect of the fixed pair on the measurement, such as Fixed parameters of fixed delay, time and temperature can be the cause of large performance variability. It is possible that the variability may be due to the variability of the ratio of cross-linked RNA to soluble RNA. , on the use of nucleic acids and other biological indicators in the (four) biological samples: Geometry and methodological knowledge based on the in situ 33 method. In situ hybridization (ISH) has been developed to overcome the limitations of flow cytometry (7) (iv) Cyt〇metry; FCM), karyotyping, and molecular genetics when it comes to immobilization. However, such probes used in ISH are not specific for the genes that are interrupted or altered by deletions, translocations, inversions or amplifications. Therefore, if the sequence analyzed is not properly expressed in the sample, it may not be possible to (4) detect genetic abnormalities. In addition, the number of variables (ie, targets) that can be measured is quite low, and usually 130267.doc 200907067 is not possible to automate. In addition, the ISH technique utilizes freshly isolated cells or cultured cells, rather than fixed paraffinized tissues. To date, previous hybridization studies have been used on cell lines, dissociated fresh tissue, or frozen tissue sections. However, it is often not possible in a clinical setting to operate a biopsy tissue in a timely manner, since the only accessible tissue is paraffin-embedded tissue. Furthermore, the use of fixed paraffinized tissue is advantageous because the tissue structure is preserved. For example, nucleic acids are susceptible to nuclease action. The in situ hybridization of the immobilized tissue can only be performed by repeating the probe of the nucleic acid sequence because the immobilization prevents detection of a single copy sequence. Therefore, a single deletion of a nucleic acid sequence (e.g., a SNP) cannot be detected. The in situ method can be used to measure DNA and RNA from a fixed tissue, which images the tissue and quantifies hybridization and tissue binding probes from the images. Detection can be performed by fluorescence (FISH) or luminescence, and the method can measure DNA (e.g., copy number) or RNA.

Hellborg (Ann. One., 2007) describes PCR and microarray measurements as an alternative to fluorescence in situ hybridization (FISH) detection of RNA. However, PCR-dependent techniques and microarray technology have limitations. For example, extraction by PCR and assays are costly and cumbersome (Shibutani et al, Lab Invest 2000, 80: 199-208). Another important aspect is that the microarray acts on FFPE, where the soluble RNA is of good quality. Microarrays can therefore be used to measure soluble pools. However, these techniques do not enable the rapid and quantitative measurement of RNA from self-immobilized samples. This is especially true when the target is insoluble. It is used to detect related biological targets and organisms in fixed tissues. 130267.doc -10- 200907067 The method of the object will be advantageous. Single-array with low density and high density and both fixed (to capture probes) or programmable (printing error plus additional stylized/capture combinations) or to measure multiple genes in each sample = ( Such as printing in microwells or microbead arrays, including "microbeads" in solution, or hunting and imaging of nucleic acids, either by immobilization or unfixed to the surface, or by mixture Form or individual dry form detected in each of the anti-hybrid characters (such as using gel, electrophoresis, chromatography, mass spectrometry, sequencing, or PCR with nucleic acid _ ankle needle in conventional microdisk assays (or Other methods of amplification) or self-immobilization of samples by hybrid capture or other methods that may be used by those skilled in the art. The reagents and methods described herein allow for self-fixation of tissue samples from infected hosts and infectious agents (different types) In the same experiment, the oligonucleotides were measured from the same sample and even in the same well of the ArrayPlate. Measurements of different forms of nucleotides from immobilized samples (two single samples) can be performed and Products (including, for example, diseased samples versus normal samples, therapeutic samples versus control samples) or the same sample (such as mRNA and DNA ' or microRNA and RNA, or ribosomal RNA and mRNA, or mitochondrial RNA and mRNA, or Comparisons between any combination of ° et al. The present invention may also allow the use of aptamers or other probes to measure proteins. The invention also relates to the simultaneous use of appropriate probes for the measurement of proteins and nucleotides. In another aspect, the invention relates to the hybridization (or binding) of a probe to a cross-linked (and soluble) Rna and subsequently removes and measures the probe or probe/target molecule, even if the target molecule may be damaged or broken Or splitting, but the probe or probe complex is intact or filled with 130267.doc 200907067. Also, in the case of bDNA, the probe complex can be formed on the cross-linked target RNA, and The method is then used to release the complex, even though the treatment may result in destruction of the RNA template, but the combined complex remains sufficiently intact to allow its measurement outside the tissue. The probe binds to the paternal or surface dry molecule ( In any method, such as an oligonucleotide or, for example, rNa) and a linker molecule, or associated with a cross-linking or surface-bound target molecule, the probe is reduced to an analyzable amount relative to the dry molecule, followed by self-organization In addition to and in addition, the invention further provides compositions, methods, and methods for performing one or more biological or chemical assays on any type of single or multiple arrays, including high density arrays measuring hundreds of thousands of stem molecules. The present invention further provides compositions, devices, and methods for performing multiple biological or chemical assays in parallel, 1 permitting multiple samples containing an insoluble target (eg, multiple patient samples to be screened in a diagnostic assay or High yield analysis of a number of potential musical or therapeutic agents to be tested in a drug discovery method. A combination is provided to measure one or more of the dried samples. This combination includes - a surface comprising a plurality of dual release regions. The regions may be referred to as test regions and may be two: at least two of which are substantially identical. Each surface contains at least two, more than ... 2 . Or 2. More than one (e.g., at least about two (10), etc.) such substantially identical zones 6 864 or introduced containing "potentially contained" or otherwise defined for use in biological or chemical arrays. (Such as the space of the sample and the standard word does not mean that the sample T is detected in the sample to detect the sample in the sample or the measurement (10) the test is not 3, the grain target or the undetected is not the J sentence. In the general sense, the present invention 130267.doc 200907067: and = whether the standard dry is included in the sample, regardless of whether it is two = one contains the general "anchor", each of which combines a bifunctional linkage: a sub-knife, a money function The linker molecule has a combination of the second part of the probe having the specificity of the first-order specific to the mismatch, and the probe is placed in contact with the sample containing the - or the plurality of labels. The situation reacts with the detection molecule, and then the inquiry device asks, and the detection target molecule and the probe are opposite to the shore in the test area, thereby generating a verification result. The reaction is further: the present invention is particularly suitable for high Method and combination of yield bioassay: In other preferred embodiments, the present invention can be used for direct screening of drug discovery. For example, high yield assays can be performed simultaneously with numerous (eg ^0:) 96-well micro. In the middle of the plate. Each hole of the plate can be connected by using a secret Arrays in which, for example, 16 different tests are performed. Also, 1 〇 0 disks (96 holes per disk and 16 tests per hole) may not be used for a total of tests; for example, It can simultaneously test 16 different parameters or verifications for each of the 9th, 2nd candidate music orders. The high-yield Si匕-test-only test provides a much more (four) information. t, it is possible to test in a single initial high-yield cotton J:, music substance, selectivity, specificity and / or non-toxic production S method requires extensive follow-up test to test each of the concerns:: these parameters of the drug Several types of high yields are known in the art: verification. Simultaneously performing multiple bioassays and performing multiple assays (ie, the ability to perform at very high yields is a two-pronged advantage of the invention. 130267.doc -13- 200907067 In one embodiment, for example, a 96-well DNA binding disk made of polystyrene having a derivatized surface suitable for attachment to a first amine such as an amino acid or a modified nucleotide is used ( C〇rning c〇star), can be used for 16 different nucleotides The rendezvous is applied to the surface of each well of each disc to act as a pin. The nucleus can be covalently attached to the derivatized polystyrene and the 16 anchors can be used for all screening assays. For any particular assay, 可 can be used - Group specific linkers to plan the surface of each well to be specific to up to i 6 different targets or assay types of interest, and different test samples can be applied to each of the % wells in each disc. It can be used in the same way - the wrong group can be used more: the surface of the hole that is deflated and re-planned by the household, or it can be combined with the (4)-group connector - multi-person. This flexibility and reusability Other advantages of the present invention are embodied in a method for detecting at least one insoluble target, comprising combining a sample that may include the target, as described above. It is effective to contact the (equal) target under the conditions in which the combination is combined. A further embodiment is a method for determining a manifestation of a ship comprising cultivating a sample comprising at least two Wei molecules as a stem with a combination as described above, wherein at least one probe of the combination is a nucleic acid (eg Oligonucleotide 4 acid), the nucleic acid is at least - and terry (i.e., selective) to the insoluble RNA under conditions effective to specifically hybridize the (R) RRa stem to the (4) probe. Another embodiment is a method for identifying (four) (or conditions) for regulating the expression of sputum, which is the above-described method for determining a swell, which further comprises an RNA to be present in the presence of the agent (or condition) The form of expression is compared to the form of salt produced under different sets of conditions. 130267.doc •14· 200907067 f By way of example, FIG. 1 schematically illustrates the ones in the drawings, that is, weaving, in the preferred embodiment of the invention, it is a bitter acid. Weaving is connected with a linker molecule! , the linker] contains two parts. The first part, which is specific for this purpose, is in this description an oligonucleotide that hybridizes to the misspecificity. The second part, which is a specific probe for the insoluble label of interest, here, the "RNA" in this description is an oligo which can be hybridized with the stem. "Although not shown in this figure , but each of the remaining 15 errors may hybridize to the self-linker via the (four) heterosexual moiety; each linker may contain a probe specific for, for example, mRNA different from mRNA 1 or mRNA identical to mRNA! In part, the so-called syllabus can be used to simultaneously verify the presence of up to 96* samples in the same sample (or the same day, the other 5 probes in the array specify (plan) 2 mRNA targets). Performing an assay 'Add each sample (which in this example may be an RNA extract from one of the 96 independent cell lines) to a region or well in a small volume, and The probe is incubated under conditions that hybridize to the insoluble target. To determine if mRNA 1 is present in the sample, an identifiable form and/or a detection device that can be queried for the presence of a signal at a particular location within each region is used. Under the condition that its mRNA is labeled in vivo Incubation, and if mRNA 1 is present in the sample, the detector will detect a signal from the labeled mRNA at the position defined by the error/probe complex 1 or 'in the addition to the region (well) Before or after, the mRNA can be directly labeled in vitro. Alternatively, the mRNA can be raised, for example, by making the RNA and the label "detector" Incubation with a sequence other than the sequence recognized by the probe) 130267.doc -15- 200907067 The mRNA can be indirectly labeled before or after hybridization with the probe. In the illustrative example, 15 samples can be analyzed simultaneously. Since the invention can simultaneously divide at least 20 or more (e.g., up to 1536 or more than 5%): product, it is an extremely high-yield verification system. 7 "Insoluble" as used herein refers to an entity that is heterogeneously present in a mixture or solution comprising both, relative to the solvent. " + / can also be used to characterize the properties of particulates, such as 腧 negative 11 ^,, cytosolic vacuoles, membranes and / or its aggregation is wide. Body ° "insoluble" can also be used to describe the fact that although it can be homogeneously present in solution , but an entity that is heterogeneous due to its attachment to the surface or to the residue. The term can also be used to define a substance (e.g., a precipitate) that remains after dissolution or lysis. The insoluble system as in (4) herein corresponds to any solvent. Preferably, the solvent is polar (eg 'water, saline, PBS, Krebs-Ringer buffer, reticulocyte lysate, serum, etc. and the solubility is between 4°) The temperature between C and l〇〇t is measured under the weight of J. The "insoluble" property can be measured by conventional techniques (for example, light scattering or over- or centrifugation). Insoluble includes other molecules or histochemistry or Physical cross-linking and labeling with particle physics such as organelles, membranes, surfaces, cells or subcellular bodies (such as due to internal acoustics) or chemical association. The method of the invention for analyzing "insoluble" targets Does not include techniques for the analysis of soluble targets. As used herein, "fixed" 咅转PPishan ^ U疋Μ明 has been placed in formalin, buffered formalin, polyoxymethylene or equivalent preservation solution The preserved biological sample. In some cases, the 'fixed sample will also be placed in the stone to prepare for cutting with a slice of 130267.doc -16- 200907067 and any number of U-Hau π 1 ± q Reagents other than Marin , alcohol, etc.) As used herein, "standard stem" refers to a substance whose presence, activity and/or amount is intended to be determined and which has an affinity for a particular probe. The label may be an artificial or naturally occurring material. Furthermore, it may be used in its unaltered state or in the form of aggregates with other substances. The stem may be covalently or non-covalently linked to the binding member either directly or via a specific binding substance. Examples of targets that can be used in the present invention Including, but not limited to, receptors (on vesicles, lipids, cell membranes, or a variety of other receptors); Cha-specific receptor-binding ligands, 4-footed or anti-reagents, and specific antigens & Stators react with multiple strains of anti-sputum, monoclonal antibodies and antisera (such as on viruses, cells or other substances); drugs; nucleic acids or polymorphic acids (including mRNA, tRNA, rRNA, oligonucleotides, dNA, Viral RNA or DNA, EST, cDNA, PCR amplification products derived from RNA or DNA, miRNA, siRNA, RNAi and their mutations, variants or modifications); proteins (including enzymes such as enzymes responsible for cleavage of neurotransmitters, proteases) Kinases and analogs thereof; enzyme substrates; peptides; cofactors; lectins; sugars; polysaccharides; cells (which may include cell surface antigens); cell membranes, organelles, etc. Other such molecules or other substances in the form of valency linkages, etc. As used herein, the terms nucleic acid, polynucleotide, polynucleic acid, and oligonucleotide are interchangeable. The label may also be referred to as a counter probe. As used herein, a "probe" is a substance (eg, a molecule) that is specifically recognizable by a particular target. The type of potential probe/target or target/probe binding partner includes a receptor/ligand; a ligand /anti-ligand; nucleic acid (polynucleotide) 130267.doc -17- 200907067 Interaction, including DNA/DNA, DNA/RNA, PNA (peptide nucleic acid) / nucleic acid; LNA (locked nucleic acid), enzyme, other Catalysts or other substances with substrates, small molecules or effector molecules, and the like. Examples of probes encompassed by the present invention include, but are not limited to, organic and inorganic substances or polymers including metals, chelating agents or other compounds that specifically interact with metals, plastics, cytomembrane receptor agonists, and Antagonists, toxins and venoms, viral epitopes, hormones (eg, peptides, steroids, etc.), hormone receptors, lipids (including phospholipids), peptides, enzymes (such as proteases or kinases), enzyme substrates, cofactors , drugs, lectins, sugars, nucleic acids (including oligonucleotides, DNA, RNA, PNA, LNA or modified or substituted nucleic acids), oligosaccharides, proteins, enzymes, polyclonal antibodies and monoclonal antibodies, single Chain antibody or fragment thereof. The probe polymer can be linear or cyclic. The probe can distinguish between phosphorylated and non-phosphorylated proteins based on differential activity or differential binding. Probes such as lectins can distinguish between different glycosylated proteins. As used herein, the terms nucleic acid, polynucleotide, polynucleic acid, and oligonucleotide are interchangeable. Any of the above substances as "probes may also act as "target", and vice versa. In addition, other agents may be used to help disrupt tissue to allow the probe to access the target knife, such as for immunohistochemical analysis or for "defecting" or "removing" in a fixed tissue, or for example using proteinase k or collagen or soap. Or a detergent, or use a specific buffer to make the molecules in the tissue "refolding. Any phase, 14 main work. Zhuang surface can be used in conjunction with the invention. Surface (usually solid) ^ for organic or inorganic Any of the substances or combinations thereof, including 130267.doc -18- 200907067 (by way of example only) plastics, such as polypropylene (melt) cerium oxide, quartz or sputum, II. The present ethylene 'ceramic' 矽The thickness of the mirror slide or glass cover slip. έ ° " has (for example) glass microscopy; nitrocellulose fibrin over ruthenium film; ^ membrane, such as crepe paper; diazotized fiber type gel 塾, for example, by aerogels;

(:=More...Zhao (including _, which is prepared by using a variety of normal I methods, and both light and dry condensate. When the test is performed with ^ optical detection, it is useful for light-transparent substrates) The surface may have any thickness or opacity, recombination. In contrast to, for example, conventional detection methods, the surface is a porous plastic = surface (iv) opaque disk. On a more plastic surface, for example Tissue culture plate, such as two-well plate, 96-well plate, 256-well plate, 384-well plate, 864-well plate or 1536. (eg modified disk 'such as c〇rning c〇star dna binding plate.) Anchor. Surface directly Combine (for example, in combination), or with a type of surface (such as glass), clean the mouth, and then make it (for example, in the plastic "holes in the microtiter plate), self-contact. The shape of the surface and Not critical. It can be, for example, a flat surface such as a square, rectangle or circle; a curved surface; or a three-dimensional surface such as microbeads, particles, strands, precipitates, f, balls, etc. or caregivers, microbeads , in capillary, or microfluidic surface or nanoparticle surface inclusion An area (such as is usually used for high-density arrays) or a space-supporting group -^1-; v fc is located or identifiable in multiple areas. Each area contains a set of anchors or Ί #获^朱针 (as commonly used In the array or for capturing molecules on the surface), the region separation, its physical properties and its relative positioning to one another are not critical. In the embodiment, the region can be protected by any physical medium that prevents liquids from passing through. The barriers are separated. For example, in a preferred embodiment, the regions can be porous (eg, tissue culture) discs (eg, 24-well discs, 96-well discs, 256-well discs, 384-well discs, 864 wells). Or a hole in a 1536-well disk. Alternatively, a surface (such as a glass surface) may be etched to have, for example, 864 or 1536 shallow holes. Alternatively, the surface may include areas without spaces or holes. For example, a flat surface (such as a sheet of plastic, glass or paper), and individual regions may be further defined by overlapping structures that define individual regions (eg, a piece of plastic or glass). Optionally, before individual regions are defined, Surface can One or more arrays of tin that have been incorrectly associated or associated with the linker. In another embodiment, the array of anchors within each region may be free of blank spaces on the surface of the anchor or by chemical boundaries (such as wax or poly The helium is separated from each other to prevent droplet spread. In another embodiment, the region can be defined, for example, as a tube or fluid control channel that flows through the assay, such as, for example, Beattie (1995) Clin. Chem. 4, 700-706. Tubes can be of any size, such as capillaries or wider caliber tubes; can allow liquid to flow; or can be partially or completely filled with gel (eg agar) Sugar or polyacrylamide), the compound can be, for example, transported through the gel by electrophoresis (passing, flowing, aspirating or having a space with channels (eg, linear channels) such as, for example, Alb〇ta Et al. (1998). Science 281, 1653-1656; Cumpston et al., (1998). Mat. Res. Soc. Symp. Proc. 488, 217-225; and/or Cumpston et al., (1999). Nature Said in 398, 51-54. In such a space-filled matrix, the liquid and/or molecules thereof may travel not only along the vertical direction of the tube wall, but also laterally. In a preferred embodiment, the tube is filled with a knee or space-filled matrix; the swell or space-filled substrate is activated to facilitate anchoring of the anchors and sequentially through different anchors, thereby allowing anchors to be formed within the gel An array (eg, a linear array); and continuously passed through a linker, target, or the like. The array can be linear, 2-dimensional or 3-dimensional. Multiple checks can be performed in a single tube. For example, the single-array in the (iv) tube of the m-bonding link can be reused (e.g., stripped and reused or reprogrammed) in a continuous assay of the same or different samples. In another embodiment, a plurality of tubes are used in a single assay, for example, analyzing a sample of interest in a plurality of tubes containing different arrays. The error in the tube and the error/linker association can be any of the types described elsewhere herein. Table: The area inside or on the surface can also be defined by the modification of its surface. The surface of the silicone may comprise a portion made of modified or derived plastic that acts as, for example, a site for addition of a particular type of polymer (eg, peg may be attached to a polyethylidene surface) Subsequent to carboxyl or amine groups, double bonds, and analogs thereof). Alternatively, the plastic surface may comprise a molded structure such as a protrusion or a convex ghost' which may serve as a platform for adding an anchor. In another embodiment, the region may be a gel crucible (eg, a polyacrylamide gel pad or an air cushion) that is arranged in a desired form on a surface (such as glass and stone, or sandwiched between two glass). Between the stone and the stone plate). Anchors, connectors, etc. can be fixed at night: or can be embedded in it. The gel pad on the surface of the second = his arrangement will be obvious to those skilled in the art

And it can be produced by conventional, conventional, and traditional L4. The relative positioning of the §-type region can be taken as 130267.doc 200907067, which includes, but is not limited to, parallel or vertical arrays, circles or other radial directions in square or elongated or other surfaces. Extended arrays or linear arrays, etc. In-plane spatial discrete regions of the invention exist in one or more copies. That is, there are at least one, two, preferably at least 20, or at least about 24, 5, 96, 256, 384, 864, 1536, 2025, or 2Q25 or more. The same spatial discrete (separated) area. Increase: The number of repeating zones allows for a gradual increase in the production of the test. As used herein, a substantially identical region refers to a region comprising the same or substantially identical array of anchors and/or malware/linker replicons. As used herein, the same is meant to mean that an array or region is intended to perform substantially the same function as another array or region in the context of analyzing the stem according to the present invention. The difference that does not substantially affect the function (ie, the detectability of the standard dryness) follows the principle of small (tetra) acid incompleteness (missing/insertion/substitution) or oligo-nucleic acid incompleteness (bad surface bonding). It does not significantly affect the dry-label determination within the accuracy of the assay. Of course, those skilled in the art will recognize that when only one sample is to be tested, or when a single-combined sample is tested, or when a larger n-area of the array is useful' And not all areas on the surface need to be substantially identical to each other. For example, it may be advantageous to test two different arrays J in parallel, including two sets of arrays on the early surface. For example, + D ' different sets of arrays can be arranged in alternating strips to help compare the doors, , and . In another embodiment, the implementer may wish to include an area that can be detected in a manner distinguishable from other areas on the surface of the surface and, in turn, a bamboo "6/J state 区域 area". For example, 130267.doc -22- 200907067, the localization region may comprise a nucleotide or peptide that exhibits a special form of fluorescent molecules that can be recognized by the scanning detection device as "starting point I· to align the position on the 2-sided region. The size and physical spacing of the T region are not limited. Typical regions have an area of from about 1 _2 to about 700 mm 2 , preferably from mm 2 to about 4 〇 2 , and are spaced from about 0.5 ug to about 5 mm 'and are conventionally viewed Regarding the area, the choice is two. In the preferred embodiment, the 'area spacing is about 5. For example, +, each area may contain a point having a substantially circular shape (for example, a rectangular grid of _ and Saki, such points) The diameter is about (10) micrometers and the spacing is 5 micrometers; this will cover a square area of about 20 millimeters. It includes larger and smaller area and spacing. The area can be further divided to make some or some of the areas Indentations or pits are physically separated from adjacent faults. For example, the number of fields (sub-regions) may be in the range of about _ to about _solid or below the disk. In the embodiment, the area of the hole of the hole (10) may be further 爯 or n n 乂The knives are smaller scale pores, for example from about 4 to about _, preferably from about 16 to about 31, such as from about 4 to the column. The phase of the pores and thus the pores of the pores (or a set of anchors) are physically placed into each The area of the anchored area is more inflammatory, which in turn helps to detect the target that is bound to the probe. As used herein, the term "anchor, 裨*. μ, field buckle and surface association" (for example, fixed on the surface, or covalent or non-covalent disk table U 疋 、 、, face connection) or one part of the surface for this (for example, the connection surface of the plastic surface And may be subjected to any entity or 130267.doc -23. 200907067, for example, a molecule that is associated with or linked to a linker or other substance as described herein. The portion associated with, for example, the linker molecule can be directly associated with the surface, or the coma can contain an intermediate "spacer" portion. Such a gate: a sub-health, such as a multi-substance of this technique = two In one embodiment, the 'spacer is a linear carbon molecule having, for example, about 5 to about 1, preferably about 12, c. In another embodiment, the spacer is (in the type described elsewhere herein).任任— ''' can again) nuclear rainbows that do not undergo specific interactions or associations of 盥 (for example) linker molecules. 〃 (“The terminology used in this article” is also the position of each set of cones in this paper. Known as the "same location, the number of J-glazed knives that are present in the individual anchors present at the site is limited only by the physical constraints imposed by, for example, the size of the anchor. For example, diameter The site of about Μ · μιη can contain millions of errors. For example: when the anchor and the linker have been combined and combined in a specific way, exist, the complex, and the linker The interaction may be irreversible, such as via some covalent bond; or reversible, For example, via nucleic acid hybridization. In a preferred embodiment, tin is a nucleic acid, which can be of any length (eg, nutrient acid) or of any type (eg, dna, (10) octa, naval, LNA or RNA or DNA molecules The pcR product) may be modified or substituted (for example, comprising non-naturally occurring nucleotides such as muscle bitter; via various known bonds (such as amine-based sour vinegar, distillate amine 琉 麟 麟 vinegar, A A single-stranded nucleic acid is preferred. A nucleic acid error may have any length suitable for the present invention. For example,言,销可130267.doc -24· 200907067 is an oligonucleotide having a length in the range of about 8 to about 50 nucleotides, preferably about ι, Η, 2 〇, 25 or 3 核苷酸 nucleotides. In another embodiment, the anchor can be up to about 50 to about 300 nucleotides long, or longer or shorter, preferably from about 200 to about 250 nucleotides. For example, the anchor can comprise a "spacer" nucleic acid as described above from about (9) to about 2 nucleotides, and adjacent to the spacer (example) For example, a short sequence of about 10, 15, 2, 25, or 3 nucleotides designed to interact with a linker molecule ("linker-specific sequence"). Any nucleic acid of any length or type, and which may have any of the assay bases that function in the invention. In a preferred embodiment, the spacers and/or different bits of each anchor at a single point in the region The spacers on the points are substantially the same; therefore, the errors are mainly different from each other in terms of their linker-specific sequences. The 丄1 sub-send can give cadmium an advantage' to allow for efficiency improvement. For example, such a misconnection The sub-specific portion is far from the surface, and thus = it is less physically bound and less sterically hindered when it is closer to the surface. Thus, for example, a plurality of different linkers (e.g., from about 2 to about 100) having different target specificities associate with a particular site at a particular site. As described in more detail below, 'an individual error can include (in addition to a spacer) a plurality of (for example) a fast-linker-specific sequence arranged in a linear manner in a column; this allows for a plurality of different types of linker zebras, 1, _ ^ This error is associated at a specific site. In the following 2, we discuss in detail another way in which a plurality of different types of linkers can be fused at a specific site. _ /,,,,,,,,,,,,,,, Different linker associations with different dry specificities. Due to the physical elasticity of the anchor containing the spacer, the anchor at the 疋 position can be easily combined with a plurality of different linker molecules of 130267.doc -25· 200907067, and a plurality of linkages at one anchor point: sub:=phase The adjacent anchor molecules are physically bound. The number is increased by two or two at a specific site: the advantage is that the plurality of connections are combined at the (four) point; and there is = one: in the embodiment, the same portion (for example, the same in the nucleic acid) The focus of the dry nucleic acid is not a needle. This allows the fish to use a 4 叼 核苷酸 核苷酸 核苷酸 核苷酸 具 实施 实施 实施 实施 实施 实施 实施 实施 实施 米 米 米 米 米 米 米 米 米 米 米 米 米In the other case, the plurality of linkers and the ancient Dingmen are probes that are specific to the stem (there are different (eg, irrelevant) labels. The inclusion of the plant is detected within a specific site. Another advantage of a plurality of different relatively dry π faces is that it is easier to accommodate linkers that associate with (white (or protein) associations and/or with relatively large stems or cells) ^Acid=The composition of the test base is not necessarily limited. Any composition of the error base can be taken as follows: Limit: "The condition is that the wrong system works for the purpose of the present invention, and the s 'in-region A single-stranded nucleic acid error at a mid-site or at a different site may comprise a partially or completely random sequence (e.g., a randomly generated J such as a relative amount of A, G, τ, and/or C is not limited). In one embodiment, the pin is not a "sequence isomer" (eg, a random sequence isomer), that is, a nucleus having a phase of G, C, UT but arranged in a different relative order. Glycosylate. That is, the anchor at a different location of the region, for example, does not conform to the equation: Gn Cn Am Tm ' where nAm is an integer. See, for example, the anchor shown in Figure i, which is not a random sequence isomer. In the anchor of the present invention, G and C do not need to be substantially the same, and the relative amounts of a and τ do not need to be substantially the same. In addition, the net relative amounts of G, C, VIII and D are not necessarily limited. For example, the base composition of the anchor in the region can be relatively rich in GC (ie, greater than 5〇% G+c) to 130267.doc -26-200907067, "CA and T to relatively rich AT (ie, More than 5 〇〇 / 0 T) within the fe within the change. In one embodiment the anchor system is randomly generated, for example, such that the relative amounts of G, C, A, and T are not limited. The error of the nucleic acid-containing spacer and/or the plurality of linker-specific portions does not necessarily conform to any particular limitation regarding the composition of the assay. For example, if the errors at different sites in the region have substantially the same spacer (eg, substantially the same 25-mer or _-mer), each material has a different linker Terry '(four) minutes (eg , 25-mer), riding makes the linker-specific part satisfy the special two as the number of 'A|^G is roughly equal; The number is roughly equal. The oligomers meet the equation Gn Cn Am Tm; and/or the G+C content meets the specific requirements. As a whole, tin will not meet their specific requirements. Likewise, even the linker-specific portions of tin at different sites in the region are substantially different from each other (eg, each linker-specific portion has a difference from at least about the other linker-specific portions of the region, at least about 〇 or 鸠 or _ 歹乂 歹乂 ' Taking into account the entire length of the nucleic acid, the net sequence consistency of the anchor may also be much smaller. | 々丨丨品一 „ For example, S, if each anchor contains substantially the same 25 〇 polymer interval And the 25-mer linker-specific portion (which differs from all other linker-specific portions (10)% in the region), then the error will still differ by only 10% relative to each other. i seedlings may also be peptides or proteins. And π + .... Lean white, for example, 'may be a plurality of antibody molecules or early strain antibody molecules or tablets, 4, tablets & ' or early chain antibodies or fragments thereof, Or a partial-specific binding of a linker of an anti-antibody molecule; in one aspect, the error may be a peptide, and the linker moiety to which it is bound may be an antibody or an analog thereof. In another example, 竑, the anchor can be a foreign lectin (such as to 1302 67.doc •27· 200907067 From Concanavalin A or Lectin, an organism such as Limulus, peanut, mung bean, phase〇lus, wheat germ, etc., which is specific for a particular carbohydrate. In another embodiment, the anchor may comprise an organic molecule (such as a modified or derivatized plastic polymer) that acts as a site for specific solid phase chemical synthesis of, for example, an oligonucleotide. In this case, a derivative The knee can be distributed in an array of discrete derivatization sites, which are integrally formed in the combined plastic surface during the manufacturing process. In another embodiment, the metal ions can be utilized (eg, Ni, Zn, Ca, Mg^^). w^R._ specific or preferred binding. For example, the pin may be a polyhistidine, and the tin-specific portion of the & T may be recorded, which is via the chelating agent and the IM temple heterosexual wood needle The linker or 'chelator may be a poly-histamine which may be a probe-related moiety. Alternatively, the tin may comprise an inorganic substance. For example, the pin may comprise (iv) or a metal of magnesium, and the mis-specific portion of the linker may be Preferably, the whole gossip is such as Xie Ba or called And (4) heterosexual probes: those skilled in the art (4) recognize a variety of other _: wrong 'such as - combined with their general error can also be a heterozygous structure, such as _ in the way described elsewhere Live-chain, or contain (for example)

And the duplex of the protein. The DNA of the heterosexual interaction (the portion is in direct contact with the surface: the "base portion" acid of the body anchor; preferably, the base portion also contains a 3 spacer (tetra) carbon spacer. An example towel, such as the line association (9) as described above, is hybridized to form a package: to this part, the v-point double-stranded (duplex) core 130267.doc -28. 200907067 acid w. For example, the test The base moiety can comprise a linear carbon spacer attached to the surface at the end and attached at the other end to a single-stranded oligo-acidic acid of about (10) nucleuses, preferably about 25 nucleuses (d); The second portion of the double bond may comprise a sequence that is complementary to at least a portion of the base portion (eg, about 40 nucleotides in length), followed by an optional spacer (eg, about 15, preferably about 1G) Nucleus (4)), followed by a linker-specific sequence (eg, 'length from about 8 to about 50 nucleotides, preferably about 2 〇, 25 or % nucleotides, optimally about 25 nucleotides Sequences. Using the optimization procedure known in the art, the complementary portion of the pin duplex and its linker-specific sequence can be altered. The length of the money base is composed to suit the needs of the assay. I 丨 & r- η _ For example, the sequence can be selected such that the linker can be dissociated from the money (4) molecule in the condition that the double strand (10) itself is intact (for example, Separation from (d) somatic molecular melting). Thus, if necessary, the remaining arrays of duplexes can be used to hybridize to the same genomic molecule. Alternatively, the sequence can be selected to make a hybrid/linker hybrid. Dissociating from the two complementary portions of the double bond tin under the same conditions, leaving only the portion of the moiety that is in contact with the surface. In the embodiment, at or at a specific site of the region: all or substantially all The base moieties are identical or substantially identical. Only when initially added to the complementary portion of the duplex anchor, which is a method that requires knowledge of the sequence involved in the base portion of the double bond formation, An array of base residues remaining after dissociation can be reused (5) as 'for hybridization with a linker molecule.' Manufactured by a user who is unfamiliar with the sequence of the base moiety or * can be reused by the array of dragons ability The advantages of these hybrid anchors are now used. For example, the manufacturer can prevent unauthorized reuse of the array of 130267.doc -29- 200907067. The prevention of this reuse is caused by excessive use. The performance degradation or unreliability that can be caused, for example, by using - in the embodiment 'in a specific site within the region μ. That is, for example, only for the -type linker, the specificity, part Specific in one embodiment. In another embodiment, a plurality of different tin points (referred to as "mixing) for a plurality of different rod targets and having a specific J-transport and/or alpha-specificity The site is located at a particular position 2), such as from about 2 to about 100, for example, to at least about 4 or at least about one of a plurality of errors. The advantage of the mixing site is that it allows the detection of different targets with an increased number of locations. In each of the mixed sites, there is a - in all or at least several bits: = wrong. For example, the same error at more than one site can be guaranteed and/or controlled or used for signal normalization. Although it is also advantageous for 'surface sites with only a single (non-repetitive) region), such a single "cluster, the mother of the site of the έμ 匕Α" is directly connected to the anchor Target interaction. The number of errors in the test area (ie, the group at which the individual sites are wrong) can be up to: two, preferably between about 8 and about 9 ( (more or less included), better " Between about 8 and about 300' and preferably between about 3 〇 and about _ (eg, about 64). In some preferred embodiments, for a surface having, for example, a test area (such as a mouth opening), there are about 16, 36 'such as 1 tin/test area' or for 384 test areas (eg For the surface of the hole, there are about 9, 16 or 25 anchor/test areas. In a preferred embodiment, each pin has a different specificity than all other tins in the array in the test area. However, two or more of the four fields of -Hai et al. share the same specificity and 130267.doc •30- 200907067. In an embodiment, wherein the combination of the invention comprises a = test area (e.g., about 864 '1536 or more), so the same can be handled! Test samples, which may be of interest, are parameters that test only a limited number (e.g., about 2, 4, 6 or 9) of their samples. In other words, for a combination comprising a large number of regions, it may be advantageous to have only about 2 to 9 anchors per region. In another preferred embodiment, the array can be composed of molecules that directly capture a target (such as a nuclease protected probe) without the use of cone/pig. Furthermore, an array can consist of only a few to tens of thousands of capture sites (commonly referred to as high density arrays), and there can only be one or more arrays on each surface. The physical spacing and relative positioning of the errors in the test area or on the test area (i.e., the group of anchors at individual locations) are not limited. Typically, the distance between the anchors is from about 0.003 mm to about 5 mm or less, preferably between about 〇3 //, and 々1 mm. Includes larger and smaller anchor intervals (and areas). It is understood that they may be arranged in any direction with respect to each other and with respect to the boundaries of the regions. By way of example, 5, they may be arranged in a two-dimensional orientation, such as a square, rectangle, hexagon or other array, or a circular array having anchors scattered from the center in the form of radial or concentric rings. Anchors can also be arranged in a one-dimensional, linear array. For example, a nucleotide may be hybridized to a specific position along a DNA or RNA sequence to form a supramolecular array, or linearly arranged in a structure flowing through a gel or flowing through a beta surface or flowing through a device. Hybridize with a specific location. Or the 'anchor can be arranged in a |, barcode" form. For example, the anchors can be arranged in long rows that are parallel to each other. The spacing or width between the long lines can be changed in a regular manner to produce a simple, recognizable pattern that resembles a bar code. For example, the number - 130267.doc -31 - 200907067 and the second order can be twice as large as the remaining lines. Lines can be omitted. An additional blank line can be placed after the last line to divide a test area, and the bar code pattern can be repeated in the subsequent test area. The wrong pattern does not need to be strictly aligned with the separation verification hole (test area) or the position of the droplet alone. The term "verification position" shall be used to refer to the position of the calibration surface to which the sample is applied. (The position may be determined by the position of individual droplets of the assay sample or by walls or partitions defining, for example, individual assay apertures on the porous disk. The location of the pin.) The pin pattern itself (for example, the oligo-plotted "bar code"-like pattern) is used to define the exact location of each individual error by pattern recognition, just as each row of bar codes is relative to the remaining rows. The location is identified. Therefore, the first tin does not need to be located at one edge or a corner of each verification position. The first brocade will be discovered by pattern recognition rather than relative to the position of the verification position. As long as the area used for each verification position (eg, the area of the droplet or the area of the aperture) is large enough to determine at least one complete unit containing the repeating pattern of the anchor, each verification point will be specified by the (bar code) pattern for the test position of the test position All targets' regardless of where the pattern is located in the area of the verification position. The anchors need not be arranged in each test area in a strict or even fixed pattern. For example, 'I tin can be attached to particles, (d), or the like, which assumes an arbitrary position within the test area. The location of each error can be determined by using, for example, a detectable tag. For example, linker molecules specific for each type of anchor can be labeled with different camping, illuminating, etc., and the position of the particles containing the particular linker/anchor pair can be based on signals from the linker (eg The nature of the excitation or emission spectrum is identified. Those skilled in the art can prepare a set of 130267.doc •32-200907067 linkers having a plurality of such linked tags (each having a distinguishable spectrum). Also jy, . _ , 43⁄4 can be marked directly. For example, each type of tin can be labeled with a different fluorescent spectrum than the labels on other types of anchors. Alternatively, the particles, microbeads or the like may differ from each other in size or shape or color or fluorescence or signal emission. Any of the methods described in this article can be used. For example, t can be activated by a CCD-based imaging system, a first-scan fluorescence microscope, or a fluorescence-activated cell sorter (Flu〇rescence).

Activated Cell Sorter; FACS) measures fluorescence. The error may interact with a part of the linker molecule (a mis-specific part) or become a specific association with a part of the linker molecule (an anchor-specific part). , "interaction" or, association, "in this context means two substances or compounds such as 'wrong and linker's misspecific part, probe and its target, or = and dry specificity The reporter conductors are sufficiently bonded to each other (eg, a bond, a bond, a covalent bond, or otherwise associated) to be carried out in an I month. The term "specificity" or "specifically" is used in the context of such components (eg, 'wrong and linker's misspecific portion, probe' or label # and (iv) heterosexual reporters)

. 2. In the absence of any protection technology, it is usually not combined with other components that do not want I = 2 parts. In general, parameters can be used, for example, using conventional methods in the art. m is required to achieve specific interactions:: For nucleic acids, for example, those skilled in the art can experimentally make nucleic acids (such as oligo-sulphur picrate) selected, :(: as a linker, an isotropic part) Hybridization, at the same time most:; two: non-specific hybridization of secondary knives (eg other oligonuclear linkers) 130267.doc -33- 200907067 Features (such as length, base composition and degree of complementarity). Usually, anchors, connections The DNA or other nucleic acid sequence of the sigma sigma or detector oligonucleotide will have sufficient complementarity to its binding partner to enable it to be heterozygous under the selected stringent hybridization conditions and the Tm will be above room temperature About 1 〇. 〇 to 2 〇. 〇 (for example, about 37. 〇). The average έ oligonucleotide backbone can be in the range of about 8 to about 5 nucleotides, preferably about 15, 20, 25 or 30 nucleotides. As used herein, '"high stringency hybridization conditions" means that at least 95%, preferably about 97[deg.] to about 1% nucleotide complementarity exists between nucleic acids. Any condition of hybridization will occur at (consistency). However, depending on the desired purpose, hybridization conditions that require less complementarity (e.g., about 90%, 85%, 75%, 50%, etc.) can be selected. Among the hybridization reaction parameters, the change can be a salt concentration, a buffer, a pH, a temperature, a culture time, the amount and type of a denaturant such as formamide, and the like. (see for example

Sambrook et al. (1989). Molecular Cloning: A Laboratory Manual (2nd ed.), vol. i_3, Cold Spring Harbor Press, New York; Hames et al. (1985). Nucleic Acid Hybridization, IL Press; Davis et al. '(1986), Basic Methods in Molecular Biology, Elsevir Sciences Publishing, Inc., New York). For example, a nucleic acid (e.g., a linker oligonucleotide) can be, for example, 6 and 88? Ugly-T (0.9 M NaC Bu 60 mM NaH2P04, 6 mM EDTA, and 0.05%)

The concentration of Triton X-100) in the range of from about 0. 01 μΜ to about 5 μΜ (in a preferred embodiment, about 1 μΜ) is from about 〇 1 μΐ to about 100 μΐ. Or a volume in the range of 100 μΐ or more (in a preferred embodiment, from about 1 μΐ to about 50 μΐ, optimally about 40 μΐ) is added to the test area (eg, a well of a porous disk, in a preferred embodiment) The middle is a 96-well plate or a 384-well plate or a larger 130267.doc -34-200907067 hole in the well) and is at about η: to about 37. . Hybridization of the binding partner (e.g., 'on the surface of the raised nucleotide anchor) at a temperature within the range (in a preferred embodiment, about room temperature) for from about 10 minutes to about at least 3 hours (in one In a preferred embodiment, at least about 15 minutes). Conditions can be selected to allow for high yields. In one embodiment of the invention, the reaction conditions are close to physiological conditions.

Design 1 of other types of substances or molecules (eg, polypeptides, lectins, etc.) that can serve, for example, as anchors or linker moieties, and the reaction conditions required to achieve specific interactions with their partners: In this technique: conventional and conventional (for example, as Niemeyer et al., (DM) Chest. Acids Res. 22, 5530-5539; Fodor et al. (1996), U.S. Patent No. 5,510,27 Pirrung et al., (1992), U.S. Patent No. 5,143,854). Buffer, salt concentration, pH in the incubation parameters

The value, temperature, incubation time, presence of the carrier and/or agent, or conditions that reduce non-specific interactions, and the like. For example, it can be added to a test area containing a wrong antibody (for example, a well of a porous disk, in a preferred embodiment, a 96-well disk or a 384-well disk or a larger-hole disk), such as 6 χ. The concentration of the sspE_T, PBS, and saline buffer is in the range of about 1 () pM to about ι (in the preferred embodiment, about ! nM), and the volume is from about 〇" to about 100 μΐ. Or an antibody (eg, an antigen or antibody-specific secondary antibody) in the range of 100 μΐ or more (in a preferred embodiment, about i μΐ to about 50 μB optimally about 40 丨), and 4. (: to a temperature in the range of about 451 (in a preferred embodiment, about 4. The crucible is incubated with the anchor on the surface for about 1 minute to at least about 3 hours (in a preferred embodiment, at least Approximately 15 minutes). For peptide anchors, a length of from about 5 to about 20 amino acids is preferred. 130267.doc -35· 200907067 In some embodiments of the invention, the errors in the array may be Under the reaction conditions, the mis-specific part interaction with its corresponding linker is substantially the same as the other errors in the array. This ensures a substantially uniform array of misdirected and thus probes. The anchors within the region (i.e., the group of anchors at individual sites) can be "general" groups where each anchor can interact with one or more of a variety of different connectors. A specific part, but with a different two-probe "part; therefore, a single array of general anchors can be used to plan or define a different probe. Refer to the figure! Explain the elastic properties of such an anchor test. Figure 1 schematically illustrates the One of the errors, that is, the anchor 1' is in contact with the linker, which has a specific portion containing the woven fabric and a second portion specific to the target mRNA i. Alternatively, Substitution 2, for example, linker 2, which, like linker i, comprises a pair of W-specific portions, but which comprises a pair of standard mRNA 2 instead of the second part of the standard heterologous mRNA i. Thus, Anchor 1 can be used to designate (or plan, or define, or determine) a probe for any of two or more different target mRNAs. Generate and link high resolution patterns of oligonucleotides or peptides (array) The method can be costly, time consuming, and/or physically difficult. The ability to use a pre-formed array of anchors to plan multiple probe arrays is an advantage of the present invention. Although the general anchor-defined oligo is illustrated in Figure 1. The type of glycoside probe, ^ the same tin array can also be used to plan the array of other probes (such as receptor proteins), 'Bei, if given the type of tin / linker interaction type, 5 noon Change more to possible 'for example, "inflammatory layer" or " piggyback" A more complex layer of needles (such as egg 130267.doc -36-200907067 white/antibody combination). For example, in one embodiment, a set of general anchors can be associated with a set of linkers (covalent or non-covalent Covalently) to form a "joined" array of modifications, as described in more detail below. Thus, the surface of the anchor itself according to the present invention provides novel advantages. In one embodiment of the invention, the anchor can be reversibly connected to the linker Interaction 2 due to 2 'A set of general errors can be reused to shut down a different set of probes. For example, 'oligonucleolytic acid antinoses can be linked by, for example, heating steps that dissociate two oligonucleotides The oligonucleotide moiety is separated and can then be recombined with the second linker. The ability to use an anchor array (which can be costly, time consuming, and/or physically difficult) is another option of the present invention - advantage. The error does not necessarily have to interact with the linker. For example, a misdetectable molecule (such as a camping dye) is coupled (directly or indirectly), and in turn can be used to locate the bridge within the direction of alignment between the detectors (4) The point. Alternatively, for example, the anchors of the 枋 杈 杈 杈 ’ can be detected by a known amount of knives to serve as internal quantitative objects. The term "linker" as used herein, refers to the specificity of the stem of the inclusion or error subset (''material anisotropy') (the first knife and the "possible substance". The linker The two 2 = the younger brother of the needle - part of the double official connection, and can be directly connected or warp;:: covalent bond or non-valent bond to the linker of the tint sex 2: the substance (such as spacer) connection The chemical interaction between the t-parts of the Phyllostachys pubescens L. interacts with (4) changes. For example, the second heterogeneous acid is interacting with the right anchor. The right anchor is a peptide that specifically binds to the oligonucleoside. A nucleic acid that specifically hybridizes to its effective and 130267.doc -37-200907067 under selected stringent hybridization conditions. The nucleic acid can be, for example, raised nucleotides, / brain, sputum, PNA, PCR money, or substituted Or a modified nucleic acid (eg, comprising non-naturally occurring nucleotides such as muscle bitter; via various known bonds (such as amino sulfonates, sulfonamides, phosphorothioates, methylphosphonates, amines) Formic acid vinegar) or semi-synthetic molecules (such as Liba_streptavidin conjugate, etc.). Preferably, the portion of the linker specific for the oligonucleotide anchor is from about 8 to about 5 nucleotides in length, preferably about 15, 20, 25 or 30 cores. If the anchor is an antibody, the portion of the linker with which it interacts can be, for example, an anti-antibody, an antigen, or a smaller fragment of one of them, which can specifically interact with the anchor. Substances or molecules that specifically interact with other types of anchors described above and that can serve as anchor-specific portions of the linker are well known in the art and can be designed using conventional procedures (see, eg, above). Target-specific portions of the linker The chemical nature of course varies with the target, wherein for the target the target-specific portion is a probe and it interacts with the target. For example, if the target is a specific mRNA, then the linker The specific portion of the Ba can be, for example, an oligonucleotide that specifically binds to the hiding under the selected hybridization conditions but does not specifically bind to the interfering RNA or DNA. Using a well-established method, the skilled person can experimentally determine Best _ target hybrid but not An oligonucleotide that specifically interferes with the least hybridization of DNA or RNA (see, eg, above). In general, an oligonucleotide probe for distinguishing a target mRNA present in the background of a large excess of untargeted RNA The length may be in the range of from about 8 to about 50 nucleotides, preferably about 18, 2, 22 or 25 nucleotides. For the biological background of the large background without the competition target I30267.doc •38- 200907067 The nucleoside sputum in the test; ^ u ^ Ganzi catching needle can be shorter. Using a recognized procedure (such as computer program BLAST), the sequence of the oligonuclear acid probe can be selected to make them mutually Not relevant and different from potential interfering sequences in known gene repositories. Conventional procedures (see, eg, above) can be used to determine the selection of hybridization conditions that will allow for the hybridization of oligonucleic acid probes with occupational specificity. For example. 'RNA can be grown (eg, self-grown in any container (such as a porous microtiter plate; p丨 in 1 Λ, a pore of the genus (eg, 96-well or 384-well or more)) The total RNA or mRNA extracted from the tissue or cells of the reagent treatment) is added to an agent containing, as appropriate, a substance for reducing non-specific binding (for example, about 0.5 mg/ml of degraded salmon or baby fish sperm = na, or In the test area of the oligonucleotide (see above) in the buffer of yeast (10) VIII (such as or other), and incubated for about 10 minutes to at least 18 hours at the temperature determined by the continuation test Internal (in the preferred embodiment, about 3 hours). The stringency of hybridization may be the same as or lower than the stringency used to associate the (4) heterosexual moiety with the linker. The design and use of other types of probes is also conventional in the art, for example as described above. In one embodiment, all or substantially all of the linkers that are associated with the mismatch at a particular site contain the same (one or substantially the same) probe that is specific for the mono-, target of interest. In another embodiment, one or more linkers that are mis-associated at a particular locus comprise a plurality of different probes and are therefore specific for a plurality of different scales. The probes can be positioned as a branching structure to the linker, or preferably in a linear relationship, and the σ is the same substance (for example, both nucleic acids or peptide sequences) or 130267 of various substances. Doc -39- 200907067 Combination. In fact, there is a $ 铋 needle on each link to make the target of the debt test at a specific location (4). In _ _, a plurality of probes on a particular linker are specific to the particular target of interest (eg, they are specific to a different portion of the single mRNA of interest and are specific to the cleavage, or to the corresponding mRNA Different portions of the nuclease-protected fragment are specific; this allows for increased sensitivity to the assay of the label, which is present in the sample with low abundance. The number of probes on the linker can be, for example, about four (four), preferably about two, four or one. Of course, it is also advantageous to use a linker comprising such a plurality of different probes for use on a surface containing only a single-(non-repetitive) region. The (four) heterosexual moiety-specific moiety of the linker can be joined (joined, linked) by any of a variety of valer or non-covalent bonds, the nature of which is not critical to the invention. The two parts can be joined directly or via an intermediate molecule. In the examples, wherein the two portions of the linker are all oligonucleotides, they can be joined by covalent bonds (such as phosphodiester bonds) to form a mono-secret nucleic acid. In another embodiment, wherein the heterogeneous portion is a nucleus; and the specific portion is a receptor (eg, a receptor protein) that can be joined via interaction of biotin with a streptavidin molecule . Many variations of these bonds are known (see, for example, State et al., (10) sentence NAR 22, 5530-5539). Alternatively, the two moieties can be directly joined, for example, to recruit "can be amidated and then directly linked to the peptide or protein via a guanamine bond (eg, cross-linking)' or via a guanamine bond or lipid f linkage to the membrane group Joint bonding. Methods of forming such covalent or non-covalent bonds are well known and readily optimized by those skilled in the art. A spacer sequence (e.g., a nucleic acid) can also be present. 130267.doc 200907067 lies between the pin-specific portion and the stem-specific portion of the linker. After the two substances are associated (for example, white matter, protein plus nucleus...he: use two nucleic acids, two kinds of egg complexes), you can use them; horses, ^ heart! Determine the conditions to maintain the integrity of the specific interaction but remove the non-chemical (eg, washing) derived complex: 1 ° substance 'and thus depending on the situation, and n jn except for unbound substances (such as connection + ~ The conditions for the K-complex (for example, the anchor/linker complex) are the same or the reaction is mixed with the salt according to the pressure of ^ /, a little more stringent. The treaty is washed once to ten times or ten times. More than this. Those skilled in the art will recognize that 'multiple types of tin and linker sandwiches can be produced. For example, $1 Β + will be the first set of linkers and anchors (for example, have substantially the same Array connection of the sequence $ w w # 歹 之 , , , , , , = = = = = = = = = = = = = = = = = = = = = = = 阵列 阵列 阵列 阵列 阵列 阵列 阵列 阵列 阵列Etc. In fact, the sandwich layer allows the first set of anchors (eg the same nucleus acid) to be converted into different arrays with 'different specificities' "joined" The group of linkers and anchors can be covalently or non-covalently associated with each other. Conventional techniques can be used. Conventional fabrication of the combinations of the invention - Some of the surfaces that can be used in the present invention are readily available from commercial suppliers. In a preferred embodiment, the surface is a % pore, such as a well or a (10) well microtiter plate, such as by C〇rning C_Locked modified disk. Or, containing holes and including repeated marks or "pits, the surface can be formed by micro-machining materials such as Shao or steel to prepare - mold, then A plastic or similar material is microinjected into the mold to form a structure. Alternatively, it may be assembled from a glass, a plastic material, or the like, such as a polycrystalline material. The separator may be (for example, a hole such that when the three sheets are separated from each other, the partition may be, for example, a hole forming a wall of the test hole. The substrate may be (for example: material... material (9) such as the lower part of the microdisk - the flat surface is a typical surface for biochemical verification, and the material may be flat ± material (for example, broken glass). +tan, or can form the top of the 。. P table and sub-divided shape To provide a sample of holes in the sample hole (which will be by standard procedures ', where the sub-partitions) or holes. Three pieces can be used (for example, the wafer can be installed in the west φ can be used by Xi 4 Park ~ 褒 中 中The procedure used is to join. By synthesizing the oligonucleus (4) by means of a commercial oligo-sulphuric acid synthesizer and/or a pin and a linker (4), it is easy to synthesize: a knife. It is too long to be produced by such a method, and can be generated by an amplification material (for example, PCR) using the f-knowledge program. In the present invention, the 'fourth material' is nucleated by the two preformed nucleic acid. Positioning on or in the surface of test area 2, either by photolithography or screen printing, by inkjet technology, capillary, wire mesh or fluid channel wafer placement, use Electrochemical patterning of electrode arrays, with needles or needles (4) (10)

Or k J1, followed by baking or uv irradiation on the filter (see, for example, Va et al. (1996) 'US Patent No. 5,545, 53 1; Fodor et al. (1996) 'US Patent No. 5,510,270; Zanzucchi et al. (1997), U.S. Patent No. 5,643,738; Brennan (1995), U.S. Patent No. 5,474,796; PCT WO 92/10092; PCT WO 90/15070) ERROR can be placed on top of the surface of the test area or (for example, in a poly 130267.doc -42- 200907067 In the case of acesulfamide gels, some anchors can be made to protrude from the surface and can be embedded in the surface in a manner that interacts with the linker. In a preferred embodiment 'derivatizes the preformed oligo(4)(d) at the 5' end via a free amine group. It is dissolved in a buffer (such as phosphate buffer, pH 85mi) at a conventionally determined concentration (eg, about 1 μM). Out of the river ε〇τα; and with pi ganai spray dispenser (Cartesian Technologies) to about 〇 〇 4

The rising droplets are dispensed at specific locations within the test well, the I side of the test well being the surface of the fresh, dry DNA binding disk (c〇rning c〇star). Depending on the relative rate of oligo-acidic acid linkage and evaporation, it may be desirable to control the humidity in the pores during preparation. In another embodiment, oligonucleotide anchors can be directly synthesized on the surface of the test region using conventional methods such as: photoactivated deprotection of the growing oligonucleotide strand (eg, combined site targeting) Shield ") or patterned dispensing using a nanojet dispenser by deactivating the nanodroplets of the compound. For example, deprotection of all growth sequences to be accepted for a single nucleotide can be performed and then nucleotides are added throughout the surface. In another embodiment, the nucleotide anchor is attached to the surface via a third end of the oligonucleotide using conventional methods. Peptides, proteins, lectins, chelation examples, plastic and other types of anchors or linker moieties can also be routinely produced using appropriate techniques available, and the anchors can be positioned on or in the surface (see, for example, F〇) D〇r et al., (1996) 'US Patent No. 5, 51, 270; Pirrung et al., (1992), U.S. Patent No. 5,14,3,854; Zanzucchi et al., (1997), U.S. Patent No. 5,643,738; Lowe et al., (1985), U.S. Patent No. 4,562,157; Niemeyer et al. (1994). NAR 22, 5530-5539). 130267.doc -43- 200907067 In some embodiments of the invention, the disclosed combination is used in a plurality of screening private sequences and/or for obtaining information about the probe or the amount, activity or structure of the molecule. Call the multi-array screening Array

The Plate Screen; MAPS) method or assay, and the surface of the array containing the anchor or anchor probe for the assays is referred to as the M A p s array or the M A s disc. The components, assays or screening procedures of the reaction mixture can be assembled in any order. For example, the anchor, linker, and target can be assembled sequentially; or in the presence or absence of a reporter conductor, the target and linker can be assembled into solution and subsequently contacted with the anchor. This is a method for detecting at least one of the markers, comprising: making a sample and a bifunctional linker that may contain the target, and the like Contacting under conditions of a first hybrid product of a linker, wherein the linker has a first portion specific for a pinhead acid-producing pin and a second portion comprising a probe specific for the tag, such that The first parent product is contacted with a combination under conditions effective to obtain the first hybrid product and the combined first-hetero#$& Λ /<rt di-hybrid product, wherein prior to adding the first hybrid product The combination comprises: a surface comprising a plurality of regions, wherein at least two regions are substantially identical, each region comprising: at least 8 different oligonucleotide anchors, such as a hybrid product or The second hybrid product is contacted with a labeled detection probe and the detection probe is detected. 130267.doc -44 - 200907067 Each of the assays or procedures described below can be performed in a high-yield manner in which a large number of samples are quickly and concurrently calibrated on each disc or surface (eg, depending on the number of regions in the combination) , up to about 864, 1〇36, 1536, 2025 or more). In addition, many discs or surfaces can be processed at one time. For example, in a method of drug discovery, a large number of samples (each containing a candidate drug (eg, a member of a combinatorial chemical library, such as a variant of a small molecule, peptide, nucleotide, or other substance) can be added to the In a separate region of the combination, it may be added to a biological or biochemical sample and subsequently added to a separate region of the combination, and the probe array located in the region is subjected to beta and may be assayed for each sample. The latest emergence of high-density microdisks, DNA spotting tools and methods for generating data from higher-density microdisks (such as laser technology), robotics, improved dispensers, advanced detection systems, and data management software And continuously, the method of the invention can be used to screen or analyze thousands or tens of thousands or more of compounds per day. For example, in embodiments where the probe is an oligonucleotide, the assay can be for a genetic variation or defect in a large number of samples (eg, a polymorphism or specific mutation associated with a disease such as cystic fibrosis, See for example

Iitia et al., (1992) Molecular and Cellular Probes 6,505-5 5) Presence or absence, pathogenic organisms (such as bacteria, viruses, and protozoa whose owners are animals or plants including humans) or for specific physiological states Diagnostic nucleic acid or polynucleotide screening of mRNA transcripts of disease symptoms (eg, 'binding assays or other assays'). A nucleic acid probe array comprising a portion of an EST (including a full-length replica) can be used to assess the transcriptional form produced by cells (otherwise) derived from EST. Nucleic acid probes can also detect peptide, protein or protein domains that specifically bind to a particular nucleic acid sequence 130267.doc -45- 200907067 (and vice versa). Similarly, assays in embodiments where the probe is an antigen binding molecule (e. g., an antibody) can be screened for a variant protein or for a protein expression that is a symptom of a particular physiological state or condition. In further embodiments, the combinations of the invention can be used to monitor biochemical reactions of proteins, nucleic acids, small molecules or analogs thereof, such as interactions, such as the efficacy or specificity of the interaction between an antigen and an antibody; a biochemical reaction of a receptor (such as a purified receptor or a receptor that binds to a cell membrane) with its ligand, agonist or antagonist; or a biochemical reaction of an enzyme (such as a protease or kinase) with its substrate; or transformation 9 plus or minus the amount of substrate of the product; and many other reactions. These biochemical assays can be used to characterize the nature of the probe or county or as a basis for screening assays. For example, to screen for the presence of a particular protease in a sample (eg, a protease involved in the blood port, such as proteases h and vih), a combination assay can be used to determine the combination of glory substrates specific for each protease of interest. 1 sample. If the terminator binds to the silk and decomposes the substrate, typically the substrate will fluoresce due to, for example, the cleavage and separation between the two energy-transfer pairs, and the ♦ θ _ L can be detected. number. In another example, in order to screen a sample for the presence of a specific kinase (eg, Src, glutamate kinase or hydrazine 70), a group of peptides that are selectively phosphorylated by the kinase of interest can be used. 3 There are - or a plurality of samples of the kinase of interest. Use a well-recognized, conventional substrate: the roe can be incubated in the appropriate buffer and with the necessary cofactors; (In some assays - for biochemistry 130267.doc -46 · 200907067 studies on factors that modulate the activity of kinases of interest, the concentration of each kinase can be adjusted to allow each substrate to be phosphorylated at a similar rate.) After treatment (eg, washing) the reactants under empirically determined conditions to remove the kinase and the undesired reaction components (as appropriate), the phosphorylated substrate can be made, for example, with a detectable agent (eg, Fluorescein-labeled anti-phosphotyrosine or anti-phospho-serine antibody (for example, a concentration of about 1 〇 nM, or greater than 10 nM or less than 1 〇 nM) is incubated together to detect, and the signal can be detected . In another example, a binding assay can be performed. For example, an SH2 domain (such as GRB2 SH2* ZAP70 SH2) can be assayed with a probe array of an appropriately phosphorylated peptide; or a probe array of a particular receptor can be used to screen for the presence of immunodeficiency in the serum. In addition, enzyme-linked assays can be performed in this assay format. The combination of the invention can also be used to detect mutant enzymes that are more or less active than their wild-type counterparts, or for screening for a variety of tests including herbicides or insecticides. Of course, MAPS assays can be used for quantification ( Measure, quantify, in the active ruthenium target in the sample, the restriction condition is that the probe is not completely occupied, that is, the available probe site of no more than about the coffee is bound (or reacted or hybridized) to the stem. Under these conditions, the number of markers can be quantified because the label (4) is more than 1 resulting in more binding probes. On the other hand, in the case where more than about 9% of the available probe sites are bound, there are more (d) which substantially does not increase the amount of & Any of the above types of non-targets can be quantified. In addition, it has been confirmed that even if & 纴 、, θ, history added to the binding mixture = known unlabeled lighter 'standard dry large excess exists (for example, if " to make it saturated MAPS probe array The amount of probe available in the large number is wide) 'also, change the sensitivity of the reaction' to allow quantification even so = 130267.doc • 47· 200907067. In another embodiment, the original meaning The interaction of the needles is used to screen and adjust the standard dry and the two types formed by the label without the probe. It can be adjusted by the probe's stem or ^/r 4, ° or indirect interaction. The target can be presented in a variety of forms including, but not limited to, increasing or decreasing the rate at which the target binding to the probe binds to the probe; the shovel dip, μ ^ and lower 軚, "Blank or non-competitive inhibition of the probe and the target"; or increase or decrease the activity of the probe or the stem" in which case the probe/standard interaction may be increased or decreased. These (4) may be artificial or naturally occurring substances. Furthermore, such agents may be used in their unaltered form or in aggregate form with other materials; and they may be covalently or non-covalently linked to the binding member either directly or via a specific binding substance. For example, an agent that interacts with one of the cascades of a protease that causes blood coagulation, or a mixture of proteases of interest, can be tested with a plurality of candidate agents and then tested as described above. Its activity. Other examples of reagents useful in the present invention are extremely diverse' and include insecticides and herbicides. In another embodiment, the combination of the invention can be used to screen for agents that modulate the expression of the gene. For example, an array of oligonucleotides can be used to identify mRNA species ("relevance" genes, RNA or expression patterns) in a set of genes that are associated with a particular physiological state or developmental stage or associated with a disease state. The term "related" or "relevant" means that the synthetic form of RNA is associated with the physiological state of the cell' but does not necessarily mean that the expression of a particular RNA is responsible for a particular physiological state. For example, a smaller subset of mRNAs that are expressed, up-converted, and/or down-converted in cells that serve as a model of a specific disease state, 130267.doc • 48-200907067; and expression in normal cells &, the manifestation of this change (which does not exhibit a pathological phenotype) can serve as an indication of the state of the disease ("indication" gene, RNA or expression). Terms, dependencies, and "indications" are used interchangeably. For example, cells treated with a tumor promoter (such as 十四rb〇1 myristate) may exhibit gene expression patterns seen in the early stages of mimicking tumor growth. In another cancer model, when a mouse insulinoma cell (for example, cell line TGp61) is subjected to an adenovirus, it exhibits an increased expression of, for example, 0 such as Μιρ·2, and a housekeeping gene such as GAPDH and L32. The performance remains largely unaffected. Upon contact with cells from a disease model, either directly or indirectly and in vivo or ex vivo (eg, in tissue culture), the agent indicating the expression may act as an organism for the disease (eg, human or other A therapeutic agent or drug contact of an animal patient, or a plant, (9) as in an ex vivo (test tube) expression system, and a nucleic acid, as used herein, is adjusted to mean a molecule involved in a measurable reaction or The amount and/or activity of the analog increases or decreases. The kit of the present invention can be used to inspect such reagents. For example, t, can make a series of cells (example

The disease model) is contacted with a series of agents (eg, for a period of time ranging from about 1 minute to about 48 hours or more than 48 hours) and can be used using conventional, recognized methods (eg, 'commercial kits'). Produce total RNA or extract. To increase the amount of RNA to the right, standard procedures such as RT·PCR amplification can be used (see, for example, et al., ed%) Protocols: A to Methods in Amplification, Academic 130267.doc -49- 200907067

Press’ New York). The extract (or amplification product therefrom) may be allowed to contact (e.g., incubated with it) a plurality of substantially identical arrays comprising probes for appropriate indications of rna, and may be identified as associated with indicative changes in expression These reagents. Similarly, agents that modulate expressions associated with a particular physiological state or developmental stage can be identified. Such agents may be artificial or naturally occurring substances, including environmental factors such as substances involved in embryonic development or regulation of physiological responses' or substances important in agricultural enterprises such as insecticides or herbicides. Furthermore, such agents may be used in their unaltered state or in aggregate with other materials; and they may be covalently or non-covalently linked to the binding member either directly or via a specific binding substance. A further embodiment of the present month is a kit suitable for detecting at least one of the specimens in the sample, comprising: a surface comprising a plurality of spatially discrete regions, at least two regions in the discrete regions of the space Substantially identical 'each region comprising at least eight different anchors (raised nucleotides) or one of the other types described herein and a container comprising at least one bifunctional linker molecule having (a) the at least one of the specific first part and the second part of the probe containing at least one of the material (etc.) silk scarves. The implementation of the spread, provided - as above a) The surface of the surface and the group are used to link the ruthenium linker molecule to at least one of the financial members, the bifunctional linker molecule having a specificity for at least one of the (etc.) anchors - a portion and a second M1 comprising a probe specific for at least one of the standard stems, etc., may include (10) if not limited to the description of the tin on the surface 130267.doc -50- 200907067, how much tin is present and its presence Surface connection (association, binding, etc.) linker and error And <showing and using a specific anchor as an oligonucleotide means that it can be packaged as "for example, if the complementary linker of the designed linker is::: = the practitioner can hybridize with (9), for example) Peptides, which indicate that the action will specifically interact with the peptide to form a t-phase (eg) association-linker, such as conditions and reagents (such as temperature and rheology (or other type of association). Any type of control connection may include the tffl structure and the use of the defensive, " ♦ ♦ 丨丨 、 、 、 、 、 、 、 、 做 做 做 做 做 做 做 做 周 周 周 周 周 周 周 周 周 杈 杈 周Information on the method of verification. The description may cover any of the parameters, conditions, or examples disclosed in the following::: This technique is conventionally performed by a conventional procedure. /, White is not, as in this application As described elsewhere, the 'practitioner can connect multiple types of linkers: package:- or multiple specific materials to the surface of the invention to plan any of a variety of probe arrays. In addition, practitioners can Remove a specific set of connection + day line linkers from the surface of the invention and another set A linker (identical or different from the first set) is added to the surface to allow the particular surface to be reused multiple times. This elasticity and reproducibility make purely other advantages of the present invention. In another aspect, the present invention is A method for determining which of a plurality of polymorphic acids is complementary to a particular nucleic acid, wherein each of the polynuclear tracts comprises two different oligonuclear sequences with each of the TMs: 130267 .doc 51 200907067 The nucleus of the two different oligonucleotides in the nucleus of the "probe and the :::: definition corresponds to the polynuclear * oligonucleotide, which contains: limited to the polynucleus" Passing a sample of the molecule comprising the nucleic acid to at least one region of the combination, wherein the region comprises an array of _ · ;; y; 丨 A 丨 苷 苷 , , ,, at least one of the probes Corresponding to each of the polynucleotides, the sample is incubated with the region, thereby allowing the molecule of the nucleic acid to bind to the polynuclear (tetra) acid probe (which is associated with a portion of the nucleic acid) To make one or more of the oligonuclear acid-acid probes This region of the molecule of the secret acid is incubated with a detection oligonucleotide corresponding to the polynucleotide corresponding to a particular one of the array of oligonucleotides, and the acid is Having (iv) a specific oligoacid probe or a nucleic acid molecule bound to another nucleotide probe complementary to the nucleic acid, detecting the presence of the detection oligonucleotides, and thereby identifying which polynucleosides in the array The acid-receiving nucleotide probe is complementary to a portion of the nucleic acid that binds to the probe of the specific nucleotide-specific polynucleotide, thereby identifying which polynucleotide is complementary to the particular nucleic acid, wherein the oligonucleotide is The probe array is immobilized on a combined region, wherein the combination comprises: a surface comprising a plurality of discrete, substantially identical regions of the same number as the number of polynucleotides to be studied, each region comprising: The number of polynucleotides studied is the same | Miao, each error is associated with a bifunctional linker, 130267.doc -52- 200907067 The bifunctional linker has a first part specific to the anchor and contains Such polynuclear ^: in acid At least - part of the oligonucleate. In another aspect of the invention, locating the EST or other polynucleotides further comprises removing the unbound portion of the sample between one or more steps. If not by PCR, for example, probes isolated from the cross-linked target can be expanded or modified prior to detection by a number of methods familiar to those skilled in the art. In this case the recovery probe can be expanded, labeled and amplified. Nucleic acids (e.g., standards) used in the methods of the invention, and oligonucleotides or nuclease protected fragments (described elsewhere herein) involved in the extraction of the stem can be subjected to a variety of conventional enzymatic procedures (including PCR and ligase reaction) are used to amplify such amplification methods for transcription-mediated amplification (see, for example, 仏 et al., (1993). J. Clin. Microbiol, 31,3270-3274). In another embodiment of the invention, one or more nucleic acid targets of interest are labeled with a specific (four) «(4) shirt and subjected to a renase protection program, and the MAPS disk assay has been protected against hybridization of the target of interest. Fragment. Of course, such "MAPS discs" may contain anchors that are not associated with a linker (e.g., they may be directly associated with a target or nuclease protected fragment of interest); those skilled in the art will be able to Any of the prototypes of its rights will demonstrate the advantages of nuclease protection as used with any type of probe array. If the target of interest is RNA and the protective fragment is DNA, then nuclease protection/MAPS assay (NPA) -MAPS) reduces the need for extensive processing of RNA. 130267.doc •53- 200907067 The latter may be susceptible to degradation due to contamination with nucleases and is therefore difficult to handle. Treatment of the sample with a nuclease protection program also allows the sample to have a reduced viscosity. Nuclease protection of the sample may allow for greater sensitivity and reproducibility of the assay. One advantage of the present invention is that the assay is sufficiently sensitive that amplification of the target (eg, by PCR) is not necessary for detecting the signal In the NPA-MAPS assay, the probes in the probe array are stranded identical to the nucleic acid target of interest rather than the oligonucleotides complementary to the nucleic acid target of interest in the standard MAPS assay. In the NPA-MAPS assay, the target of interest may be any nucleic acid, for example, genomic DNA, cDNA, viral DNA or RNA, rRNA, tRNA, mRNA, oligonucleotide, nucleic acid fragment, modified nucleic acid, synthetic nucleic acid or An analog thereof. In a preferred embodiment of the invention, the program is for assaying one or more mRNA targets present in a tissue or cellular RNA extract. The sample containing the target of interest is first selected in the selected Conditions (see above for a discussion of appropriate reaction conditions for achieving specific hybridization) hybridization with one or more specific protective fragments in excess. The protected fragments are polynucleotides which may be, for example, of interest One of the nucleic acid targets has specific RNA, DNA (including PCR products), PNA or modified or substituted nucleic acids. The 'specificity' protection period means fully complementary to the expected binding partner to be selected A polynucleotide that binds under conditions but does not bind to other unintended nucleic acids. The guard fragment can be at least 1 nucleotide in length, preferably from 50 to about 100, or about as long as the full length cDNA. In a preferred embodiment, the protected fragment is a single stranded DNA oligonucleotide. Protection of up to 1 target or more than 1 target can be included in a single hybridization reaction. 130267.doc -54- 200907067 A slave. In hybridization I, the sample is treated with a mixture of - or a plurality of nuclear g-enzymes to eliminate the nucleic acid outside the protected fragment. The (the) protective fragment has been associated with the nuclear-long and (by fact) nucleic acid target of interest. Partial hybridization wherein the (etc.) moiety has hybridized during nuclease protection and protected from nuclease digestion (as in a double bond hybrid). For example, if the sample contains a cell extract, the unwanted nucleic acid can be substantially destroyed in this step, such as a chromosomal group, tRNA, rRNAA mRNA other than the one of interest. Depending on the nature of the hybrid nucleic acid present in the hybridization complex and the sample, any of a variety of nuclear enzymes can be used, including, for example, membrane adenosine RNase, mung bean nuclear s chymase, S1 nuclease. , RNase a, ribonuclease τι, exonuclease HI, exonuclease Vn, RNase CLB, RNase phyM, rna enzyme or the like. RNA*H is especially useful for digesting residual RNA that binds to DNA protectors & This technique is well known for the reaction conditions of such enzymes and can be optimized empirically. Further, for example, a chemical private sequence for allowing prophylactic hydrolysis can be used. Samples may be further processed by procedures well known in the art to remove unhybridized material and/or to remove residual enzymes (e.g., phenol extraction, precipitation, sputum > e to filtration, etc.). The methods of hydrazine, followed by nuclease digestion and, where appropriate, chemical degradation are referred to as nuclease protection programs; various nuclease protection procedures have been described (see, eg, Lee et al., Ο%?) Meth. Enzymol. 152, 633 side ;ζ-等,〇983)(10) μ milk pin). The sample is treated by nuclease protection followed by an optional (option) procedure for passivating nucleases. The sample is placed in contact with the MAps probe array and subjected to the usual steps of the MAPS assay. It can be measured by, for example, a tagged target with a target terry conductor (as described in this article on Standard Finance) 130267.doc -55- 200907067, 'σ 口保濩' and 'or protection The fragment itself may be covalently or non-covalently labeled by a detectable molecule. If necessary, 'may include a normalized NPA_MAps assay—or multiple controls. For example, one or more protective fragments corresponding to nucleic acids that are expected to be present in a substantially constant amount in each of a series of samples (eg, 'constitutively produced mRNA, chromosomal group, then eight or One part of rRNA). The ability to detect and quantify internal normalized controls (e. g., chromosomal DNA) in assays for measuring nucleic acids (e. g., mRNA) that are present in variable amounts is an advantage of using protected fragments in assays. Since the amount of normalization standard can be lower than the amount of mRNA of interest, the assay can be adjusted such that the signal corresponding to the expression gene does not cover the signal corresponding to the normalization criteria. Methods of adjusting the amount of semaphores are well known and will be apparent to those skilled in the art. For example, methods described herein for balancing signal strength (eg, 'signal attenuation, fine-tuning) can be used (eg, with a blocking link; to a higher degree than the signal portion designed to detect a postal job) The label is designed to normalize the signal portion of the standard; to place a plurality of specific linkers that are specific to the normalized nucleic acid or a protective fragment corresponding to a different part of the core: At the location of the standard). The positive target (e.g., mRNA) of interest can be detected simultaneously or sequentially, for example, by any of the methods described elsewhere herein. Regulatory "Nuclear Nucleic Acids In a preferred embodiment, the guard fragments are directly labeled, for example, and labeled by hybridization to a stem specific reporter. For example, the reporter system via a ligand-anti-ligand Interaction with the protective fragment, Example 130267.doc -56- 200907067, such as 'adding the streptavidin complex.... In another example, the protective fragment is converted to X: protected oligonucleic by horseradish Peroxidase (HRP) or singularity (for example, by modifying the nucleic acid moiety of the fragment or by not being straight: having an occlusion, for example, by, for example, enzymatic or chemical treatment; In the case of cleavage, in any of the above methods, after the cleavage of the protection), before or after the sub-hybridization is labeled. I, and the corresponding linker is divided into a control nuclease protection program, which is appropriately digested as needed, and η is used. The non-hybridized nucleic acid has a heterozygous n-protected fragment to contain a prominent (non-蕻", which should be cleaved by a nuclease when the program is functioning properly. The two complementary markers can be detected by hybridization of the probe to highlight the presence of the fragment or: Or protect the highlights of the fragment The body can be measured by the covalent price of the molecule. This control can be placed in contact with the probe array or itself as part of the MAPS assay. Of course, because the difference = can be easily distinguished (for example, with different absorption spectra) Gasstone), so right: differently labeled oligonucleotides can be included in a single assay. In addition, a standard nuclease protection assay such as by gel electrophoresis can be used during the validation to confirm that the protected fragment is treated as expected. Those skilled in the art will be aware of other controls for proper nuclease digestion. For example, any of the known examples in the examples may be included in the assay (eg) nuclease-protected fragments (eg, in Plant nucleic acid assays may include protective fragments specific for animal genes that are not known to be present in plants. ' After detection, it can be eliminated (eg, denaturation, destruction, annihilation, inhibition 130267.doc -57· 200907067 System, block) detect probe (eg, HRP-labeled) signal, wash the disk to remove any resulting reagents, agents, or buffers (eg, denaturing reagents) that can interfere with the next step The protrusions can then be detected by different detection probes (eg, also by HRP markers). Signal denaturation can be used at various stages of the assay, followed by the addition of different detection probes having the same signal transduction portion. Two different fluorescent probes and dual color detection are used in the absence of denaturation or signal blockage. In one embodiment of the invention, as described above, oligonucleotide probes are used for screening A nucleic acid comprising one or more polymorphisms. In a preferred embodiment, the nucleic acid (eg, DNA, such as genomic DNA; or RNA, such as mRNA) comprises one or more SNPs. Conventional, well-established procedures can be used to implement the program For example, to screen for DNA comprising a known SNP or mRNA derived from the DNA, a "SNP-specific" protected fragment is hybridized to a sample comprising a nucleic acid that can comprise a SNP. A "SNP-specific" protected fragment in the context means a protected fragment comprising a SNP that alters a base or (if the mRNA is to be analyzed) a reverse complement of the sequence. The sample is then treated with one or more appropriate nucleases which, under appropriate conditions, can digest unhybridized single-stranded nucleic acids and cleave the duplex at a mismatch (eg, single base mismatch) site ( A duplex) nucleic acid (eg, a DNA-DNA hybrid, a DNA-RNA hybrid, or an analog thereof). Suitable nucleases include, for example, S1 or RHA enzyme oxime. If a nucleic acid comprising a SNP is present in a sample and hybridizes to a SNP-specific protective fragment, the protected fragment will be preserved intact in the digestion procedure and can undergo a MAPS assay and be detected by a probe specific for the sequence of the protected fragment or Detection of oligonucleotide detection. Nucleic acids that do not contain a SNP will be cleaved at the mismatch site between the SNP specific 130267.doc-58-200907067 heterologous protected fragment and the corresponding wild-type sequence in the nucleic acid. Where necessary, conventional methods (e.g., heat denaturation, enzymatic cleavage, etc.) can be used to remove a portion of the guard fragment located distally or proximally of the cleavage site. The assay can be designed such that the cleavage molecule (or a portion thereof) will not bind to the linker, or = the cleavage molecule (even if part of it binds to the linker) will not be detectable by the ELISA or the nucleus Glycoside detection. Another embodiment is directed to detection that can be applied, for example, to detecting SNPs of genomic DNA. In an embodiment of the invention, various combinations of different types of stems in the sample, e.g., warship, Wei' intracellular proteins, and secreted proteins, can be assayed by a single probe array. In addition to the various high-volume tests described above, many other high-yield tests will be apparent to those skilled in the art. One of the advantages of the oxime needle assay is the ability to include multiple "control" probes in each probe array that is subjected to the same reactants as the actual experimental probe. For example. Each region in the array can contain positive and/or negative controls.

The term "positive control clock, 丨A needle is used herein to mean, for example, to substantially interact with a target or interact with it in a quantitative or qualitatively known manner: Control probe for the (η internal) standard of the target interaction. - Columns' This probe controls the efficiency of hybridization. The term, negative control is used herein to refer to the needle. Properly and secondly control the probe to interact with the target 5. This probe controls the hybridization specificity. As an example of the type of control that can be used, the use of the medium-nuclear probe array is used to screen one of the diseases of the lang lang * έ日β月# η ', and the performance of the soil Pharmacy test. As an internal normalization control of 130267.doc, 59· 200907067 such as the number of lysed cells of each sample, the recovery of m β MA — mRNA or the variation of hybridization efficiency, the probe τ „^ Τ卩One or more basal levels or constitutive housekeeping genes (such as structural genes (eg, (iv) proteins, tubulin, or others) or occupational binding proteins (eg, transcriptional regulators or empires) specific probes, I ^ The performance is expected to be unrecognized by the reagents to be tested. To determine whether the reagents to be tested produce unwanted side effects (:: = death or toxicity), the probe array may contain two weeks of death known as cells (progressive) Partially induced genes in the process of cell death) are specific or

Probes induced under conditions of cellular damage (eg, thermal shock I', , shock protein) or cytotoxicity (eg, ρ450 gene). The sensitivity of other control probes to "fine tune" assays may be included in t. Example: = Consider a reagent that modulates the production associated with a particular disease state. If the previous analysis indicates that one of the relevant mRNAs in this group (ie, mRNA-A) is produced in a higher amount than the mtp, 匕, etc., such that the signal is overlaid with other mRNAs, the linker may 々-r is a child, the singer is to use the "fine-tuning" to qualify for two: = plus, to block the linker (which contains the specified - A ^ each specific oligonucleotide sequence) Dilute the sexual connection in the temple W" lacks the sensitivity of the probe-specific sequencing I. Health: thus reducing the sensitivity of the detection of his mRNA. Those skilled in the art can measure the appropriate ratio of blocker = linker to unblocked linker by conventional, conventional methods. In the other steps of the test, the specific element of the sexual element (4) of the "micro-non-active k-substrate dilution" "inactive•, target-specific explicit release ° + case and 5 'the fineness of the case m (d) heterosexual reporters with the same target-specific portion (eg, 130267.doc -60-200907067 nucleotide sequence), but no signal transduction entity or a signal transduction entity with a non-activated or inactive form As used herein, the term "signaling entity" refers to a label, label, molecule or any substance that emits a horn or is capable of producing such a signal, for example, a luminescence molecule ' luminescent enzyme or is not disclosed herein. Any of a variety of signal transduction entities. In a particularly preferred embodiment, "fine tuning" can be performed in the step of contacting the over-labeled complex with the beta connector. A set of price-measuring connectors can be designed (for example) to fine-tune the sensitivity of individual stems in the assay. For example, if a specific (4) is known to be present in the sample at an extremely high level, the labeled detection link of the standard can be diluted by an empirically-measured ''blocking detection linker'), The linker comprises a heterosexual moiety of the temple (eg, a nucleic acid-suppressing sequence), but does not include a moiety specific for the reporter reagent, or a specific component that includes a dry-specific moiety and a pre-combined reporter reagent reagent That is, it does not contain a part of the test conductor, and the part may not exist, or prevent (for example, blocking) interaction with the reported conductor reagent (such as a miscellaneous parent). This fine tuning is sometimes referred to herein as Signal "Attenuation,. The sample tested in the assay of the present invention may comprise any of the above-mentioned standard stems or otherwise. The liquid sample to be assayed may have any volume suitable for the size of the test area, which is at about (10) Nai Raise to about (10) microliters. In a preferred embodiment, #1 I + V,: & T liters of liquid is applied to 1536-well microdroplets. It can be used for a variety of high-throughput analyses. Any of the methods (such as 'by dispensing, The sample is placed in contact with the probe array based on inkjet dispensing or by using a replication needle. Conditions that are effective for up to ten other stable interactions (eg, salt concentration, 130267.doc -61 - 200907067 positive, temperature) , incubation time, etc., see above) to cultivate the sample. These conditions can be routinely determined. After incubation, the sample can be treated (eg, washed) as appropriate using empirically determined conditions to remove unbound standard bar to enable specificity. The sexual interaction remains intact, but the non-specific binding substance is removed. For example, the sample may be washed about once to ten times under the same or slightly more stringent conditions as used to achieve the probe/standard dry binding. Or more than ten times. A sample containing dry RNA (for example, lake A, rRNA, (10) A 'viral RNA or total (four)) can be prepared by any of a variety of procedures. For example, the live tissue of the mRNA to be (iv) can be cultured. The substance is applied to the surface of the surface, such as the individual wells of the microtiter plate. After obtaining the desired cell density, the cells can be subjected to &,,,,,,,,,,,,,,,, Agent) Depending on the assay, the reagent of interest can be used in a variety of ways (for example, by copying a needle guard (such as a 96 or 384-pin tool from Beckman), by aspiration or borrowing Inkjet dispensing) is added to the cells' and cultured with cells for any suitable period of time (eg, between (four) minute spots for about 48 hours). Conventional, well-established methods can be used (eg, commercially available kits for preparation from in vitro or in vivo sources) The total hidden, mRN A-specific extract of the tissue or cells. In one embodiment, the cells are solubilized (or infiltrated) in the presence of a hexa-nucleus-protected fragment. In the absence of other purifications of, for example, other cell components, the crude lysate is used directly (for example, in the wells of a microtiter plate). If the cell line is solubilized in the absence of a nuclease-protected fragment, such protective fragments may subsequently be added to the lysate as appropriate. 130267.doc-62-200907067 In a preferred embodiment, for example, wherein nuclease protection The fragment is detected by contacting the cell of interest (eg, a cell on the surface of a well of a microtiter plate; a tissue or a cell in an entire organism sample; or an analog thereof) with an aqueous medium (dissolving solution). Prepared, the aqueous medium comprises a surfactant or detergent (for example, from about 1% to about 5% w/v and an agent (for example, formamide (for example, about 8% 'about 60%, handsome hydrochloric acid)胍 ('J is, force 〇. 1 M-about 6 M), guanidinium isothiocyanate (for example, about 105 about 8 1^) or urea (for example, about 4% _ about 46%, ^^ or About 7 Rivers, which may act as a chaotropic agent alone or in combination with one or more other reagents. The aqueous medium may be buffered by any standard buffer. In a preferred embodiment, the buffer is about 0.5-6X SSC, more preferably about 3X SSC. As the case may be, the aqueous medium may also contain an appropriate concentration (for example, about (M-2.0 mg/ml) Preferably, the tRNA is about 〇 5. The nuclease protected fragment can also be added to the aqueous medium prior to its addition to the cells. The optimal concentration of each protected fragment can be determined empirically using conventional methods. In embodiments, the concentration of each protected fragment is from about 3 pM to about 300 pM, more preferably about 30 pM. The cells are grown in an aqueous solution until the cells become infiltrated and/or lysed, and the DNA and/or mRNA are from the cells. Release into an aqueous medium. The cells are in an aqueous medium at an empirically determinable temperature (for example, from about 37 to about 5 〇c, preferably from about 90 ° C to about 11 5 ° C). The incubation may be empirically determined (eg, from about 1 minute to about 60 minutes). For example, in one embodiment 'where DNA and RNA are released from the cell in a denatured form capable of binding to the protective fragment, The cells are cultured in an aqueous medium at a temperature of about 9 Torr to about 115 ° C, preferably about 1 〇 5 ° C, about i 130267.doc • 63 - 200907067 knives to about 60 minutes, preferably about 5 Minutes to about 2 minutes. If necessary, for example, 'when needed in the absence of RNA When the dna is checked, any one of the conventional ribonuclease enzymes can be bred. The selection of appropriate ribonuclease and the optimization of the digestion conditions are conventional and easy to be determined by skilled workers. & In another embodiment, it may be at about 9 〇 ° C to about i 〇〇, preferably about 95, in the presence of one or more protective sheets. The mRNA is prepared in an aqueous medium in a period of from about 5 minutes to about 2 minutes, preferably about 1 minute. In this case, the mRNA is substantially released from the cell in a denatured form capable of interacting with the protective fragment, and (10) is substantially internal or attached to the cell' or is not available for the probe due to its double-stranded nature, or Forms that are capable of binding to the prosthetic fragment (eg, undenatured) are released from the cell. Without wishing to be limited to any particular mechanism, it appears that as the nucleic acid self-dissolves/infiltrate the cell, it is 'sufficiently denatured to allow it to bind to the protective fragment to form a stable duplex" the duplex is resistant to endogenous or exogenous agents. Or enzymatic degradation, and proteins (eg, nucleases) within the cell are denatured and/or non-activated. After preparing the nucleic acid of interest by the above procedure, the sample can be diluted to an appropriate volume such that the aqueous medium does not inhibit exogenously added proteins (such as nucleases (eg, S1 nuclease), proteases (eg, polymerases required for pCR reactions) or The function of a binding protein (eg, streptavidin)). The amount of the diluent as described above and the identity and amount of the component to be used in the aqueous solution can be determined empirically using conventional methods. For any of the methods of the invention, the target can be detected by any of a variety of procedures well known in the art and/or described elsewhere herein (eg, for use in 130267.doc • 64. 200907067 detection of nucleic acids) The enzyme protected fragment) is labeled. For example, a target molecule can be directly or indirectly coupled to a chemical group that provides a valence signal, such as a chemiluminescent molecule, or an enzyme or fluorescent molecule that catalyzes the production of a chemiluminescent molecule (such as twilight Or cy5), or a time-lapse fluorescent molecule (such as one of the chelated lanthanide metals), or a radioactive compound. Alternatively, the target may be specifically reported to be a conductor (eg, an antibody, an oligonucleotide as shown in Figure i, or a probe and a target as described above) after it has reacted with the probe. Any of the general types of molecules described). One type of fluorescent molecule can be an "up-conversion phosphor", i.e., fluorite that absorbs and excites at long wavelengths (e.g., IR), followed by a shorter wavelength (e.g., visible light). Since the up-converting phosphor absorbs longer wavelengths than most of the latent interferents f present in a typical sample to be analyzed, the up-converting phosphor causes the material in the sample to be compared to the light absorber that absorbs the shorter wavelength. The resulting interference is reduced. The narrow emission spectrum of most upconversion phosphors also allows the simultaneous detection of a large number of different upconversion spheroids. Up-conversion phosphors are well known and well known in the art and include, for example, rare earth metal ions, such as yttrium (Yb), yttrium (Er), yttrium (Tm), especially in the form of oxysulfide salts. And 镨 (Pr)). Up to the rib species or (10) or more of the above-mentioned independently convertible upconversion type optical bodies have been described (for example, see one (10)

Agent Detection and Identification, April 27-30, 1999, DARPA, Biological Warfare Defense, Defense Sciences Office). The phosphor can optionally be attached to any surface such as microspheres or latex beads. Like other fluorescent markers, up-converting phosphors can be transferred by energy to a label that is close enough to the linker, target, or reporter conductor. 13Q267.doc -65- 200907067 (or close enough to the linker, target, or The detection of the mark on the conductor can be transferred to detect. Furthermore, as with other signal transduction entities disclosed herein, up-converting phosphors can be used to determine the amount of target and can be used in any of the various procedures described herein (eg, to detect nucleic acids). Do a protection clip). Of course, up-converting phosphors can also be used to detect targets that are knife-coated on the surface in any other way, for example, targets that directly bind to the surface (including nucleic acid-protected fragments), and differently on the surface. The target of the acid is directly bound to the target, or via a bifunctional linker to the surface of the probe that is substantially flat = or anchored (different or substantially identical) in any desired tissue or form. Any surface can be used, for example, through a system or a solid surface (such as microbeads). The microbeads used in any of the assays of the invention may be of any type, such as made of any material, magnetic and/or non-magnetic; and the microbeads used in the mono-assay may have substantially the same or different sizes and/or Or shape. A variety of more complex sandwich detection procedures are also available. For example, the target can be associated with a first portion that is specific for a pair of the target and a first (ie, the same) reporter reagent (eg, labeled polynucleotide, antibody, or pot analog) The two parts of the bifunctional molecule hybridize. The difunctional molecules can be designed such that any desired number of common reporter conductors are available for use in each assay. For any of the methods of the present invention, a variety of complex sandwich type detection programs can be used for labeling. For example, the stem can be bifunctional (or more) with a portion of the first part that is specific to the target and a pair of "reporter reagents" "the second 130267.doc-66 - 200907067 Functional) molecules ("detect linkers)) interact (eg, hybridize). The term "specific" has the meaning as used herein with respect to, for example, the interaction of a probe with a stem. As used herein, the term "conductor reagent" refers to any one of a marker polynucleotide, an antibody, or a molecule of the general type described herein, and a probe of the general type described herein. The sub-sets can be identified by any of the methods described above, for example, in conjunction with the probes and the labels (interacting or associating with their respective binding partners). The detection linker may also comprise other sequences', e.g., sequences specific for the target, but different from (without overlapping with) the target specific portion of the corresponding anchor binding linker. Any sequence present in the detection linker can serve as a recognition sequence for the detection probe or reporter reagent. In a preferred embodiment, the detection linker is a polynucleotide. The detection linker can be designed such that any desired number of common conductors "-type μ can be used in the assay. For example, a set of detection linkers can be designed such that each detection linker is specific for a different target, but contains a combination of the same (common) reported conductor reagent or a limited number of reported conductor reagents. Site. The ability to use a limited number (eg, ") to report multiple conductors in a single-test provides the advantage of reduced cost and reduced background. When the :' detection linker/reporter reagent combination can be used to detect any of the two: the standard dryness 'e.g. as described above with respect to the type of target arrangement detectable by the up-conversion type phosphor. The best implementation of P, the 彳 彳 linker can be edited :::: procedural period has been detected by the nuclease protection fragment from the control, the highlight of the sequence of the nuclear (four) two ... Μ 先 first mark. In this example, 130267.doc •67· 200907067, the detection linker contains a pair of standard dryware that is specific to the common control protrusion sequence, and in the "part of the case and in the case of the case, the detection linker is at the beginning of the test. = two on - preferred enzyme-protected fragments. If necessary, if all of the nucleic acid out sequences on the nucleus = cleavage have been cleaved from the nuclease-protected fragment, then = the control part of the blunt-protected fragment will hybridize to the cleavage-protected fragment. The light-specific portion of the H-linker will not bind and will remain Can be used to enter! = control the protruding surface 'if for example due to the nuclease protection program period: step =. In addition, the control of the salient-specific sequence is not self-protected. The cleavage of the 70-nuclease nuclease moiety is incompatible with the control of the specificity and will not be used for further hybridization. In a comparison = compliant & heterozygous: enzyme-protected fragment and binding _ linker complex:; another "nucleus two: reported: body reagent hybridization, the reporter reagent contains a letter (for example, fluorescent dye, hapten (four) The conducting entity produces any other A-detectable signal or signal knives of the entity, as described herein, and the specific portion of the singular-specific portion (eg, acid). The reporter reagent will preferentially interact with the nuclease. The protected fragment ", the two columns of the cleavage of the 遴 遴 &&; 】 】 】 】 】 】 】 】 】 】 】 】 】 】 】 】 】 】 】 】 】 大 大 】 】 】 大 大 大 】 】 】 】 】 】 】 】 】 大 大It will be apparent to those skilled in the art that many other variations of the refreshing detection procedure can be routinely determined to be useful for achieving binding or other stable interaction conditions for the use of specific reporter conductors or for the use of conductors. .doc -68· 200907067 月心干ΛΛ The method of measuring the linker complex (see above) 〇 Example, can be between about 15 ° C and about 45 ° ( (preferably about room Use under temperature). CT' target will be firefly Phototrophic nucleotide reporter (in a buffer such as SSPE-T or other buffers having a degree of about 10 Μ Μ to about i μ Μ or about ! _ above, preferably about 15 minutes to 2 hours or more) Preferably, the minute is up to 60 knives.) After incubation, the sample can be treated (eg, washed) as appropriate using empirically determined conditions to remove unbound (tetra) heterosexual reporters to keep the specific interaction intact, but removed Non-specific binding substance. For example, the sample may be washed about once to ten times or more than the same or slightly more stringent than the target/reporter binding. Reporting the conductor label can provide another specificity to the initial hybridization reaction! · The biolayer, for example, in the case where the target-specific oligonucleotide reporter is targeted to the sequence portion of the target nucleic acid different from the probe oligonucleoside S, Or, in the case where the probe and the reporter antibody recognize different antigenic epitopes of the target antigen, in addition, the target-specific reporter label allows, and adjusts, the sensitivity of the reaction. For example, if it is a correlation expression Partial Targets are expressed in very low amounts and can be enhanced by hybridizing a binding target to several (eg, about two to about five or more) target-specific oligonucleotide reporter conductors. Signal 篁 (signal amplification), each of the target-specific oligonucleotide reporters specifically hybridizes to a different portion of the dry mRNA. The ability to independently detect two types of labels allows for another MA ps assay One type of control. Some of the linkers specified for a particular anchor site (eg, about 10 〇/. to about 100%) may have a label attached to one end (eg, fluorspar). For example, rhodamine (rhodamine) ) or Cy5 fluorspar can be attached at 130267.doc •69- 200907067 at the end. Such modified linkers are referred to as "control linkers". ^Linker and counter linker conjugate 6 and merging = after incubation with the resulting probe array, band = fluorine can be used to terry ^ or Another called the speculative mark (such as chemiluminescent mark)), " civilian conductor (or the standard can be directly labeled by fluorite or other price tag two and can determine the ratio of the two signals. The existence of the control linker Allowing the number of functional errors (eg, tin that can interact with the contacts) within the test area and between the test areas (ie, the ability of the array to combine the stems for the purpose of normalizing the signal), Acting as a basis for quantifying the binding identity helps to locate the anchor site and/or provide a positive control, for example, in the absence of a signal due to the absence of a target in the sample. In one embodiment of the invention Two different labels (eg, fluorophores) can also be used to test two different groups of molecules. The ability of a molecule to recognize the presence of a target by the spatial resolution of the signal allows the use of a single type of label for different target molecules. Independent investigation The ability of a label (eg, a fluorescent label (such as luciferin and rhodamine) that emits at a distinguishable wavelength, or a different up-converting phosphor) allows for other flexibility of the method of the invention. For example, two or two Each of the above standard stems may be directly or indirectly labeled by its own, unique detectable label, except for example, to identify the position of the target on the surface or the size of the bead positioned by the target. Externally (or instead), this allows detection of the target based on features specific to the target, such as the color of the emission. In another embodiment of the invention, it can be independently at a single site within the region Detecting the multiplicity of the target. For example, two or two 5, 6 or more can be detected at a site defined by a single group (substantially identical) 130267.doc • 70· 200907067 That is, you can use _ 2, 3, 4, two specificity, and the part of the opposite sex. If you use the - group (for example, this combination, the gas is at the ^ ^ point and the member of the group of anchors is allowed to be Combine four different standard dryness at the site. The distinguishable label (direct or indirect) label can independently determine the presence of each of the four labels at the site. In the region, for example, an array of five anchors (a set of errors) can be used in the above case. To detect up to 20 different targets. Classes (not in a single-site, but evenly distributed, the Ge type of tin distribution is not in any desired manner) on a solid surface (such as microbeads or flow through) On the device), the number can be independently produced: _ eg, up to 80 or more than 8); and other aspects (such as bead size or scattering) can be used to provide information about the nature of the stem or the target group Multiple linkers with different target specificities (eg, in the range of about two to about 5 or more) and errors at the locating point (a set of substantially identical anchors or "mixed sites" The association of ") (sometimes referred to herein, hybrid linker") forms the basis of other embodiments of the invention, which will be apparent to those skilled in the art. For example, an anchor at a particular locus can be combined with a mixture of linkers specific for a plurality of different guard fragments, each of which corresponds to a different portion of the nucleic acid of interest (eg, mRNA) (for the nucleic acid of interest ( For example, different parts of mRNA are specific). The presence of such multiple different linkers at a single point allows for a significant increase in the sensitivity of detecting a target of interest (eg, mRNA) (eg, 130267.doc 71 200907067, eg, a low abundance in the sample) Big. Each point can be designed such that the number of linkers corresponding to different portions of the postal money specifying the site is inversely proportional to the abundance of the mRNA in the sample (in a manner that can be determined empirically). For example, if it is found that in a preliminary experiment, RNA is present in the sample in a manner that is excessively large in excess of the mRNA of interest - then the relative number of linkers corresponding to the different portions of the two mRNs can be adjusted. The relative intensities corresponding to the respective mRNA2 signals are made substantially identical. That is, the signal strength can be adjusted such that the signal does not cover the signal corresponding to the first: in a manner of: adjusting the assay to allow simultaneous detection of a plurality of mRNAs present in the sample in widely varying amounts, the balance corresponding to each The signal intensity of mRNA. In another embodiment of the invention, as described above, a particular site may comprise a plurality of unrelated or different stem or guard segments specific for the connection eight to allow detection of a greatly increased number of stems with a single tin array Or protect the slaves. For example, if each of the 35 锚 anchor arrays contains a linker specific to one or more different targets, the array can be used to detect as many stems as possible, and the arrangement allows detection to be detectable. The array of low density standard stems is converted into an array that detects high density targets. Multiple molecules (e.g., protective fragments) that bind at a single site can be detected, for example, sequentially or in parallel using the detection methods described elsewhere in this application. (For a detection of a linker, and a report of a conductor reagent, for example, see above; > part of a noisy sandwich type detection method.) In one embodiment, for example, with a first detection The system (eg, detecting a linker/reporter reagent, or a detection probe specific for it) detects a first target at a particular site (eg, protection 130267.doc • 72-200907067 fragment); Use a conventional procedure (eg, an enzyme that produces a chemiluminescent signal to change the pH to non-activate the first beauty and a conductor reagent) to remove or deactivate the younger detect linker/reporter reagent to the second target Dry the specific second 贞^; and at the same site, make the price probe (4) (four) the second "# 接 / report conductor reagent or ring. Lili ^ Find the temple, perform multiple cycles as needed - in another embodiment, the first, ^ .'i,| 4^ λ- _ 疋 press / report conductor reagent or detect 铋 needle added to The group 〃 'θιΙ -7 甲仁 is added to the second debt test link / reported conductor reagent or detected Park. L ^ wood meter is not removed or not live 1 in this embodiment By subtracting the amount of signal corresponding to the first-standard light: the amount of signal corresponding to the second target is determined. In another embodiment, the first sub-reporter reagent or detection probe is added to the second detection link/reporter or the debt detection probe substantially simultaneously to the combination n as described above (for example) The differential detection can be used to detect individual detections as described elsewhere herein. In any of the methods of debt testing described herein, the debt test linker comprises a portion that is specific for the same or different reporter reagents. For example, if four standard stalks are associated with a linker at a particular site, the valence linkers specific for each of the four standard stems may each contain a specific reagent for the different reporters. Part of sex. Thus, after all four of the tethered linkers in the set have been crossed with the standard bar, four different reporter reagents can be used to detect the target sequentially or simultaneously as described above. Other detection methods and combinations of the above methods will be apparent to those skilled in the art. Of course, the "hybrid linker" is also advantageous for use in surfaces that contain a single (non-repeating) area. In another embodiment of the present invention, the one or more targets of interest are specifically 130267.doc • 73- 200907067. The anchor of the opposite sex is not associated with the linker and is directly associated with the target or the target. The target or the target may in turn interact with one or more detection linkers or one or more assay probes. Targeted or unlabeled targets can be detected by any of a variety of conventional and conventional procedures in the art (see, for example, F〇d〇r et al., (1996), U.S. Patent No. 5,510,270; Pirrung et al., (1992), U.S. Patent No. 5,143,854; K〇ster (1997), U.S. Patent No. 5,6,579, U.S., et al., (10)7) U.S. Patent No. 5,653,9 (d); Hlr (1996) U.S. Patent No. 5,565,322; (10) et al. (10) 7), U.S. Patent No. 5,670,322; Lipshutz et al., (1995)

BioTechniques 19, 442-447 ; Southern (1996). Trends in Genetics !2, 11 (Μ15) Methods of detection include enzyme-based reading, colorimetry, SPA, automated radiography, mass spectrometry, electrical methods, debt Absorbance or luminescence (including chemiluminescence or electroluminescence) and detection of light scattering from, for example, microparticles used as labels. In addition, it can be imaged, for example, by charge coupled device (CCD) or fluorescent microscopy (eg, scanning and confocal fluorescence microscopy), or by attaching a scanning system to a CCD array or photomultiplier tube, or Fluorescent markers can be detected by using array-based detection techniques (eg, the surface electrical power of each of the 1 〇 micron portions of the detectable region can be made sufficiently south, the surface plasma resonance can be used). Alternatively, the array may contain a label (eg, one of a pair of energy transfer probes, such as luciferin and rhodamine), which can be transferred by energy to the label of the tenth dry or reported conductor ( It can be detected by the linker, the stem or the mark on the conductor. Many fluorescent-based ray systems have fluorescence intensity 130267.doc •74· 200907067 degree fluorescence deflection (FP), jet lag, camping light resonance energy transfer and uniform time release type H 疋 (Homogeneous time -released fluorescence ; HTRF). The analysis of the repeated bar code pattern can be achieved by pattern recognition (by finding the appropriate point or line of each specific mark mark relative to the position of other points or lines with respect to other specific points or lines), and then quantizing the mark strength. Bar code recognition devices and computer software systems for knife resolution or one-dimensional arrays are conventionally produced and/or commercially available (see, for example, Rava et al., (丨 996), U.S. Patent No. 5,545,531). Another method that can be used for home detection is 2_photon fluorescence, which includes by intimately bonding to the surface of the array (eg, by being closely attached to the substrate on which the array is formed or by being included in tin or a linker or The use of other components in the binding complex to be intimately juxtaposed to enhance the endogenous nature of the components bound to the surface of the array or to bind the fluorescent light of the scorpion scorpion 筻 筻 枓 枓 。. Other fluorescent

Methods or utilities include lifespans, values, polarization, energy transfer, and the like. For example, the method of δ ′ 3 Hai allows simultaneous 丨曰 八 八 上 η T u t彳 劂 and distinguishes multiple targets within the same site.

The target 'and in some cases can distinguish between the gentleman's furnace T knife, the 'mouth' and the unbonded mark, which eliminates the unbound mark from the array wash and the west m flat j, and This helps to quickly measure reversible or weak interactions by the array. The helmet of the present invention is manufactured and used. They are well-known and well-known techniques, including the preparation of surfaces or regions such as the described 砉丁叮4; synthetic or purified and linked or assembled such as 钽,,*, as described herein. ^ Α I know the linker, the probe and the substance that detects the probe; and detect and analyze the labeled substance as described in + human τ herein. except

For example, reference to the method of t i /, A 1 dog disclosed in the above cited references, for example, is assigned to Affymax, Affymetrix, Nan〇gen, pr〇t〇gene, 130267.doc -75- 200907067

Patents of Spectragen, Millipore and Beckman from which products suitable for use in the present invention are available; standard tutorials in molecular biology and protein sciences, including those cited above; and C〇zette et al., (1991), USA Patent 5,063,081; Southern (1996), Current Opinion in

Biotechnology 7, 85_88; Chee et al, (1996) Science 274, 610-614; and Fodor et al, (1993). Nature 364, 555-556. The various features and attendant advantages of the present invention will become more fully understood from the <RTIgt; The same reference symbols indicate the same or similar parts. A nuclease protection assay is used to test for fixed tissue (eg, formalin-fixed, stone-embedded formalin-fixed or Preservcyte preservation solution, alcohol-based fixative), but may be bound to (iv) nuclear phytic acid using hybridization or binding probes. Any tests that are subsequently recovered in a quantitative manner. In addition, those skilled in the art can envision a method of measuring cross-linking nucleoside acid, wherein the probe is biosynthetically produced. Any type can be used as the sample material as long as the oligonucleotide is not excessively degraded. &acid is the oligonuclear acid extracted from the tissue (presumed to be cross-linked oligo nuclei * = the measurement of the oligo-nucleic acid of the upper main solution 1 , the sample is dissolved and then separated from each other The ball is separated and then the nuclear chymase protection probe is added to &gt; / and the error is treated separately by the protocol described below. This sputum is called a quantitative nuclease protection assay (N Huan) 彳 - can be used The resulting 130267.doc -76- 200907067 probe is separated from the dry nucleus and the other hybridization and biosynthesis measurements are taken. The schematic diagram of this assay is shown in Figure 1. The general experimental protocol is from the granule of the formalin fixed tissue. Analysis of the supernatant by lysing the lysate. Dissolve the sample, centrifuge and then measure the pellet and supernatant compared to the matched frozen sample. The nuclease protection assay measures total RNA regardless of whether it is associated with the globule It is not extracted from the autolyzed bacterial solution (crosslinking) or self-organized extraction supernatant (soluble). Therefore, the nuclease protection probe/target oligonucleotide duplex hybrid appears in the globule. A sample is shown, and Figure B shows another example, which shows the supernatant. The ratio of oligonucleotides in the pellet can vary from sample to sample. 13 This method II measures two total pools to measure total oligonucleotides in the mouth of the sample. Mi> New in crying large B-cell lymphoma Correlation between old paraffin blocks. The fixed samples that have been stored for 18 years have the same number of results as the fresh fixed samples prepared from the tissue blocks that were frozen when the original samples were fixed 18 years ago. And then plotted to determine the R2 correlation coefficient between the 18-year fixed and fresh fixation measurements. The use of migraine large B-cell lymphoma cells to verify the number of identical gene expressions measured by self-fixation, fresh, and cold-weaving Cell pellets are cold before dissolution; either in QCT, or directly dissolved in fresh sample form, or embedded in paraffin and fixed and subsequently dissolved. The use of data (for housekeeping gene normalization) by pairwise comparison to determine the fixation Samples for fresh samples, fixed samples for cold samples and cold; East samples for fresh samples between Han 2 correlation coefficients. Therefore * knife group group of Japanese temples, no matter how the sample is tested (fresh , cold; East or 130267.doc -77- 200907067 whether it is fixed and paraffin-embedded, the results are almost the same. Target oligonucleotide specific measurement. RNase and DNase experiments confirmed ArrayPlate debt test RNA, Instead of DNA, the tissue that has been fixed by Dendrobium in the Dendrobium is dissolved and denatured at 95 ° C, and then divided into individual samples, which are then treated with DNase, RNase or buffer. After this treatment, The sample is subjected to thermal denaturation to destroy DNase and RNase, and then a nuclease protection probe is added and a nuclease protection protocol is performed. The image confirms the same result between the untreated sample and the DNase treated sample, and is performed by RNA The total loss of signal produced by the enzyme treatment confirms the specific measurement of RNA from the fixed tissue using this protocol.

Comparison of ArrayPlate qNPA nuclease protection assays and immunohistochemistry results. A specific example is a nuclease protection assay, and more specifically a quantitative nuclease protection assay. Describe the hybridization of a nuclease-protecting probe to RNA that is cross-linkable to tissue and soluble RNA. The unhybridized probe is disrupted by S1 treatment and the probe is reduced to a stoichiometric amount proportional to the amount of target oligonucleotide (crosslinking oligonucleotide and soluble oligonucleotide). Addition of the nuclease protection probe under heating is released from the oligonucleotide template and tissue so that it can be measured by a stylized ArrayPlate. The remainder of the scheme is implemented in a standard manner as described in more detail in the method. The rat liver was cut in half and half frozen and the other half fixed in formalin. The frozen tissue was weighed, dissolved and then each test sample was diluted. The fixed block was cut into 5 micron sections and the sections were tested in the form of 2 slices, 1 slice, % slice or % of slices per sample. The measurement was performed using a nuclease protection assay that measures RNA. Indicates the measured gene. The reproducibility of the measurement results was evaluated by calculating the % CV of each gene and the flatness of all genes 130267.doc -78 - 200907067. Vaginal smears were collected from hpv positive individuals and stored in Preservcyte. Positive diagnosis was performed by PAP smear and hybrid capture tests. Arrays are designed to measure host RNA, viral RNA, and viral DNA. Viral DNA is measured using a probe that hybridizes to the sequence in the untranscribed portion of the gene. The test sample was repeated (n=8) and the data was not normalized to the housekeeping gene. The genes measured in the diffuse large B-cell lymphoma tissue experiment are listed in this table. For the figure shown, an array is used. When diffuse large B-cell lymphoma (DLBCL) is called DB cell strain (American Type Culture Collection, Manassas, VA), it is an MHC II-negative DLBCL cell line, or DB transfectant DB-CIITA-3.1, which includes A CIITA expression vector that induces the expression of major histocompatibility class II genes including HLA-DRA, HLA-DRB, HLA-DPA, and HLA-DQA (Glinsmann-Gibson, 2006). The cells were grown to a density of 4 million per ml in RPMI with 10% fetal calf serum. 16 million cells were centrifuged to produce cell pellets, which were 1) made into formalin fixed paraffin-embedded by conventional overnight tissue treatment on a TissueTek instrument and subjected to stone embedding by undergoing fixation in fumarin for 4 hours. (FFPE) substance, or 2) instantaneous freezing of liquid by instantaneous freezing in liquid nitrogen quenched in isopentane with or without an embedding medium (optimal cutting temperature or OCT, Sakura Finetechnieal Co, Torrance, CA). The rat liver was similarly frozen or prepared as a FFPE substance. The FFPE clinical organization used to test the efficacy of ArrayPlate includes 1 benign lymph node (reactive follicular hyperplasia) and 2 DLBCL (1 is central cytopathic, 1 130267.doc •79-200907067 is immunoblastic), which does not exist Multiple instant frozen blocks with ffpe blocks. A new FFPE% was generated by defrosting the chilled bead from the previous instant of benign lymphoid tissue in 1989, which was then immediately fixed in 4% Formalin and processed as described below. The tissue was fixed at approximately 5 x 5 x 5 parts. Frozen sections and FFPE sections were cut to a thickness of about 5 microns. For nuclease protection assays, samples were placed in HTG lysis buffer (25 μl/slice), briefly vortexed, heated at 95 ° C for 1 〇 minute, briefly vortexed, and then at -70° Freeze at C until analysis. The ArrayPlate Nuclease Protection Assay (Martel, 2〇〇2) has been previously described. In Jan. 5, the frozen sample is tested after the cells or tissues have been dissolved, denatured, and infiltrated by heating in a buffer as described above. However, it can also be tested immediately after dissolution, and the probe can be added while dissolved, or the sample can be frozen prior to the 95 °C heating step. The details of dissolution and heating may vary depending on the target molecule to be measured. For example, in the case where the target oligonucleotide is DNA, it may be necessary to heat the lysate to 1 〇5. Other methods of solubilization and destruction which are well known or commonly used by those skilled in the art to measure different types of molecular or self-fixing tissue recovery activity can be used. In particular, in the case of such experiments measuring the nucleotide target, it is at 6 〇. Subsequent probes specific for the gene of interest are incubated with the sample for 6 hours to form a specific probe-RNA duplex. Then the unhybridized probe and RNA are digested by s丨 nuclease. Second, alkaline hydrolysis destroys the mRNA from the duplex, leaving a complete probe at a concentration proportional to the amount of specific mRNA originally present. After neutralization, the sample is then hybridized to the detection disk. The detection disk is made up of a set of 16 unique anchors in a 4x4 grid on the bottom of the hole in the 96-well disk. 130267.doc •80·200907067 DNA oligomer shape. This universal array # is designed by the addition of 16 linker probes containing a sequence that binds to the gene on one end and one of the mis-aggregates on the other end. The sequence. After hybridization, the sample probe is coupled to the disk by a linker probe. Add a detection linker (but this can be added simultaneously with the sample added to the stylized AmyPMe). It contains a sequence at one end that binds to the probe on the end that is not bound by the linker probe, and contains a binding at the other end. A common sequence of probes is detected. A detection probe is added after k, which is combined with all detection linkages. The detection probe contains an enzyme that acts on a substrate for the subsequent addition of a chemiluminescent peroxidase. The disc is imaged from the bottom by 〇mix imager and analyzed using Vuescript (HTG), which calculates the average pixel intensity of all elements to determine the performance of each gene 4 °. If the data has been normalized, the expression will be directed to the housekeeping gene tbp Normalize at any level of 1〇〇〇. For the DLBL experiments, the genes measured were the genes listed in Figure 1 'but the data were not array 2 (Figure 16 and Figure 7). Due to the heterogeneity of cell composition in human tumor samples, we include probes designed to measure the tumor composition of B, .' field cells (CD19, CD20), T cells (CD3), and tissue cells (CD68). Finally, based on the previously published study to evaluate the utility of different endogenous i. biogenetic genes as official genes, 'select two housekeeping bases P and PRKG1, which identify the two genes at low or in different types of lymphoma. Moderately stable performance (L〇ss〇s, 2〇〇3). The two tube genes are repeated in each of the three arrays. IHC is performed on the protein product of the gene present on the array, for which we 130267.doc -81 - 200907067 have a clinical IHC assay routinely performed in paraffin. These include CD20, BCL2 and HLA-DR. All staining was performed by a Ventana Benchmark XT instrument (Ventana Medical Systems Incorporated, Tucson, Arizona) (VMSI) with Ventana 1_view detection. Standard clinical laboratory staining procedures with on-board antigen retrieval are used. Monoclonal antibodies against CD20 (VMSI, pure L26), BCL2 (VMSI, pure B4-2/100/D5) and HLA-DR (Biogenix, pure LN3) were used. Photographs were taken by Labophot-2 microscope (Nikon, Melville, NY) using a 10X eyepiece and a 40X/0.65 objective. Images were acquired and digitally acquired using a SPOT-RT 2.2.0 color camera and SPOT Advanced 4.0.9 software (Diagnostic Instruments, Sterling Heights, MI), which was then inserted into PowerPoint 10 (Microsoft, Redman, WA) for processing. Results: To confirm that the nuclease protection was carried out in situ in the fixed sample, a formalin-fixed paraffin-embedded (FFPE) rat liver (5 μm section) was used as a starting material. Tissue sections were treated with 30 μM lysis buffer. After solubilization, a nuclease protection probe was added to the sample followed by thermal denaturation (95 ° C, 10 minutes). The sample was centrifuged and the supernatant removed. Tissue pellets were treated with 30 μM lysis buffer and incubated with nuclease protected probes. After thermal denaturation (95 ° C, 10 minutes), the beads and supernatant were subjected to qNPA assay using ARRAYPLATE, which provided the matrix. Frozen tissue was used as a control. This test circumvents the artifacts of mRN A extraction from the formalin fixed substance. These results are presented in Figure 2. Supernatants and granular tissue sections were compared to matched frozen tissue. The results show that the target oligonucleotide can be measured from the small sphere 130267.doc -82- 200907067. In Figure A, the majority of the I-knife is from the small ball, not the supernatant. In contrast, there is also a measurement of nuclear acid from the supernatant in the product (Fig. B). In this experiment, dry oligo reads RN^ but it can also be, for example, dna, micro (10) or ribosome.疋 疋 养 养 养 与 与 与 与 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊 聊The same pool (its, Y ΚΛΙΛ,,, the master knows and is visible in the tissue), and the sum of the measurement results (each Bu, the main field, the moon liquid is not separated from the small ball) to provide a total sample The vouchers are a measure of bitterness. Figure Α and β, then 1 搂 _ and ϋ T, then the difference between the samples of type 6 is dark: perhaps due to the difference in the fixed side φ 兰 _ 刁 tea, even if the sample is otherwise the same, the parent coupon The rate of nuclear sputum can be changed. The ratio of & 社 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 Therefore, only the measurement of the target oligonucleoside acid in the sample can not be accurately measured by measuring the parent pool or only measuring Jin, 'six, a τ + , and smashing the cold pool. «Figure (C) provides a detailed description of the protocol used in this study. As a result, the nuclease protection step in the real qNPAk(R) occurs in situ, and the damage step can occur ex situ, in situ, or both. Analysis of gene expression in samples supplemented with formalin-fixed paraffin-embedded colon tissue embedded in sulphate-fixed rocky soil in a lysis buffer as previously described and supplemented with buffer alone or supplemented with rn The buffer of A enzyme 4 is supplemented with a buffer filled with DNase. The pellet was subjected to a qNPA assay as previously described. Test about 4 slices per well. These studies demonstrate the applicability of techniques for measuring RNA in tissues. The results of this experiment are shown in Figure 3. The RNase treatment, which removes the dry RNA from the homogenization-like ασ, causes the live label to be lost from the sample. The DNa enzyme was used at 130267.doc -83- 200907067 and the sample did not have any significant effect on the qNPA assay. Quantitative Nuclease Protection Assay Allows Quantitative Assay To assess the quantitative nature of the nuclease protection assays used herein, ffpe sections were titrated against the same cold bulb tissue using the qNPA assay. The rna is separated from the frozen tissue and loaded onto a slide. Load the FFPe slices in a similar manner. A schematic representation of the ArrayPlate mRNA assay for FFPE colon is provided in Figure A. The results of this study, which are presented in Figures 4 (A) and (B), illustrate the correlation of frozen tissue to gene expression in FFPE liver. A representative example of a representative dog is shown on the right side of Figure (B). As can be seen from this figure, individual performance profiles of various genes as identified from frozen samples or fixed samples are visually well correlated with each other. To further determine the amount of such results obtained from different biological samples, regression analysis of the performance profiles of various genes in the F F p E section tissue was performed by frozen tissue. Measure the same-group gene for all samples and map the data to the housekeeping gene

Normalization and subsequent mapping to determine the r2 correlation coefficient between each pairwise comparison. The results of the regression analysis are shown in Figure 5(B). A strong correlation was observed between the two samples. These results essentially indicate that equal quantitative results are obtained from fresh fixed tissues as well as frozen, tissue. Although it has been found that the use of fresh fixed tissue dissection is fixed and subsequently stored for many years, the measurement results by in situ hybridization (where the ship is marked and visible in the weave) are comparable, but for pCR or non-original This is usually not the case for the method of hybridization. This limitation is useful because I is in a large amount of material in a stored material that can be used in other ways to retrospectively identify and validate biologically labeled filament genes, or for development and 130267.doc -84 - 200907067 validation monitoring , prognostic or diagnostic tests, or used to combine safety and gene expression, or to understand disease processes. The lymph node tissue obtained from cancer patients was tested 18 years ago. A part of it has been fixed for histology at that time and another part has been cold; The patient was diagnosed with diffuse large B-cell lymphoma. It will be cold; the eastern block will be fixed to provide a fresh solid = sample for comparison with the 18-year fixed material. A set of genes was measured from each sample, and the results are shown in Fig. 6. The graph shows the relative luminescence values after the normalization of the housekeeping gene. The R2 correlation coefficient is 〇 · 9 3, which indicates that it has been fixed from fresh fixed tissue and previously fixed! The eight-year organization is basically equal quantitative results. To measure the relationship between the dependence of the qNPA signal on the amount of starting material f, = the informality obtained from the various samples (for each sample size 'n=4) as previously characterized by the gene expression The eight signals are compared for comparison. For samples containing at least G.5 FFPE sections per well, a linear relationship between sample size and qNPA signal was obtained. These results were consistently observed in all of the genes analyzed. The results of this study, the results, and the type of genes analyzed are presented in Figure 7.曰 To quantitatively evaluate fresh gene expression; gene expression in bundle samples of tmpE liver samples, comparative analysis of 16 genes using qWA assay. The performance of most of these genes between the two sample preparations (ie cold; east to FFpE), 'can not be area / knife. The results of this study are presented in Figure 8. The method described allows sensitivity measurements to be taken to raise nucleotides. Figure 9 shows the nuclease protection assay of the different number of approximately 5 micron thick slices from the frozen rat liver dilution from the frozen rat liver dilution 130267.doc -85 · 200907067 result. Note that using only 1/4 of the tissue, all genes are measurable. In addition, the reproducibility of the measurement results was excellent, and the average CV of all the measured genes was 7. /. . This reproducibility is superior to the reproducibility of genetic measurements of frozen matched samples. Finally, 1/4 of the sheets produced a similar number of luminescence counts for each gene and the total number was similar to 19 盹 frozen tissue, providing an indication of how sensitive it can be to measure RNA from fixed tissue using this method. Measurement of rna from fixed tissue by PCR and other hybridization-based methods has proven to be extremely insensitive, requiring large amounts of tissue. It is possible that the sensitivity of the nuclease-preserving method is the result of measuring total RNA (not only the soluble pool), and thus this same sensitivity will be obtained by any method of measuring the cross-linking of the target oligonucleotide. As shown in Table (B) of Figure 9, a variety of target genes can be reliably detected using this technique. The reproducibility of the measurement results is excellent, providing an average CV of less than 20% for all measured genes. Other nucleotide acid target types can be measured and samples fixed using other types of solid methods can be successfully tested. Clinical vaginal samples were collected and stored in preservcyte prior to testing. The nuclease protection assay measures host cell RNA, viral Rna, and viral DNA. To distinguish between viral DNA and RNA present in the sample, the nuclease protection probe is designed to hybridize to sequences in the untranscribed portion of the gene. The data is unnormalized and the relevant tethers are determined from the measurements of 8 replicate samples. Confirmation of the ability to measure host RNA, viral RNA and viral DNA, confirm the measurement results of various types of oligonucleotides from fixed samples, and simultaneously measure the target oligonucleotides from the same sample in the same ArrayPlate The ability of glycosides. The figures describe the concept of using a cross-linked oligonucleotide target and a soluble target associated with 130267.doc -86 - 200907067 and then separable and measured by the search for the clock . A specific example is a nuclease protection assay, and more specifically a quantitative nuclease protection assay, (iv) other techniques can be devised by the skilled artisan (including the use of probes produced by biosynthesis or hybridization via oligodeoxynucleotide) Other methods are associated: the method of the needle) to produce the same measurement of the cross-linked oligonucleotide target. Description can be fixed using the same standard protocol as fresh or frozen tissue: Oligonucleotide in the weave. The nuclease-protecting probe is shown to hybridize to rna, Wrna which can be cross-linked to the tissue. (d) Treatment of the probe to a disco oligonucleotide (crosslinked oligoacid and soluble oligo(4)) in proportion to the amount of the chemical device. The nuclease protection probe is added under heating to release the probe from the oligo, template and tissue&apos; to make it measurable by a side other than the in situ method. However, it is noted that the probe (or one or more of a large set of probes) can be first measured in situ. As described in more detail in the method, the rest of the standard scheme is used. It is possible to measure the probe continuously in situ and after tissue dissolution. Once the probe has been washed out or after treatment with S1 nuclease, the label-free association probe can be visualized and quantified in situ using, for example, fluorescein: = or luminescence. : is specific to one or two or more probes that can be distinguished, for example, by use of probes that are not long, and that the in-situ measurements of the 3 can be used for normalization or otherwise Help Description—The measurement of a large set of probes that are manufactured after dissolution. In this way, “construction” information can be related to the measurement of genes, even in space: loss. In the case of a single washing of the m« needle before the original (four) amount, the probe or the probe may be externally set to have a non-target hybridization sequence that is not complementary to the oligo-splicing detection probe 130267.doc-87. 200907067 The protrusions, and thus can be labeled by the detection probe. Alternatively, the in situ probe can be labeled directly via a detection molecule or a group capable of binding to a detection molecule or complex, such as biotin, and then followed by a wash and/or si step prior to in situ measurement. This label does not affect the ability of the nuclease protection probe to hybridize to its target oligonucleotide. There are many situations in which the ability to immobilize a sample prior to measuring the target oligonucleotide will be an advantage. For example, if blood is collected directly into the fixative, and the i of the dry-collected acid is stored, the time between sampling and testing the sample becomes less important. Processing becomes easier. In addition, immobilized samples can be otherwise manipulated by methods that disrupt or lyse cells and tissues, resulting in alteration or loss of target oligonucleotides, such as by filtration, to simplify sample preparation. This is also the case for many types of cell 'tissue or whole organism samples and environmental samples or samples collected away from the test point. Example 2: Clinical Studies Diffuse large B-cell lymphoma (DLBCL) is the most common invasive lymphoma, accounting for almost 40% of all lymphomas. The International Prognostic Index ( ιρι) score for DLBCL helps to classify patients according to clinical characteristics (Shipp, 1993). However, even within the grade, patient outcomes are still variable. Due to variable patient outcomes, it has long been suspected that DLBCL can actually cover more than one disease. The results of extensive gene expression profiling (GEP) experiments in recent years have confirmed that DLBCL is not a uniform disease entity and that differences in GEp subtypes can be associated with patient survival. Three different groups of genes have been identified based on studies from three different study groups. 130267.doc -88 - 200907067 The first group used the LymphoChip to study 240 DLBCLs. The wafer is a spot-coated oligonucleotide microarray containing a plurality of genes known to be expressed by B or T lymphoid cells, genes involved in an immune response, or genes represented by lymphoma and white disease cell lines. (Alizadeh, 1999). The researchers found four gene expression characteristics that are extremely relevant to patient survival, called the “spontaneous center”, “class II major histocompatibility (MHC)&quot;, &quot; lymph node π and "hyperplasia." In the case of the addition of the gene ΒΜΡ6, representative genes from the four characteristics were used to generate a 17-gene result prediction score that provides other prognostic values independent of the clinical sputum score (Rosenwald, 2002). The other group used the Affymetrix High Density Oligonucleotide Array Platform and Supervised Learning Taxonomy to generate DLBCL result prediction scores with the best accuracy obtained using a set of 13 genes (Shipp, 2002). Another group of researchers performed a metaanalysis of the literature on genes previously reported to be significantly associated with DLBCL survival. Using quantitative RT-PCR, the researchers evaluated a series of 66 DLBCLs and determined the six most prognostic genes (LM02, BCL6, FN1, CCND2, SCYA3, and BCL2) (Lossos, 2004). For a total of 34 genes previously identified as significantly associated with patients with DLBCL in high-profile papers, only two genes (BCL6 and FN1) were identified in the Lymphochip and RT-PCR papers. Through the analysis of the Lymphochip dataset, the identification of several redox-associated genes that are extremely relevant to patient survival has been confirmed and a redox fraction including representative genes, supermanganese dismutase and peroxidase has been generated (Tome, 2005). All four of these papers used a quantification of key sets of gene expression for genes associated with DLBCL survival. Each produced a small genome with a strong correlation with the results of a particular case group 130267.doc -89- 200907067. All techniques used relying on transient cold-cluster materials as a basis for analysis make it difficult to genomic or extend the spectrum with a wider patient group. Therefore, it is especially useful to compare these genomes for the determination of such prognosis using methods that can be applied to paraffin-embedded samples. The next step in the clinical utility of sex gene research. Quantitative nuclear protection assays are expected to quantify mRNA without the difficult steps of mRNA extraction and subsequent quantitative RT-PCR, and thus can be applied to tissues that have been stabilized by fossilization and rocky soil. This method relies on the ArrayPlate assay previously described for medical applications (Martel, 2002). In this assay, The tissue is dissolved in a 96-well plate. The probe designed for the gene of interest is incubated with the lysate to allow hybridization of the specific probe-RNA duplex. The unhybridized probe and RN A are digested and the mRNA is assayed using the assay. Separation from the duplex, leaving a complete probe at a concentration proportional to the amount of specific mRNA originally present. The remaining probes are then transferred to the use of linkers and detection probes and chemiluminescence detection and quantification In a second 96-well plate, the platform is easy to implement and has the potential to handle high sample volumes. Using this technique, develop cell structures for all 36 genes of interest as well as housekeeping genes and to determine samples (B cells, B cells and Verification of the gene for the macrophage gene. Several studies were carried out to test and confirm the efficacy of the assay for frozen material and stone-embedded material. Paraffin blocks from the previous analysis in the paper by Rosenwald et al. It is then used to determine if the technique can similarly quantify prognostic significant genes and whether this will be relevant to patient outcomes. The overall aim is to develop and confirm that it will be available for use with DLBC All patients of L, without limitation, the predictions predicted by the results of a few lucky ones with available transient frozen tissue. Frozen samples and paraffin 130267.doc -90- 200907067 Embedding samples, new paraffin blocks and paraffin blocks of almost 20 years old To compare and evaluate the relationship between the results and the patient's risk of death in a group of 40 patients previously disclosed. These studies confirm the usefulness of this technique, particularly when applied to preservatives, which have profound implications in the field. Application prospects Patient and cell strain materials, preparation of FFPE and frozen blocks: To validate the design of the ArrayPlate, DB cell strain (American Type Culture Collection, Manassas, VA), which is an MHC II-negative DLBCL cell line, was used. In addition, the newly generated DB transfectant DB-CIITA-3.1 is also utilized, which includes CIITA expressions that induce the expression of major histocompatibility class II genes including HLA-DRA, HLA-DRB, HLA-DPA and HLA-DQA. Vector (Glinsmann-Gibson, 2006). The cells were grown to a density of 4 million per ml in RPMI with 10% fetal bovine serum. 16 million cells were centrifuged to produce cell pellets, 1) by fixation in fumarin for 4 hours, conventional overnight tissue treatment with TissueTek instrument and paraffin embedding to make FFPE material, or 2) by The instantaneous cold bed material (optimal cutting temperature or OCT, Sakura Finetechnical Co, Torrance, CA) was instantaneously frozen in liquid nitrogen quenched in isopentane with or without an embedding medium. According to hospital files selected for multiple transient frozen blocks and paraffin blocks with almost 100% tumors, the FFPE tissue used to test ArrayPlate efficacy includes 1 benign lymph node (reactive follicular hyperplasia) and 2 DLBCL·(1 Central mother cell, 1 is immunoblastic). New paraffin 130267.doc -91 - 200907067 was produced by thawing to ice-cold, followed by immediate fixation in 4% formalin and treatment of the previously transient frozen mass from benign lymphoid tissue in 1989. The arrangement of the arrays is depicted in Figure 11. To investigate whether the ArrayPlate results for FFPE samples are sufficiently similar to the GEP findings for transient frozen tissue, samples previously analyzed by GEP were used. Previously, 45 transient frozen samples have been used in the LLLPD study of DLBCL. Of these, 24 were included in the NEJM paper by Rosenwald et al. describing the clinical utility of OPS in reflecting the survival rate of patients with recurrent DLBCL. In these cases, 18 paraffin blocks were used. Paraffin blocks for 21 cases outlined by LLMPP but not included in reborn DLBCL were also used, including 9 transformed cases and 12 relapsed DLBCL cases.

Sample preparation of ArrayPlate Frozen sections and FFPE sections were cut to a thickness of 5 microns and immediately placed in laboratory buffer (25 μl/slice), briefly vortexed, heated at 95 ° C for 1 〇 minute, and briefly vortexed And then frozen at -7 ° C until analysis. Use 1 tissue section per well on the ArrayPlate. The samples were obtained from incisional biopsies fixed in approximately 5 x 5 x 5 mm sections. It is important to cut the paraffin block into a thin slice because the FFPE structure that has not been sliced gives poor results.

ArrayPlate Verification The ArrayPlate assay has been previously described (Martel, 2002). Briefly, after the cells or tissues have been dissolved, denatured, and infiltrated by heating in a laboratory buffer as described above, the frozen samples are transferred to a laboratory for analysis. In the laboratory, the probe specific for the gene of interest is incubated with the sample at 60 ° C for 6 hours to form a specific probe-RNA duplex, followed by digestion of the unhybridized probe and RNA by S1 nuclease. . The DLBCL gene is listed in the table of Figure 10 130267.doc - 92 · 200907067. Second, alkaline hydrolysis is used to separate the mRNA from the duplex, leaving a complete probe at a concentration proportional to the amount of specific mRNA originally present. After neutralization, the sample is then hybridized to the detection disk. The detection disk was formed from a set of 16 unique anchor DNA polymer aggregates in a 4χ4 grid that was spotted on the bottom of the 96-well disk. This universal array is designed for the gene of interest by the addition of 16 linker probes containing a sequence that binds to the gene of interest at one end and a mis-oligomer at the other end - The sequence of the person. Three separate sets of linker probes were used for the gene of interest at a rate of 3 wells per assay. After hybridization, the sample probe is bound to the disk by a linker probe. Add Detect: Connection: 'The sequence at the end contains the sequence #2# on the end not bound by the linker probe and the constant = column on the other end contains the bound (four) probe. A _probe is then added which binds to all of the detected linkers = an enzyme that acts on the last added chemiluminescent peroxidase substrate. , room) by the 〇ΓΓΓ 〇ΓΓΓ 盘 盘 盘 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 v 且 v v v v v v v v Hole 3 has some genes for the alizarin expressed in the pores of the pores. If the range is too high, then the signal is not known to the sister = the known amount of nuclear acid (four) speculative financial sample probe, but not. w violent nuclear bitter acid can be combined with A m mouth plate. If the range is too low, the η η gene uses multiple sample probes to increase the signal, by binding the same column to the sample, and the same sequence binds to the different orders in the linker 130267.doc -93· 200907067 Gene selection As described in the last paragraph of the introduction, key genes identified as prognostic in four previously important DLBCL papers were used. These genes represent 36 genes of interest (Lossos, 2004; Rosenwald, 2002; Shipp, 2002; Tome, 2005). Due to the heterogeneity of cellular composition in human tumor samples, probes designed to test the tumor composition of B cells (CD19, CD20), T cells (CD3), and tissue cells (CD68) are also included. Finally, based on previously published studies evaluating the effects of different endogenously expressed genes as housekeeping genes, two housekeeping genes, TBP and PRKG1, were selected, which identified the two genes at low or medium levels in different types of lymphoma. Stable performance (Lossos, 2003). The two housekeeping genes are repeated in each of the 3 wells used to generate the assay. The oligo dT probe was added to assess the amount of mRNA in the sample (since the oligo dT probe should detect all mRNAs with poly-A tails). However, for technical reasons, this probe is non-functional and has not been further utilized. Cytochrome oxidase probes are also included initially because they are encoded in mitochondrial DNA and should be expressed in high amounts. This was originally a combination of DNA and RNA, and thus gave a very bright and usually supersaturated signal and therefore not further considered, but it can be used to discern whether there is an insufficient assay substance, or if it has completely disappeared, and the sample is excessively degraded in use. . These genes are listed in the table of Figure 10. Immunohistochemistry and photography (IHC) IHC is performed on the protein product of the genes present on the array. Clinical IHC assays have been routinely performed for these genes. These include CD20, CD3, CD68, BCL2, BCL6 and HLA-DR. All staining was performed by a Ventana Benchmark XT instrument (Ventana Medical Systems Incorporated, Tucson, Arizona) (VMSI) with Ventana 130267.doc • 94- 200907067 I-view 4 speculation. Standard clinical laboratory staining procedures with on-board antigen retrieval are used. Use anti-CD20 (VMSI, pure L26), CD3 (VMSI, pure PS1), CD68 (VMSI, pure KP1), BCL2 (VMSI, pure B4-2/100/D5), BCL6 (pure IG191E/AB) and HLA- Monoclonal antibody to DR (Biogenix, pure LN3). Photographs were performed by using a Labopite-2 microscope (Nikon, Melville, ΝΥ) using a 1 〇χ eyepiece and a 4 〇χ / 0.65 objective lens. Images were acquired and digitally acquired using a SPOT-RT 2.2.0 color camera and SPOT Advanced 4.0.9 software (Diagnostic Instruments, Sterling Heights, MI), which was then inserted into PowerPoint 10 (Microsoft, Redman, WA) for processing. Methods: Statistical analysis was performed on 18 cases, the results from ArrayPlate and Affymetix/Lymphochip analysis of gene expression. For each gene, the Spearman rank order correlation of each pairwise comparison of the three research methods was obtained. For a particular comparison, no analysis of less than 10 available observations is performed due to capacity considerations. The median total correlation between the available genes for each pairwise comparison is calculated; for all three pairwise comparisons, only genes with available data are included to avoid bias. The results of the single variable analysis (hazard ratio, 95% confidence interval, and p-value) as measured by ArrayPlate for total survival gene performance were obtained from Cox DR. Regression models and life tables. J Royal Stat Soc B 1972; B34: 187-220). 130267.doc •95- 200907067 Clinical studies on gene expression in diffuse large B-cell lymphoma (DLBCL) cells are depicted in Figures 12-25. Results The test performance of frozen tissues was compared to FFPE: for all 44 genes of interest, specific probes and linker probes were designed. For the eight genes, the signal decreases with undetectable competitive oligonucleotides. For the four genes, the signal must increase with the additive quantitation of multiple probes (as shown herein). TBp has a stronger and more uniform luminescence result between the two housekeeping genes, PRKG1 and TBP. Therefore, all data is normalized to TBP and manually set to 1 〇〇〇 (data not shown). The assay performance was linearly reduced to 0.125 mg per well. RNase and DNase treatment confirmed that the assay was only specific for rnA detection. The supernatant and cell lysate assays determined that RNA was not self-organized (only positive results using cell lysates). This may explain the good correlation between frozen tissue and FFPE tissue because RNA extraction from tissue is not necessary. The CV for the quadruple operation of the cold bundle benign lymph nodes ranged from 8% to 15% (data not shown). For the lysate for instant freezing (R2 = 〇.989), instant freezing for FFPE (R2 = 0.919), lysate for FFPE (R2 = 0.994), the comparison between different types of tissue preparations is excellent (data) Not shown). Confirmation of ArrayPlate technology for RT-PCR and IHC: As confirmed by quantitative RT-PCR, ArrayPlate results accurately reflect HLA-DRA, HLA-DRB, HLA-DQA and HLA-DPA in DB cell line transfectants 1-5 Increase in mRNA (data not shown). The coefficient of variation for the four experiments of FFPE for these embedded cell strains ranged from 7% to 9% (Fig. 130267.doc-96·200907067 4A). These ArrayPlate results correlate well with immunohistochemical staining patterns of prognostic significant genes and gene products (Figures 5A-5D). Correlation between new paraffin blocks of the same sample and preserved paraffin blocks: According to the same biopsy, the correlation between the new paraffin block and the old paraffin block was excellent (Fig. 4B). The new stone ant block was taken from the cold H-woven portion that was cold at the time of biopsy, quickly defrosted, fixed in the formalin and embedded. The results were compared with the same type of rocky soil that was produced by the phase-by-mesh inspection at the time of the patient's surgery 18 years ago. This result indicates that the test can be applied to aged preservation materials. Correlation between different mRNA quantification techniques. Four other cases of #FFpE Dlbcl were analyzed by ArrayPIate analysis. 39 cases were successfully analyzed. One case had no signal at all. In situ hybridization with the P〇iyT probe confirmed that this case did not have any comparison of the results of the remaining 39 cases with the previous Affymetrix and competitive GEp array results using Spearman Rank Statistics. , as shown in Table 2. Since not all gene results are available for all platforms i', as shown in the table, there are frequent missing data. The median correlation of ArrayPlate to Affymetrix was 〇 52, the median correlation of ArrayPlate to Lymphochip was 〇, and the median correlation of Affymetrix to Lymphochip was 〇 78. It is understood that the latter correlation is higher because the two analyses are performed on aliquots of RNA obtained from the same frozen tissue block, while the ArrayPlate analysis is performed on different blocks (but from the same sample). In summary, for this type of technology, these have excellent correlations. 130267.doc -97- 200907067 The risk ratio of prognostic genes compared to other GEp technologies was assessed by ArrayPlate. The results of 44 genes were then compared to 39 cases successfully analyzed using ΑΓ^ρ_ compared to survival. First, a single variable analysis of gene expression to patient survival is performed. However, the absence of the gene species was significantly associated with the presence, which was attributed to the low number of cases in this study group (because in the larger group of patients all of these genes were associated with survival). Calculate the death risk ratio for each gene. For risk ratio &gt; 1, there is an increased risk of death and for risk ratio &lt; 1, there is a reduced risk of death. For each gene, the risk ratio is usually in the same direction as the larger data set in which it is significant. A comparison of consistency (risk ratio is higher or lower than 丨). For Affymetrix's comparison of Lymphochip data, 29 of the 33 genes were consistent (88%) for the two methods, and 3 of the 4 genes for the ArrayPlate for Affymetrix (75). %)' For ArrayPlate, for Lymphochip, 27 of the 34 genes were consistent (79%). Usually the quantitative measurement of gene expression is not well correlated with the measurement of the content of protein products. This figure demonstrates that measurement of the gene expression of the formalin-fixed tissue measuring total RNA (in this case protected by nucleases) results in quantitative correlation with the amount of protein product measured in situ by immunohistochemistry. Fixed tissue samples from three patients who had been diagnosed with diffuse large B-cell lymphoma or benign reactive lymph nodes were measured by nuclease protection assay. The content of the three genes (HLA-DR, Bel 2 and CD 20) is described by a histogram. For each patient, the protein product of these genes was measured by immunohistochemistry (IHC) 130267.doc -98- 200907067. Describe the staining load 4 and the relative quantitative content of each protein marker (high performance, medium performance, low performance). Note that IHC protein content is related to gene expression. Diffuse large b-cell lymphoma diagnosis is based on histology and in situ measurements. Such data suggest that it may also be based on the use of spatial expression (spatial c〇ntext) that has been lost, but quantitatively measures the total oligonucleotide content of the assay for self-organized measurement of gene expression, or may use such methods to obtain other prognostic And diagnostic information. Example 3: Use of qNPA technology in research and/or diagnostics The present invention can be used in basic scientific research and diagnostic applications. For example, drug inducible changes in gene/protein expression can be routinely analyzed using the methods described above. Representative examples are described below. Rifapicin-mediated gene regulation The aforementioned qNPA technique was used to analyze the regulation of gene expression by rifampicin (an antibiotic with known hepatotoxic effects). Human or canine native hepatocytes were cultured with 〇_丨〇 4^1 rifampicin and qNPA was used to analyze the levels of major transcripts of various metabolic enzymes. These results are presented in Figures 26-27. As shown in Figure 26 (A), the gene expression studies provide a pharmacological measurement (e.g., ECm value) for the enzyme-induced manner of rifampicin. In addition, comparative studies can be performed on human and canine samples. In humans, cytochrome P450 enzymes Cyp3 A4, Cyp2C9, CyP2B6, GSTA2 and SULT2A1 were induced by rifampicin. Cyp2D6 and PXR are inhibited, indicating a poor drug response. However, the inhibition rate of these enzymes was less than 50% (panel A of Figure 27). In canine liver cells, CyplAl, Cyp2D15 and Cyp2A337 genes are up-regulated. However, unlike human cells, rifampicin treatment did not result in the inhibition of the genes shown in Figures 26 and 27, as shown in Figure 26 and Figure 27 (8). In the case where the domain of the present invention is clarified, the use and conditions can be used. In view of the foregoing, it will be apparent to those skilled in the art that the present invention may be modified and modified without departing from the spirit and scope of the invention. Therefore, the foregoing specific embodiments are to be considered in all respects, and are not intended to / In the foregoing and examples, unless otherwise stated, all temperatures are listed in degrees Celsius without modification, and all parts and percentages are by weight. A similar success can be obtained by repeating the foregoing examples by using one of the present inventions or specific reactants and/or operating conditions in place of the reactants and/or operating conditions of (4) of the foregoing examples. The entire disclosure of all of the claims, patents, and publications cited in the drawings, and the entire disclosures of U.S. Provisional Application No. 60/920,814, filed on March 30, 2007, and the entire disclosure of The entire disclosure of U.S. Provisional Application Serial No. 61/018, the entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire content BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the flow for detecting an insoluble target (e.g., cross-linked RNA) in a biological sample. Figure 2 (A) illustrates a quantitative nuclease protection assay for gene expression in a biological sample 130267.doc -100- 200907067 (qNPA). The fixed tissue was dissolved and the mRNA content in the granular tissue sections and supernatant was quantified using the ARRAYPLATE using the qNPA assay. A frozen tissue sample was used as a positive control. B) Describe the ratio of the amount of RNA in the supernatant to the pellet of the second dissolved sample. C) Provided A) specific examples for the analysis of RNA targets in formalin-fixed paraffin-embedded tissues. Figure 3 illustrates the applicability of this technique for measuring RNA in tissues. Figure 4 (A) is a schematic representation of the ArrayPlate mRNA assay for FFPE colon. (B) A titration of frozen liver and a section of paraffin-embedded paraffin embedded. Representative measurements are shown in the inset (right). Figure 5 (A) illustrates comparable gene expression in frozen liver and fixed liver. Figure (B) illustrates the correlation of frozen liver to gene expression in FFPE liver. Figure 6 shows the same quantitative results from fresh fixed samples for storage of fixed samples for 18 years. Figure 7 shows the linearity of the ArrayPlate mRNA assay for FFPE liver. Figure 8 illustrates the relative gene expression of frozen liver in FFPE liver. Figure 9 (A) illustrates the reproducibility of the ArrayPlate mRNA assay for FFPE liver and the sensitivity of the cold sample to the fresh sample; (B) Demonstration of qNPA diagnostics in the measurement of viral nucleic acids. Figure 10 shows a list of genes analyzed in a diffuse large B-cell lymphoma (DLBCL) gene expression assay. Figure 11 illustrates the layout of genes in the ARRAYPLATE DLBCL gene performance assay. Figure 12 illustrates the compatibility of assays for various media. The performance of the various genes measured on the freshly prepared samples, the formalin-fixed paraffin-embedded 130267.doc-101 - 200907067 samples and the OCT frozen samples was analyzed using the qNPA assay. Figure 13 illustrates the bidirectional correlation between the freshly prepared samples, the formalin-fixed paraffin-embedded samples, and the OCT frozen samples for the performance of various genes. Gene expression analysis was performed using the qNPA assay. Figure 14 illustrates the testing of clinical biopsy tissue blocks using ARRAYPLATE analysis. Three different biopsy samples were analyzed using three different biomarkers. Figure 15 illustrates the reproducibility of the Ar ray Plate mRN A assay of a clinical biopsy tissue block. Figure 16 (A) illustrates the results of gene expression of array 1 (see Figure I5). Panel (B) shows the average normalized signal of gene expression in four biopsy samples. Figure 17 (A) illustrates the results of gene expression of array 2 (see Figure 15). Panel (B) shows the average normalized signal of gene expression in four biopsy samples. Figure 18 illustrates immunohistochemical (IHC) analysis of antigen expression in diffuse large B-cell lymphoma samples (case 1). The samples were stained with hematoxylin and eosin (H&amp;E) and analyzed for HLA-DR, BCL-6, BCL-2, CD-20 and BCL-68 expression. Figure 19 illustrates immunohistochemical (IHC) analysis of antigenic expression in diffuse large B-cell lymphoma samples (case 2). The samples were stained with hematoxylin and eosin (H&amp;E) and analyzed for HLA-DR, BCL-6, BCL-2, CD-20 and BCL-68 expression. Figure 20 illustrates an immunohistochemical (IHC) analysis of antigen expression in benign reactive lymph nodes (LN) in Case 3. The samples were stained with hematoxylin and eosin (H&amp;E) and analyzed for HLA-DR, BCL-6, BCL-2, CD-20 and BCL-68 130267.doc -102-200907067. Figure 2 1-23 summarizes the results of Figures 18-20 and shows immunohistochemical analysis (IHC) of HLA-DR, BCL-6, and BCL-2 expression in three biological samples. The discovery of IHC was associated with gene expression analysis using biopsy samples from the ARRAYPLATE qNPA assay. Figure 24 shows the correlation between the expression of HLA-DR, BCL-6 and BCL-2 at the levels of protein (analyzed by immunohistochemistry) and RNA (analyzed by quantitative nuclease protection assay [qNPA]). Figure 25 expands the study of Figure 24 to include CD20 and CD3 antigens. Figure 26 provides a representative example of the utility of a quantitative nuclease protection assay (ARRAYPLATE) for conducting gene expression studies. Rifampicin-induced regulation was measured using qNPA assays (A) human hepatocytes or (B) expression of various cytochrome P45 〇 isoforms in canine liver cells. Figure 27 summarizes the results of Figure 26 by presenting the data in an expanded form. The gene significantly induced/inhibited by 5 μ of rifampicin is provided in the right panel. Figure 28 provides a representative example of a custom designed array and the genes contained therein (with GenBank accession numbers). Figure 29 illustrates the results of gene expression of custom arrays 1 and 2 in control (untreated) and experimental (cl〇fibrate treated) PC-3 samples (n=3). The prior art has mentioned the need to extract RNA and dissolve it for PCR and microarray methods. The reference also indicates that the quality of this soluble extracted RNA is critical to the quality of the rcr or microarray results. Therefore qNPA is unique because no extraction is required. 130267.doc -103-

Claims (1)

200907067 X. Patent Application Range: 1 · A method for detecting at least one insoluble label in a biological sample, comprising: (i) ligating the sample with at least one nuclease that specifically binds to the target Molecular (NPM) contact, (ii) exposing the sample to one or more reagents under conditions effective to remove any unbound NPM, (iii) separating NPM from the target as appropriate, and (iv) detecting Measure the presence of the NPM. 2. The method of claim 1, comprising detecting the NPM in a combined or free form. 3. The method of claim 1, wherein the insoluble standard is fixed and/or crosslinked. 4. The method of claim 1, wherein the insoluble target is a peptide, a protein or a nucleic acid. 5. The method of claim 4, wherein the nucleic acid molecule comprises a ribonucleic acid (RNA) molecule or a deoxyribonucleic acid (DNA) molecule, or an antisense nucleotide, which may contain a non-natural base. 6. The method of claim 5, wherein the RNA is information RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), siRNA, and antisense RNA or viral RNA (vRNA). 7. The method of claim 5, wherein the DNA is genomic DNA (gDNA), mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA) or viral DNA (vDNA) or transfected DNA. The method of claim 1, wherein the NPM comprises a nucleic acid or an aptamer that specifically binds to the target. 9. The method of claim 8, wherein the target is a protein, a peptide, a polynucleotide, or a protein capable of hybridizing to the nucleic acid or aptamer NPM. 10. The method of claim 9, wherein the target is a nucleic acid. 11. The method of claim 8, wherein the NPM comprises a DNA molecule. 12. The method of claim 11, wherein the NPM is a single-stranded DNA (ssDNA) or a branched DNA (bDNA) molecule or contains LNA or PNA or other non-natural test group. 13. The method of claim 1 wherein the NPM is a nucleic acid that specifically binds to the target, and step (ii) comprises treating with a nuclease to effectively remove any unbound NPM. The method of claim 13 wherein the target is a nucleic acid. 1 5. The method of claim 13 wherein the target is an RNA molecule, siRNA or antisense RNA which may contain a non-natural base. The method of claim 15, wherein the target RN A molecule hybridizes to the intact NPM molecule or a portion thereof. The method of claim 13, wherein the NPM is single-stranded DNA (ssDNA) or branched (bDNA) DNA. 18. The method of claim 13, wherein the nuclease is DNase, RNase or a combination thereof. 19. The method of claim 13, wherein the NPM is a DNA molecule and the nuclease is a DNase. 20. The method of claim 13, wherein the nuclease is an S1 nuclease. 130267.doc 200907067 21. The method of claim 1, wherein the biological sample is fixed. 22. The method of claim 1, wherein the biological sample comprises an agent that crosslinks the target molecule. 23. The method of claim 21 wherein the immobilization comprises treating the sample with ethanol, formalin, dithiobis(succinimide propionate) (DSP). 24. The method of claim 1 wherein the target is crosslinked. 25. The method of claim 24, wherein the target is bis(sulfosuccinimide) (sodium octanoate) (BS3), diammonium iminoate (DSS), penta- Amber succinimide (dsg), disuccinimide tartaric acid (DST), glutaric acid or a derivative thereof are crosslinked. 26. The method of claim 1, which is for detecting at least one insoluble nucleic acid target in a fixed biological sample, comprising: (i) sufficient to facilitate the target and at least one nuclease protective molecule (NPM) The sample is contacted with the NpM under conditions in which the nuclease protecting molecule is a nucleic acid molecule that specifically hybridizes to the nucleic acid target, and (ii) the sample is rendered under conditions effective to remove any unbound NpM Exposure to one or more nucleases, (Hi) optionally separates the bound NPM from the target, but removes the NPM from binding to the tissue, and (iv) detects the presence of the NPM. 27. A method of quantitative assay comprising: immobilizing at least one insoluble reference in a biological sample (1) under conditions sufficient to facilitate binding of the stem to at least one nuclease protecting molecule 130267.doc 200907067 (NPM) Contacting the sample with the npm, wherein the nuclease protecting molecule specifically hybridizes to the nucleic acid target, (11) exposing the sample to one or more reagents under conditions effective to remove any unbound NpM, (ill The NPM is separated from the target as appropriate, but the NPM is removed from binding to the tissue, and (iv) the presence of the NPM is detected. 28. 29. 30. 31. 32. 33. 35. 35. 36. The method of claim 27, wherein the insoluble standard is a cross-linked nucleic acid. The method of claim 28, wherein the insoluble nucleic acid is a cross-linked mRNA, miRNA or VRNA. The method of claim 27, wherein the NPM is SSDNA or bDNA or aptamer. The method of claim 27, wherein the reagent in step (ii) comprises a nuclease. The method of claim 27, wherein the NPM is DNA and the reagent in step (ii) comprises DNase. The method of claim 27, wherein the reagent in step (Η) comprises 81 nuclease. The method of claim 27, further comprising amplifying the bound NPM prior to detection. The method of claim 27, further comprising separating the bound NPM from the target base using a base and/or heat. A method of detecting at least one of a soluble and insoluble form of a biological sample, comprising: 130267.doc 200907067 (I) sufficient to facilitate binding of the target to at least one nuclease protective molecule (NPM) The sample is contacted with the NpM, wherein the nuclease protecting molecule specifically binds to the target, and (II) $ is effective to remove any unbound NPM to expose the sample to one or more reagents, (III) separating the NPM from the target as appropriate, and (iv) detecting the presence of the NPM. 37. A method of finding a target, wherein the target molecule is detected without extraction. The method of claim 1, wherein the target molecule is detected without being dissolved. 39. The method of the present invention, further comprising preparing the NPM using the target molecule as a template for biosynthesis. The method of claim 1, further comprising assembling the NPM onto the target molecule (e.g., using a PCR-type primer). 41. A method of claiming, comprising detecting the sputum with a probe that specifically binds to the portion or a portion thereof. 42. The method of claim 26, which comprises detecting the sputum with a probe that specifically binds to the sputum or a portion thereof. 43. The method of claim 27, which comprises detecting the sputum with a probe that specifically binds to the sputum or a portion thereof. 44. The method of claim 36, which comprises detecting the sputum with a probe that specifically binds to the sputum or a portion thereof. 45. The method of claim 27, which comprises using a probe ❹ mNPM that hybridizes to the sputum or a portion thereof specificity 130267.doc 200907067, the chemistry of the towel from the hetero (4) needle system and the content of the insoluble standard in the sample Measurement related. 5 迷 46· A detection—at least one solid-label method in a biological sample' comprising: a square of knowledge (1) under conditions sufficient to facilitate binding of the target to at least one nuclease-protecting molecule (ΝΡΜ) Contacting the sample with the hydrazine, wherein the nuclease protecting molecule specifically hybridizes to the target, and (8) is effective for removing any unbound surface to expose the sample to one or more nucleases, (1) 丨) The condition separates the binding enthalpy from the target and (iv) detects the presence of the sputum. 130267.doc