EA011415B1 - Solution for lysis of cells or providing their permeability and methods of detecting a nucleic acid target - Google Patents

Abstract

The present invention proposes a solution for cells lysis for providing their permeability. The solution comprises formamide and sodium dodecyl sulfate in 0.5-2X SSC used a buffer. The use of the solution provides an effective release of nucleic acid target and not preventing its detection. There proposed embodiments of methods for detecting the nucleic acid target, the methods are useful for concurrently performing multiple, high throughput, biological or chemical assays, using repeated arrays of probes.

Description

This application submits a US patent application number 09/337325, filed June 21, 1999, which submits a US patent application number 09/218166, filed December 22, 1998, which submits a US patent application number 09/109076 filed July 2, 1998. This application claims the priority of provisional application No. 60/068291, filed December 19, 1997.

Field of Invention

The present invention relates, for example, to compositions, apparatus and methods for providing simultaneous multiple biological or chemical analyzes using repeating probe arrays. Many areas for each of them include a number of close anchor molecules. The anchors are connected with bifunctional linkers, each of which contains a section specific to at least one of the anchors and a section that is a probe for the target of interest. The resulting set (matrix) of probes is used to analyze the presence of one or more target molecules that specifically interact with the probes. The invention also relates to a solution for cell lysis or permeability, which is used in the framework of these methods. The invention relates to various fields characterized by the nature of molecular interaction, including, but not limited to the development of pharmaceuticals, molecular biology, biochemistry, pharmacology and medical diagnostic technology.

BACKGROUND OF THE INVENTION

Various biological and chemical analyzes use many molecular probes located on the surface, or "chips." Assays are performed to determine if the molecules of interest interact with one of the probes. After the probes are exposed to the target molecules under the selected test conditions, it is determined using detecting devices whether the target molecule interacted with this probe.

These systems are suitable for various screening methods in order to obtain information about either probes or target molecules. For example, among other things, they were used to screen peptides or potential drugs that bind to receptors of interest; for screening samples, for example, for the presence of genetic mutations, allelic variants in a population or a particular pathogen or strain of a pathogen among many others; to study gene expression, for example, to identify mRNA, the expression of which correlates with a certain physiological state, stage of development or a painful state, etc.

Description of the invention

The present invention provides compositions, apparatus and methods for simultaneously conducting a variety of biological and chemical analyzes and allows for high-throughput analysis of a plurality of samples, for example, a plurality of patient samples for screening in a diagnostic test or a plurality of potential drugs or test drugs for drug development. The invention also provides a solution for cell lysis or permeability, which is used in the framework of these methods. A combination suitable for detecting one or more targets in a sample is provided. This combination comprises a surface including a plurality of spatially separated regions, which may be called test regions, and which may be wells, at least two of which are substantially the same. Each surface includes at least two, preferably at least twenty or more, for example at least 25, 50, 96, 864 or 1536, etc. similar basically identical areas. Each test area defines a space for introducing a sample containing (or potentially containing) one or more targets and contains a biological or chemical population. (Similar phrases like “target containing sample” or “target detection in a sample” are not intended to exclude samples or determinations (detection attempts) that do not contain or not detect a target. In a general sense, this invention includes the totality to determine whether whether the target in the sample, regardless of whether it is detected or not detected). This population (matrix) includes close "anchors", each associated with a bifunctional linker, which has a first section specific to the anchor, and a second section that contains a probe specific to at least one of the target (s). The combination of the present invention is brought into contact with a sample containing one or more targets that optionally interact with a detecting molecule (s), and then is detected by a detecting device that detects reactions between target molecules and probes in test regions, thereby obtaining analysis results.

The invention provides methods and compositions, in particular suitable for staging biological analyzes with high throughput.

The invention provides a solution for cell lysis or permeability, which contains formamide, sodium dodecyl sulfate (8-8) and 88C as a buffer. The concentration of formamide varies from about 8 to about 60% (vol./about.), The concentration of 8-8 is from about 0.01 to about 0.5% (vol./about.). The buffer is represented as 0.5-6X 88C. The use of such a solution ensures the release of the target from the cells and does not interfere with

- 1 011415 analysis to identify the target nucleic acid.

In particularly preferred embodiments, the invention can be used for high throughput screening in drug development. For example, a high throughput assay can be performed simultaneously on many (e.g. 100) 96-well microplates. Each well of a tablet may have, for example, 36 different tests conducted therein using a combination (matrix) of about 36 pairs of anchors and linkers. That is, on 100 96-well plates, and each with 36 tests per well, a total of 345,000 tests can be set, for example, each of 9,600 different potential drugs can be tested simultaneously for 36 different parameters and tests. High-performance analyzes provide significantly more information for each candidate for a drug than tests that allow you to analyze only one parameter at a time. For example, it is possible in one initial high throughput screening test to determine if a potential drug is selective, specific and / or non-toxic. For low-throughput methods, extensive, consecutive tests are required to determine such parameters for each potential drug of interest. Several types of high throughput screening tests are described, for example, in Examples 15-17. The ability to simultaneously conduct a wide range of a wide variety of biological analyzes and simultaneously put many tests (i.e., with high throughput) are two important advantages of the invention.

In one embodiment, for example, using 96-well DNA binding plates (Cohshid CoMag) made from polystyrene with a derivatized surface for binding primary amines, such as amino acids or modified oligonucleotides, a series of 36 different oligonucleotides can be applied to the surface of each well of each tablet for functioning as anchors. Anchors can be covalently attached to derivatized polystyrene, and the same 36 anchors can be used for all screening tests. For any particular analysis, this series of linkers can be used to make the surface of each well specific for 36 different targets or tests of interest, and different test samples can be added to each of 96 wells in each plate. The same series of anchors can be used repeatedly to reprogram the surface of the holes for other targets and tests of interest, or it can be used repeatedly with the same set of linkers. Such flexibility and reusability represent additional advantages of the invention.

One embodiment of the invention is a combination suitable for detecting one or more target (s) in a sample, which includes, before adding said sample:

a) a surface containing many spatially separated regions, at least two of which are basically the same, where each region contains

b) at least eight different oligonucleotide anchors, each associated with

c) a bifunctional linker that has a first region specific for the oligonucleotide anchor and a second region that includes a probe specific for said target (s).

Another embodiment of the invention is a combination suitable for detecting one or more target (s) in a sample, which includes, before adding said sample:

a) a surface containing many spatially separated regions, at least two of which are basically the same, where each region contains

b) at least eight different anchors, each associated with

c) a bifunctional linker that has a first portion specific to the anchor and a second portion that includes a probe specific to said target (s).

Another embodiment of the invention provides a method for detecting at least one target, which comprises contacting a sample, which may contain the target (s), with the combination described above, under conditions effective to bind said target (s) to the specified combination. Another embodiment of the invention provides a method for determining the character of RNA expression, which comprises incubating a sample containing at least two RNA molecules as a target (s), with the combination described above, where at least one probe of the combination is a nucleic acid (e.g. oligonucleotide), which is specific (i.e., selective) to at least one of the target RNA, under conditions effective for the specific hybridization of the target RNA (s) with the probe (s). Another embodiment provides a method for identifying an agent (or condition (s)) that modulates the nature of RNA expression, which represents the method described above for determining the nature of RNA expression, further comprising comparing the nature of RNA expression in the presence of said agent (or condition (s)) with the nature of RNA expression under various conditions.

As an example in FIG. 1 and 2 show a combination of the invention and a method for its use for detecting a target mRNA. The surface of the invention shown in FIG. 2, contains 15 identical test areas; and in a particularly preferred embodiment of the invention, each of these test regions represents a well in a microtiter plate. Each of the test areas contains

- 2 011415 six different anchors numbered 1-6. In FIG. 1 schematically illustrates one of such anchors, anchor 1, which in the most preferred embodiment of the invention is an oligonucleotide. An anchor 1 is attached to the linker molecule, linker 1, which includes two sections. The first region that is specific for the anchor in this figure is an oligonucleotide that specifically hybridizes with the anchor. The second region, which is a probe specific for the target of interest, in this case mRNA target 1, in this figure is an oligonucleotide that hybridizes with its target. Despite the fact that this is not shown in the figure, each of the remaining five anchors can hybridize with its own linker through a site specific to the anchor; each linker includes a probe portion specific, for example, to an mRNA different from (or the same) mRNA 1. This combination shown in the figure can be simultaneously used to analyze 15 different samples for the presence of mRNA 1 (or simultaneously 15 mRNA targets that are specific (programmed) 5 other probes in total). To test, each sample, which in this example can be an RNA extract, for example, from one of 15 independent cell lines, is added in a small volume to one of the regions, or wells, and incubated under conditions effective for hybridization of the probe and target. In order to determine whether mRNA 1 is present in the sample, a detection device is used that recognizes the types and / or can detect specific positions in each region for the presence of a signal. If cell lines are incubated under conditions in which the label is included in the mRNA molecule under the conditions ίη νίνο, and if mRNA 1 is in the sample, the detector will detect a signal coming from the labeled mRNA in the position determined by the anchor / probe complex.

1. Alternatively, a label can be directly introduced into the mRNA under ίη νίίτο conditions before or after addition to the region (well). Alternatively, as shown in FIG. 1, mRNA can be marked indirectly before or after its hybridization with a probe, for example, by incubating RNA with a labeled “detecting” oligonucleotide (target-specific oligonucleotide reporter), which is complementary to a different sequence than that recognized by the probe. In the presented example, 15 samples can be analyzed simultaneously. Since at least 20 or more, for example 1536 or more samples can be simultaneously analyzed according to this invention, this is an analytical system with a very high throughput.

In the sense in which this term is used here, “target” refers to a compound whose presence, activity and / or amount is desired to be determined, and which has affinity for a given probe. Targets can be artificially produced or natural compounds. They can also be used unchanged or in the form of aggregates with other substances. Targets can be linked, covalently or non-covalently, to a binding member, either directly or using a specific binding compound. Examples of targets that can be used according to this invention include, but are not limited to receptors (on vesicles, lipids, cell membranes, or various other receptors); ligands, agonists or antagonists that bind to specific receptors; polyclonal antibodies, monoclonal antibodies and antisera that react with specific antigenic determinants (such as viruses, cells or other substances); drugs, nucleic acids or polynucleotides (including mRNA, tRNA, rRNA, oligonucleotides, DNA, viral RNA or DNA, E8T, cDNA, PCR amplified products derived from RNA or DNA, and their mutants, variants or modifications); proteins (including enzymes such as those responsible for the breakdown of neurotransmitters, proteases, kinases, and the like); substrates for enzymes; peptides, cofactors; lectins; sugars; polysaccharides; cells (which may include surface antigens); cell membranes; organelles, etc., as well as other molecules or other compounds that may be in the form of a complex, linked by covalent or cross bonds, etc. In the sense in which they are used here, the terms nucleic acid, polynucleotide, polynucleic acid and the oligonucleotide are interchangeable. Targets can also be attributed to anti-probes.

In the sense in which the term “probe” is used here, it represents a compound, for example, a molecule that can be specifically recognized by a specific target. Types of potential probe / target or target / probe binding components include a receptor / ligand; ligand / antiligand; nucleic acid (polynucleotide) interactions, including DNA / DNA, DNA / RNA, PNA (peptide-nucleic acid) / nucleic acid; enzymes, other catalysts, and other compounds with substrates, small molecules, or effector molecules, etc. Examples of probes that are suspected to be included in the scope of the present invention include, but are not limited to, organic and inorganic compounds or polymers, including metals, chelating agents, or other compounds that specifically interact with metals, plastics, agonists and antagonists of cell membrane receptors, toxins and poisons, viral epitopes, hormones (for example ep, opioid peptides, steroids, etc.), hormone receptors, lipids (including phospholipids), peptides, enzymes (such as proteases or kinases), enzyme substrates, cofactors, drugs, lectins, sugars, nucleols

- 3 011415 new acids (including oligonucleotides, DNA, RNA, PNA, or modified or substituted nucleic acids), oligosaccharides, proteins, enzymes, polyclonal and monoclonal antibodies, single-chain antibodies or their fragments. Polymer probes can be linear or cyclic. Using probes, phosphorylated and non-phosphorylated proteins can be distinguished either through differential activity or differential binding. Using probes, such as lectins, glycosylated proteins can be distinguished. In the sense in which they are used here, the terms nucleic acid, polynucleotide, polynucleic acid and oligonucleotide are used interchangeably. Any of the compounds described above as “probes” may also serve as “targets” and the like.

Any mating surface can be used according to the invention. The surface (usually solid) may be of various organic and inorganic materials or combinations thereof, including, by way of example only, plastics, such as polypropylene or polystyrene; ceramics; silicone; silica (melting) silica or glass, which may have a thickness of, for example, a microscope slide or a cover glass; paper such as filter paper; diazotized cellulose; nitrocellulose filters; nylon membranes; or polyacrylamide or other type of gel bases, for example, aerobases or air granules made from an airgel, which is, for example, a highly porous solid, including a film, which is prepared by drying the crude gel using any of a variety of conventional methods generally accepted in such cases. When optical detection tests are used in the formulation of the methods, light-transmitting substrates are quite applicable. The surface may be of any thickness or transparency, which are compatible, for example, with conventional detection methods. For example, the surface may be a thick bottom, transparent or opaque tablet. In a preferred embodiment, the surface is a plastic surface of a plurality of wells, for example, for tissue cultivation, for example, a 24-, 96-, 256-, 384-, 864- or 1536-well plate (for example, a modified plate, such as a DNA binding plate production Sotshid So81at). Anchors can be attached, for example, connected directly to the surface, or can be attached to one type of surface, for example glass, which, in turn, is brought into contact with a second surface, for example, a “hole” made of plastic in a microtiter plate. The shape of the surface is 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 a granule, particle, thread, sediment, tube, sphere, etc.

A surface includes areas that are spatially disjoint and addressable or identifiable. Each area includes a series of anchors. How the regions are separated, their physical properties and their relative orientation with respect to each other, is not decisive. In one embodiment, the regions can be separated from each other by any physical barrier that is resistant to liquids. For example, in a preferred embodiment, the regions may be wells in a multi-well plate (eg, for tissue culture), for example, in a 24-, 96-, 256-, 384-, 864- or 1536-well plate. Alternatively, the surface may be an engraved glass surface such that, for example, there are 864 or 1536 separate shallow wells. Alternatively, the surface may include areas without partitions or holes, i.e., for example, be a flat surface, for example, a piece of plastic, glass or paper, and the individual areas can be further divided by a structure lying on top (e.g., a piece of plastic or glass) that will be separate separate areas. Optionally, the surface may already include one or more sets of anchors or anchors associated with linkers, before dividing into separate areas. In another embodiment, the set of anchors within each region can be separated from each other by control spaces on the surface on which there are no anchors, or by chemical separation lines such as wax or silicones, to prevent droplets from spreading.

In yet another embodiment, the regions can be distinguished in the form of tubes or fluid-controlling channels, for example, designed for flow tests, as disclosed, for example, in HCS e! a1. (1995), C11P. S11st. 4, 700-706. The tubes can be of any size, for example, they can be capillaries or wider tubes so that liquids can flow, or they can be partially or completely filled with a gel, for example, agarose or polyacrylamide, through which compounds are transported (pass through, flow through, pass under pressure through), for example, by electrophoresis; or through channels with matrix-filled space, for example, linear channels described, for example, in A1b0! a e! a1. (1998) 8c1eise 281, 1653-1656; Sitrsoi e! a1. (1998), Ma !. Kek. 8os. 8m Rgos. 488,217-225; and / or Sitrooy e! a1. (1999), Ya! Ig 398, 51-54. In such a space-filling matrix, the liquid and / or molecules can move not only in the direction perpendicular to the walls of the tube, but they can also diffuse in the lateral directions. In a preferred embodiment, the tube is filled with a gel or a space-filling matrix; the gel or matrix filling the space is activated for binding with anchors, and various anchors pass sequentially, which allows the formation of a set (for example, a linear set) of anchors inside the gel; and linkers, targets, etc. also pass sequentially. Totality

- 4 011415 can be linear or 2- or 3-dimensional.

In one tube, you can put many tests. For example, one set of anchors or anchors associated with linkers in a tube can be reused (for example, scraped and reused, or reprogrammed) in sequential tests with the same or different samples. In another embodiment, multiple tubes are used in one test, for example, a sample of interest is analyzed in multiple tubes containing different populations. The anchors or anchor / linker complexes in the tubes can be any of the types described here.

Areas inside or on etc. surfaces can be separated by modifying the surface itself. For example, a plastic surface may include areas made of modified or derivatized plastic, which can serve, for example, as sites for adding specific types of polymers (for example, PEG can be bonded to a polystyrene surface and then derivatized at a carboxyl or amino group, double bonds , aldehydes and the like). Alternatively, the plastic surface may include molded structures, such as protrusions or bulges, which can act as pads for adding anchors. In another embodiment, the regions can be gel substrates, for example polyacrylamide gel or aerobase substrates, which are arranged in the desired order on a surface such as glass, or are placed between two surfaces, such as glass and quartz. Anchors, linkers, etc. can be immobilized on the surface of such bases or can be immersed inside. A person skilled in the art will obviously understand various other arrangements of gel bases on surfaces, and which can be obtained by conventional methods generally accepted in such cases. Test areas can be located in any relative orientation, including but not limited to parallel or perpendicular populations within a square or rectangle, or other surface, with radial propagation of populations within a circle or other surface, or linear populations, etc.

Spatially separated areas of the invention are in multiple copies. That is, there are at least two, preferably at least twenty or at least about 24, 50, 96, 256, 384, 864, 1536, 2025 or more, etc. basically the same, spatially separated (separate) areas. The increasing number of repeatable areas allows for analyzes with significantly higher throughput. In the sense in which this term is used here, basically the same areas refer to areas that include the same or basically the same sets of anchors and / or anchor / linker complexes. In the sense in which this term is used here, basically the same means that the population or region is intended to perform basically the same function as the other population or region in relation to the analysis of the target of this invention. Differences that do not significantly affect the function, i.e., the detectability of targets, are compatible with small changes in nucleotides (gaps / inserts / substitutions) or changes in oligonucleotides (weak binding to the surface), etc., which does not significantly affect determination results.

Those skilled in the art will obviously appreciate that not all areas on the surface should be identical to each other. For example, if a test is conducted in parallel with two different rows of populations, then it may be beneficial to include both rows of populations on the same surface. For example, two different series of populations can be arranged in alternating order in the form of stripes to facilitate comparison between them. In another embodiment, it may be desirable for a practitioner to include areas that can be detected in a manner different from other areas on the surface and use them as “registration area (s)”. For example, the detection region may include oligonucleotides or peptides that have various types of fluorescent molecules that can be recognized by a scanning detection device as a “starting point” for locating regions on the surface.

The size and physical placement of the test areas are not limited. Typical areas have an area of from about 1 to 700 mm ² , preferably from 1 to about 40 mm ² , and they are located at a distance of from 0.5 to about 5 mm from each other, and are usually selected depending on the area. In a preferred embodiment, the regions are approximately 5 mm apart. For example, each area may include a rectangular grid, for example, with 8 rows and 6 columns of roughly rounded anchor spots, which are about 100 microns in diameter and 500 microns apart; such an area will occupy an area of approximately 20 mm ² . Areas with a larger and smaller area and distance between them are included.

Regions can also be further subdivided so that some or all of the anchors of the region will be physically separated from neighboring anchors using, for example, depressions or depressions. For example, the number of subsections (subdomains) in a region can be in the range of about 10 to about 100 or more or less. In one embodiment, the region that represents the well in the 1536 well plate can further be subdivided into smaller wells, for example from about 4 to about 900, preferably from about 16 to about 36 holes, thus forming a plurality of holes within the holes. See FIG. 4. A similar surface with recesses reduces tolerance

- 5 011415 necessary for the physical placement of one anchor (or group of anchors) in each planned space (locus), and the size of the areas containing the anchors is more uniform, which facilitates the detection of targets that are associated with the probe.

The term “anchor”, as used herein, refers to any substance or compound, for example, a molecule that binds (for example, immobilizes on or attaches covalently or non-covalently) to a surface or portion of such a surface (eg, a derivatized portion of the surface plastic), and which may specifically interact or bind to a linker or other compound described above. A portion of the anchor that binds, for example, to the linker molecule, may be directly attached to the surface, or the anchor may include an intermediate “spacer”. Such a spacer may be any substance, for example, any of various substances that are generally accepted in the art. In one embodiment, the spacer is a linear carbon-containing molecule having, for example, about 5-20 C atoms, preferably 12 C atoms. In another embodiment, the spacer is a nucleic acid (any of the types described herein) that does not specifically interact or bind, for example, linker molecule.

The term "anchor" in the sense in which it is used here also refers to a group of basically identical anchors. See, for example, FIG. 7, which schematically shows the test area, including 3 anchors (A, B and C), each of which is in multiple copies (“group”). The position of each group of anchors is called a “locus” here. As is well known to specialists in this field, the number of individual anchor molecules located in the locus is limited only by physical obstacles that arise, for example, as a result of the size of the anchors. For example, a locus that has a diameter of, for example, about 25-200 microns, may contain millions of anchors.

In the sense in which this term is used here, an “anchor / linker complex” exists if the anchor and linker are molecularly bonded in a specific way. The interaction with the linker can be either irreversible, such as through certain covalent bonds, or reversible such as through hybridization of nucleic acids.

In a preferred embodiment, the anchor is a nucleic acid, which may be of any length (eg, oligonucleotide) or type (eg, DNA, RNA, PNA or a PCR product of an RNA or DNA molecule). The nucleic acid can be modified or substituted (for example, incorporate unnatural nucleotides such as inosine; combine by known bonds such as sulfamate, sulfamide, phosphorothionate, methylphosphonate, carbamate, etc. or obtain a semisynthetic molecule such as a DNA streptavidin conjugate etc.). Single stranded nucleic acids are preferred.

The nucleic acid anchor may be of any length compatible with the invention. For example, the anchor may be an oligonucleotide of about 8 to about 50 nucleotides in length, preferably 10, 15, 20, 25 or 30 nucleotides. In another embodiment, the anchor may be from about 50 to about 300 nucleotides long, or longer or shorter, preferably from about 200 to about 250 nucleotides.

For example, an anchor may include from about 150 to about 200 nucleotides of a spacer nucleic acid, as described above, and a shorter sequence adjacent to the spacer, for example, from about 10, 15, 20, 25, or 30 nucleotides, which is intended for interactions with the linker molecule (“linker-specific sequence”). Such spacers may be of any length or type of nucleic acid, and may have any base composition that is functional for the invention. In a preferred embodiment, the spacers of each of the anchors at the locus and / or anchors at various loci within the region are substantially the same; Thus, anchors differ from each other mainly in their linker-specific sequences.

Spacers can give anchors an advantage to increase their performance. For example, linker-specific sections of such an anchor are located farther from the surface and, therefore, they have less physical obstruction and are less sterically obstructed than if they were closer to the surface. This facilitates, for example, the linking of many different linkers (for example, from about 2 to about 100) with different specificity for the target, with anchors at this locus. As will be discussed in more detail below, a single anchor can include (in addition to the spacer) many linker-specific sequences arranged, for example, in a linear order; this allows many different types of linkers to communicate with at least one similar anchor at a given locus. Also discussed below in more detail is another way in which many different types of linkers can be associated with anchors at a given locus: in a “mixed locus”, where each of two or more anchors is connected to a different linker with different target specificity. As a result of the physical flexibility of anchors, including spacers, anchors at a given locus can easily bind to many different linkers without physical difficulties on the part of molecules of adjacent anchors. The advantage of linking multiple linkers to anchors at a given locus is that it becomes possible to detect a large number of targets at a particular locus. In one embodiment, many

- 6 011415 linkers linked at a given locus have probes that are specific for different regions of the same target nucleic acid of interest (for example, for different oligonucleotide sequences within a nucleic acid). This allows amplified target detection compared to single probe detection. In another embodiment, the plurality of linkers have probes that are specific to different, non-close targets. This allows you to detect many different targets within a specific locus. An additional advantage of anchors, including spacers, is that they can easily include linkers that are associated with relatively large molecules, such as proteins, and / or which are associated with relatively large targets, such as proteins, membranes, or cells.

The base composition of the nucleic acid anchor is not strictly limited. Any base composition of anchors is suitable provided that the anchors are functional for the purposes of the invention. For example, single-stranded nucleic acid anchors at a locus or at different loci of a region may include partially or completely arbitrary sequences (for example, randomly generated sequences, for example, without limiting the relative amounts of A, C, T and / or C). In one embodiment, the anchors are not “sequence isomers” (eg, “arbitrary sequence isomers”), i.e. oligonucleotides containing the same amount of C, C, A and T, but which are located in different relative order. That is, the anchors, for example, in different loci of the region do not satisfy the equation C _p C _p A _t T _t , where η and t are integers. See, for example, the anchors shown in Example 1, which are not arbitrary sequence isomers. In the anchors of the invention, the amounts of C and C should not be approximately the same as the relative amounts of A and T. In addition, the total relative amounts of C, C, A and T are not particularly limited. For example, the composition of anchor bases in a region can range from a relatively high SS content (i.e., above 50% C + C) to an equal content of C, C, A, and T, and to a relatively high AT content (i.e. above 50% A + T). In one embodiment, the anchors are obtained arbitrarily, for example, without limitation with respect to relatively high amounts of C, C, A, and T.

Anchors, including a nucleic acid spacer and one or more linker-specific sites, are unlikely to be consistent with any particular limitation on the composition of the bases. For example, if the anchors located at different loci in the region have spacers that are basically the same, for example, represent basically the same 25-dimensional or 200-dimensional oligonucleotide, then each anchor has a different linker-specific group (for example, 25-dimensional) even if a linker-specific groups satisfy certain requirements (e.g., the number of A and C is approximately equal, the number T and C is approximately equal; oligonucleotide corresponds to the equation C _n C _n A _m T _m, and / or the content of C + C sp vletvoryaet specific requirement), then the whole anchor will not meet these specific requirements. Similarly, even if linker-specific groups of anchors at different loci of the region are mostly different from each other (for example, each linker-specific group has a sequence that differs by at least about 20 or 50%, or 80% from each other linker- specific group in the field), the general identity of the sequences of anchors, taking into account the full length of the nucleic acid, can be significantly less. For example, if each of the anchors includes basically the same 25-dimensional spacer, and a 25-dimensional linker-specific group, which is 100% different from every other linker-specific group in the region, then the anchors will still differ from each other only on 10%.

An anchor can be a peptide or a protein. For example, it may be a polyclonal or monoclonal antibody or fragment thereof, or a single chain antibody or fragment thereof that specifically binds to a linker site representing an antigen or antibody; additionally, the anchor may be a peptide, and the portion of the linker that binds to it may be an antibody or the like. In another embodiment, the anchor may be a lectin (such as concanavalin A or agglutinins from organisms such as Ul1i8, peanuts, golden beans, Rya8e1i8, wheat germ, etc.) that is specific for a particular carbohydrate. In another embodiment, the anchor may include an organic molecule, such as a modified or derivatized plastic polymer, which may be useful, for example, as the basis for specific solid-phase chemical synthesis of an oligonucleotide. In this case, the derivatized plastic can be distributed in the form of a set of disconnected, derivatized loci, which generally form the plastic surface of the combination in the manufacturing process. In another embodiment, the anchor has the advantage of specific or preferred binding between metal ions, for example, N1, Ζη, Ca, MD, etc. and certain proteins and chelating agents. For example, the anchor may be polyhistidine, and the anchor-specific portion of the linker may be nickel, which is coupled by a chelating agent for nickel to a target-specific probe. Alternatively, the chelating agent may be an anchor, and polyhistidine may be the site of the probe. Alternatively, the anchor may include an inorganic compound. For example, he

- 7 011415 may include a metal, such as calcium or magnesium, and the anchor-specific portion of the linker may be the preferred chelating agent, such as, respectively, EDTA or EGTA, which binds to the target-specific probe. Those skilled in the art will obviously appreciate that a wide variety of other types of molecules can serve as anchors, as such, are common types already mentioned in connection with probes and targets.

An anchor can also be a hybrid structure, such as DNA duplex or duplex, including, for example, DNA and protein, which specifically interact by any means described herein. For example, the “main group” of a duplex anchor (a site that directly contacts the surface) may include an optionally modified single-stranded nucleic acid; preferably the main group also includes a spacer, for example a linear carbon-containing spacer, as described above. In one embodiment, the second single-stranded nucleic acid binds (e.g., hybridizes) to this main group to form an anchor that includes at least partially double-stranded (duplex) nucleic acid. For example, the main group may include a linear carbon-containing spacer that is attached to the surface at one end and at the other end is connected to a single-stranded DNA oligonucleotide of about 10-100 nucleotides, preferably about 25 nucleotides, and the second duplex group may include a sequence that complementary to at least a portion of the main group (e.g., about 40 terminal nucleotides), then an optional spacer (e.g., from about 5-15, preferably about 10 nucleotides), then nker-specific sequence (e.g., a sequence length of about 8-50 nucleotides, preferably about 15, 20, 25 or 30 nucleotides, most preferably about 25 nucleotides).

The relative length and composition of the bases of the complementary sections of the duplex anchor and its linker-specific sequence (s) may be different in order to meet the test requirements using optimization methods adopted in this field. For example, sequences can be selected so that linkers can dissociate (for example, split when melted) from the duplex anchor under conditions in which the duplex anchors themselves remain intact. Then, the remaining sets of anchor duplexes can, if desired, be reused for hybridization with the same or different linkers. Alternatively, sequences can be chosen so that both the anchor / linker hybrids and two complementary duplex anchor regions dissociate under identical conditions, leaving only the main groups in contact with the surface. In one embodiment, all or substantially all major groups at a particular locus or at all loci of a region are the same or substantially the same. The sets of the main groups remaining after such dissociation can be reused (for example, for hybridization with linkers) only if the complementary sections of the anchor duplex come back, in a way for which it is necessary to know the sequence of the main group that takes part in the formation of the duplex. The possibility of producing sets of anchors that can or cannot be reused by a user who does not know the sequence of the main groups represents the advantage of using such hybrid anchors. For example, a manufacturer may prevent unauthorized reuse of its assemblies. Preventing such reuse can, for example, prevent the performance or unreliability problems that result from heavy use.

In one embodiment, the group of anchors at a given locus within a region is substantially the same (for example, they are specific to the "anchor-specific" portion of a linker of the same type or for only one target). See, for example, FIG. 7. In another embodiment, a plurality of different anchors having specificity for a plurality of different linkers and / or for a plurality of different targets may be in a particular locus called a “mixed locus”, for example, a plurality of about 2 to about 100, for example at least about 2, at least about 4, or at least 10. The advantage of mixed loci is that it is possible to detect a large number of different targets at a particular locus. In one embodiment, each mixed locus contains one anchor that is the same at each or at least several loci. For example, an anchor that is the same at more than one locus can be used to guarantee quality and / or control, or to normalize the signal.

Of course, “mixed loci” are also advantageous for surfaces having only one (non-repeating) region. Anchors in each of the loci of a similar separate region can interact with linkers or directly with targets of interest.

The number of anchors (i.e., groups of anchors at individual loci) in the test region can be at least two, preferably in the range of about 8 to about 900 (with more or less), more preferably about 8 to about 300, and most preferably about 30 to about 100 (e.g., about 64). In some preferred embodiments, there are about 16, 36, 45, or 100 anchors / test area for a surface with 96 test areas (e.g., wells) or about 9, 16 or 25 anchors / test area for a surface with 384 test areas ( for example, holes). In the most preferred embodiment, each anchor in the test area has times

- 8 011415 personal specificity compared to every other anchor in the aggregate. However, two or more anchors may have the same specificity, and all anchors may be the same. In one embodiment in which the combination of the invention comprises a very large number of test areas (e.g., about 864, 1536 or higher), as a result of which it is possible to process a large number of test samples at the same time, it may be interesting to test these samples with only a limited number of parameters ( for example, about 2, 4, 6, or 9). In other words, for combinations involving a very large number of regions, it may be advantageous to have only about 2-9 anchors per region.

The physical location and relative orientation of the anchors (i.e., groups of anchors in a separate locus) in or on the test area are not limited. Typically, the distance between the anchors is from about 0.003 to about 5 mm or less, preferably from about 0.03 to about 1. Larger and smaller distances between the anchors (and their areas) are possible.

Anchors can be placed in any orientation relative to each other and the boundaries of the area. For example, they can be arranged in a two-dimensional orientation, such as a square, rectangular, hexagonal or other population, or circular assemblies with anchors emanating from the center in radial directions or in the form of concentric circles. Also, the anchors can be arranged in the form of a one-dimensional, linear population. For example, oligonucleotides can be hybridized with specific regions in a DNA or RNA sequence to form a supramolecular population or linearly in a flow gel or on the surface of a flow device or structure within a flow device. Alternatively, the anchors may be recessed into a “strip-like” formation (see FIG. 6). For example, anchors can be in the form of long strips parallel to each other. The distance between or the width of each long line can vary on a regular basis to get a simple, recognizable pattern that resembles a grid, for example, the first and third lines can be twice as long as the rest, lines can be skipped, etc. An additional blank line can be placed after the last line to separate one test area, and the grid can be repeated in subsequent test areas.

The location of the anchors should not be in strict accordance with the position of the divided analytical wells (test areas) or individual analytical drops. The term "analytical provisions" will be used in relation to the analytical surface where samples are applied for analysis. (These can be determined, for example, by the position of individual droplets of a sample for analysis, or by the position of wells, or separators that define individual wells for analysis on a multi-well plate). The very nature of the location of the anchors (for example, the "strip-like" nature of the location of the oligonucleotide anchors) is used to determine where exactly each individual anchor is located by the nature of recognition, since each line of the lattice is recognized by its position relative to other lines. Therefore, it is not required that the first anchor be on one edge or in one corner of each analytical position. The first anchor will be detected by the character of recognition to a greater extent than by the position relative to the analytical position. Since the area used for each analytical position (for example, the area of the drop or the area of the hole) is large enough to include at least one whole unit of repeating sets of anchors, then at each point of analysis the sample will be tested by the position of the analysis for all targets defined by the (lattice) of the population, where the population is within the area of the analytical position.

Anchors are not required to be placed in a strict or even fixed order within each test area. For example, each anchor can be connected to a particle, granule or the like, which take an arbitrary position within the test area. The position of each anchor can be determined using, for example, a detectable tag. For example, in a linker specific for each type of anchors, you can enter a different fluorescent, luminescent, etc. label, and you can determine the position of a particle containing a certain pair of linker / anchor, by the nature of the signal coming from the linker, for example, from the excitation spectrum or emissions. Specialists in this field can prepare a number of linkers with various such attached tags, each having a distinguishable spectrum. Alternatively, anchors can be tagged directly. For example, in each type of anchors, you can enter a label that will fluoresce with a characteristic spectrum that is different from the labels of anchors of other types. Alternatively, the particles, granules or the like may vary in size or shape from each other. Any tagging and detection method described here can be used. For example, the fluorescence intensity can be determined by using an SDS-based visualization system using a scanning fluorescence microscope or a cell sorter with excitation of fluorescence.

An anchor can interact or specifically bind to one site - an anchor-specific site - a linker. In this case, the terms “interact” or “bind” mean that two substances or compounds (for example, an anchor and an anchor-specific portion of a linker, a probe and its target, or a target and a target-specific reporter) bind (for example, bind, bind, hybridize, combine, anneal, covalently bonded

- 9 011415 or somehow connect differently) with each other sufficiently to conduct the intended analysis. In this case, the term “specific” or “specific” means that two components (for example, an anchor and an anchor a specific region of a linker, a probe and its target, or a target and a target-specific reporter) selectively bind to each other, and in the absence of any protection , but, as a rule, not with other components that are not intended to be associated with these components. The parameters necessary for specific interactions can be determined by conventional methods, for example, using methods generally accepted in the art.

A person skilled in the art can experimentally determine the properties of nucleic acids (such as length, base composition and degree of complementarity) that will help the nucleic acid (e.g., oligonucleotide anchor) hybridize with another nucleic acid (e.g., anchor-specific linker site ) under the conditions of selected rigidity, while minimizing nonspecific hybridization with other compounds or molecules (for example, other oligonucleotide linkers). Typically, the DNA or other nucleic acid sequence of an anchor, a linker portion or the detection oligonucleotide will have sufficient complementarity with regard to binding partner, facilitating the hybridization of selected stringent hybridization conditions, and the value of T _m will be about 10-20 ° C above room temperature ( for example, about 37 ° C). In general, an oligonucleotide anchor can have a length in the range of about 8 to about 50 nucleotides, preferably 15, 20, 25, or 30 nucleotides. In the sense in which this term is used here, “highly stringent hybridization conditions” means any conditions under which hybridization will occur when there is at least 95%, preferably from 97 to 100% nucleotide complementarity (identity) between nucleic acids. However, depending on the desired goal, it is possible to choose hybridization conditions for which less complementarity is required, for example, at the level of about 90, 85, 75, 50%, etc. Hybridization reaction parameters that may vary are salt concentration, buffer, pH, temperature, incubation time, amount and type of denaturing agent, such as formamide, etc. (see, for example, Sambo e! a1. (1989), Moiociagi Closid: Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa). Lysyuyu Ak1y NyuPy1 / ayyup. 1b Rzhekk; Ωην еκ e! A1. (1986), Vakyu Meyuyyk ίη Mo1esi1ag Vyuodu, E1keu1g 8s1epsek Riyykypd, 1ps., Exe Uogk). For example, nucleic acid (e.g., linker oligonucleotides) can be introduced into a test region (e.g., a well of a multi-well plate — in a preferred embodiment 96 or a 384-well or more plate) in a volume ranging from about 0.1 to about 100 μl or more ( in a preferred embodiment, from about 1 to about 50 μl, most preferably about 40 μl) at a concentration in the range of from about 0.01 to about 5 μM (in the preferred embodiment, about 0.1 μM) in a buffer such as 6X 88PE -T (0.9 M NaCl, 60 mM NaN ₂ PO ₄ , 6 mm E DTA and 0.05% Triton X-100) and hybridize with a binding partner (e.g., oligonucleotide anchor on the surface) for about 10 minutes to about at least 3 hours (in a preferred embodiment, at least for 15 minutes) at a temperature in the range of about 4 to about 37 ° C (in a preferred embodiment, at about room temperature). Conditions can be selected for high throughput. In one embodiment of the invention, the reaction conditions can be approximated to physiological conditions.

The construction of other types of compounds or molecules (for example, polypeptides, lectins, etc.) that can serve, for example, as anchors or sites of linkers, and the reaction conditions necessary to achieve specific interactions with binding partners are common and generally accepted a given area (for example, as described in Meteueg e! a1. (1994), M.1c1. Ac1 Century 22, 5530-5539; Euoge! a1. (1996), in US patent No. 5510270; Ripede e! a1. (1992), in US patent No. 5143854). Incubation parameters are buffer, salt concentration, pH, temperature, incubation time, the presence of a carrier and / or agents or conditions that contribute to the reduction of non-specific interactions, etc. For example, in a test region (eg, a well of a multi-well plate, in a preferred embodiment of a 96- or 384-well or more plate), which contains antibodies as anchors, anti-antibodies (eg, antigens or an antibody specific secondary antibodies) can be introduced into a volume in the range of about 0.1 to about 100 μl or higher (in a preferred embodiment, about 1 to about 50 μl, most preferably about 40 μl) at a concentration in the range of about 10 pM to about 10 nM (in the preferred embodiment, about 1nM) in a buffer, for example, such as 6X 88PE-T, PB8 or saline, and incubated with anchors on the surface for about 10 minutes to at least about 3 hours (in a preferred embodiment for at least 15 min) at a temperature ranging from 4 to about 45 ° C (in a preferred embodiment, about 4 ° C). For peptide-based anchors, a length of from about 5 to about 20 amino acids is preferred.

In some embodiments of the invention, each anchor in the aggregate can interact with the anchor-specific portion of the corresponding linker to the same extent as the other anchors in the aggregate, under the selected reaction conditions. This can ensure that the anchors accurately provide a largely uniform set of linkers and therefore probes.

- 10 011415

Anchors (ie groups of anchors at individual loci) within the test area can be a “related” series, i.e. each anchor of which can interact with one or more different linkers having a site specific to a similar anchor, but differing in areas of the “probe”; thus, one set of related anchors can be used to program or define different series of probes. The flexible nature of such a related set of anchors can be seen when referring to FIG. 1 and 2. In FIG. 2 shows a surface including 15 test regions, each of which contains a combination of 6 different anchors, which in this example are oligonucleotides. In FIG. 1 shows one of the (oligonucleotide) anchors, anchor 1, which is in contact with a linker 1, including one region specific for anchor 1, and a second region specific for mRNA target 1. Alternatively, it can be replaced, for example, with linker 2, which like linker 1, it includes a region specific to anchor 1, but which includes a second region specific to mRNA target 2 instead of mRNA target 1. So, anchor 1 can be used to define (or program, or establish, or define) probes for each of two or more different mRNs , is achieved. The method of obtaining and attaching a high-resolution series (aggregate) of oligonucleotides or peptides can be expensive, time consuming and / or physically difficult to implement. The ability to use a predefined set of anchors for programming a wide range of sets of probes is another advantage of this invention.

Although the related anchors shown in FIG. 2 determine the nature of the oligonucleotide probes; a set of identical probes can also be used to program a set of different probes, for example, receptor proteins (see, for example, FIG. 3). It is clear that many permutations are possible, as a result of which a number of types of anchor / linker interactions are obtained, for example, even more complex probes of the sandwich or multilayer type, such as protein / antibody combinations. For example, in one embodiment, the related series of anchors can be linked (covalently or non-covalently) to a number of linkers to produce a modified set of “conjugated” anchors, as described in more detail below. Thus, the surface of the anchors of the invention in itself provides new advantages.

In one embodiment of the invention, the anchors can interact reversibly with the linkers, so a number of close anchors can be reused to program other rows of probes. For example, the oligonucleotide anchor can be separated from the oligonucleotide portion of the linker, for example, by heating, which leads to the dissociation of two oligonucleotides, and then you can again bind it to the second linker. The ability to reuse sets of anchors, the preparation of which can be costly, time consuming and / or physically difficult to accomplish, is another advantage of the invention.

An anchor does not have to interact with the linker. For example, an anchor can be linked (directly or indirectly) to a detectable molecule, such as a fluorochrome, and thereby it can serve to establish the localization of a spot inside the grid, for example, to distinguish between a test surface and a detector. Alternatively, a label with a known amount of detectable molecule can be introduced into the anchor, thereby it will serve as an internal marker for quantification, for example, for calibration.

In the sense in which it is used here, the term “linker” refers to a bifunctional compound that contains the first section (or group, or region) that is specific to the selected (constructed) anchor or subgroup of anchors (“anchor-specific”), and a second portion that includes a probe specific for the target of interest (“target-specific”). The two linker sites can be connected via a covalent or non-covalent bond, and can be linked directly or via an intermediate group (e.g., a spacer).

The chemical nature of the anchor-specific linker site, of course, is a function of the anchor or anchors with which it interacts. For example, if the anchor is an oligonucleotide, then the portion of the linker that interacts with it may be, for example, a peptide that specifically binds to the oligonucleotide, or a nucleic acid that can hybridize efficiently and specifically with it under selected stringent hybridization conditions. The nucleic acid may be, for example, an oligonucleotide, DNA, RNA, PNA, a PCR product, or a substituted or modified nucleic acid (for example, including unnatural nucleotides, for example, such as inosine; linked via various known bonds, such as sulfamate, sulfamide, phosphorothionate, methylphosphonate, carbamate; or a semisynthetic molecule such as a DNA streptavidin conjugate, etc.). Single chain groups are preferred. The linker site specific for the oligonucleotide anchor may have a length in the range of about 8 to about 50 nucleotides, preferably about 15, 20, 25, or 30 nucleotides. If the anchor is an antibody, then the portion of the linker that interacts with it may be, for example, an anti-antibody, antigen, or a smaller fragment of one of these molecules that can specifically interact with the anchor. Compounds or molecules that specifically interact with other types of anchors described above, and which can serve as an anchor-specific linker site,

- 11 011415 are well known in this field, and can be obtained using conventional methods (for example, see above).

The chemical nature of the target-specific linker site, of course, is a function of the target for which it is a probe and with which it interacts. For example, if the target is a specific mRNA, then the target-specific linker region may be, for example, an oligonucleotide that specifically binds to the target but does not interact with RNA or DNA under the selected hybridization conditions. A person skilled in the art, using methods generally accepted in the art, will experimentally determine the properties of an oligonucleotide that will optimally hybridize to a target with minimal hybridization with non-specific interfering DNA or RNA (for example, see above). Typically, the length of the oligonucleotide probe used to detect a target mRNA against a large excess of non-target RNA can be from about 8 to about 50 nucleotides, preferably about 18, 20, 22 or 25 nucleotides. An oligonucleotide probe for use in biochemical analysis, in which there is no significant background of competitive targets, may be shorter. Using the methods adopted in this field (for example, the computer program VL8T), one can select sequences of oligonucleotide probes that are interconnected and differ from potentially interfering sequences in the well-known genetic database. In the usual way, you can select hybridization conditions that allow specific hybridization of the oligonucleotide probe with RNA to occur using methods generally accepted in the art (for example, see above). For example, a target RNA (e.g., a total fraction of RNA or mRNA isolated from tissues and cells cultured (and optionally treated with an agent of interest) in any container, such as wells of a multi-well microtiter plate (e.g., 96 or 384 wells or more), is introduced to a test area containing a combination of oligonucleotide probes (see above) in a buffer such as 6X 88PE-T or others, optionally containing an agent to reduce non-specific binding (for example, about 0.5 mg / ml of destroyed herring or salmon sperm DNA, il and yeast RNA) and incubated at an empirically determined temperature for a period of time ranging from about 10 minutes to at least 18 hours (in a preferred embodiment, about 3 hours). The stringency of the hybridization conditions can be the same or lower than the stringency used to bind anchors to an anchor-specific site of linkers Designing and using other types of probes is also common in the art, for example, as discussed above.

In one embodiment, all or substantially all linkers associated with anchors at a given locus comprise the same (or substantially the same) probe that is specific to a particular target of interest. In another embodiment, one or more linkers associated with anchors at a given locus includes many different probes and is thus specific to many different targets. These probes can be positioned in the linker as part of a branched structure or preferably can be arranged in a linear order, and they can be the same substance (for example, all are nucleic acid, or all are peptide sequences) or combinations of different substances. In practice, the presence of multiple probes in each linker increases the number of targets that can be detected at a particular locus. In one embodiment, in a plurality of probes in a given linker, all are specific for a particular target of interest (for example, they are specific to different regions of a particular mRNA of interest or specific to nuclease-protective fragments corresponding to different regions of that mRNA); this increases the sensitivity of the assay with respect to a target, for example, a target that is in a low concentration in the sample. The number of probes in the linker may be, for example, about 2-50, preferably about 2, 4 or 10.

Of course, linkers that include so many different probes are also advantageous for use with surfaces that contain one (non-repeating) region.

Anchor-specific and target-specific sites of the linker can be combined (connected, connected) by any of various covalent or non-covalent bonds, the nature of which is not relevant to the invention. Two sites can be connected directly or using an intermediate molecule. In one embodiment, in which both portions of the linker are oligonucleotides, they can be joined by covalent bonds, such as phosphodiester bonds, to produce one, colinear nucleic acid. In another embodiment, in which the anchor-specific site is an oligonucleotide and the target-specific site is a receptor, for example, a receptor protein, the two sites can be connected by the interaction of biotin and streptavidin molecules, an example is shown in FIG. 3. Many variants of such bonds are known (for example, see Noeteueg c1 a1. (1994), ΝΆΚ 22, 5530-5539). Alternatively, the two sites can be connected directly, for example, the oligonucleotide can be amidated and then directly linked (for example, crosslinked) to the peptide or protein via an amide bond, or attached to the membrane component via an amide bond or attached to a lipid. Methods for forming such covalent and non-covalent bonds are common, and one skilled in the art can easily optimize them.

- 12 011415 Spacer sequences (e.g., nucleic acid) may also be located between the anchor-specific and target-specific sites of the linker.

After binding of two compounds (for example, by incubating two nucleic acids, two proteins, a protein plus nucleic acid or others) to form a complex (such as, for example, an anchor / linker complex), the resulting complex can optionally be processed (for example, washed) to removal of unbound compounds (e.g., linkers) using conditions that can be easily determined empirically, leaving specific interactions intact, but removing non-specifically bound substances. For example, reaction mixtures can be washed from one to ten times or more under similar or somewhat more stringent conditions that were used to obtain the complex (for example, the anchor / linker complex).

One skilled in the art will obviously appreciate that it is possible to obtain various types of sandwiches for anchors and linkers. For example, to the set of anchors (for example, from anchors having basically the same sequence), you can attach the first group of linkers, each of which has a first group specific to the anchor, and a second group specific to one of the second group of linkers, and so on. In practice, this second layer of “sandwich” allows you to turn the first group of anchors (for example, the same oligonucleotides) into another population having a different group of specificities, “conjugated” anchors. The various groups of linkers and anchors can be linked together covalently or non-covalently, as desired.

The combinations of this invention can be produced in the usual way using conventional technology.

Some surfaces that can be used according to the invention are industrially available from various sources. In a preferred embodiment, the surface is a 96-, 384- or 1536-well microtiter plate, such as modified plates made by Sotspd Soiat. Alternatively, a surface including wells, which in turn contain depressions or "recesses," which can be made by microprocessing a compound such as aluminum or steel to form, then microinject plastic or similar material into the mold to form a structure such as presented in FIG. 4. Alternatively, a structure such as that shown in FIG. 4 made of glass, plastic, ceramic or the like, for example, of three parts such as those shown in FIG. 5; the first section, called the hole separator (Fig. 5A), which will form the separation between the sample wells; a second section, called a sub-unit (Fig. 5b), which will form subsections or indentations within each test well and a third section, called a base (Fig. 5c), which will form the base of the plate and the bottom surface of the test wells. The separator can be, for example, a piece of material, for example silicone, with holes located on it so that each hole will make up the walls of the test well when three pieces are connected. The sub-separator may be, for example, a thin piece of material, for example, silicone, in the form of a sieve or a fine network. The base may be a flat piece of material, such as glass, for example, in the form of the bottom of a conventional microplate used for biochemical analyzes. The upper surface of the base may be flat, as shown in FIG. 5c, or may have depressions that will be located with a subdivider to provide subsections, or wells within each sample well. The three parts can be connected by conventional methods, for example, the methods used to assemble silicone wafers.

Oligonucleotide anchors, linkers or detectors can be synthesized using conventional technology, for example, using an industrially available oligonucleotide synthesizer and / or ligation of sub-fragments that were synthesized in this way. Nucleic acids that are too long for convenient synthesis using such methods can be obtained by amplification, for example, by PCR using conventional methods. In one embodiment of the invention, predetermined nucleic acid anchors, such as oligonucleotide anchors, can be positioned on or within the surface of the test region using any of the methods generally accepted in such cases, including photolithography or chemical bonding, ink placement, capillaries, trellis or channel chips, electrochemistry using electrodes, contacting with a pin or a rod, or denaturation followed by UV irradiation on filters (for example, see Kata e! A1. (1996), US patent No. 5545531; Robot e! A1. (1996), US patent No. 5510270; 2apphiss1 and e! A1. (1997), US patent No. 5643738; WGeppap (1995), US patent No. 5474796; patent application \ UO 92/10092; patent application XVO 90/15070). Anchors can be placed at the top of the surface of the test area, or, for example, in the case of a polyacrylamide gel base, they can be immersed inside the surface so that some anchors protrude from the surface and are accessible for interaction with the linker. In a preferred embodiment, the predetermined oligonucleotide anchors are derivatized at the 5'-end with a free amino group; dissolved at a concentration that is usually easily determined empirically (for example, about 1 μM) in a buffer such as 50 mM phosphate buffer, pH 8.5 and 1 mM EDTA and dispensed using a pipette-dispenser (Sayeyai Tesypod1e8) in drops of about 10. 4 nanoliters in specific positions inside the test well, whose top is on top

- 13 011415 nost is a fresh plate for DNA binding (Sogshid Juice! Ag). Depending on the relative rate of attachment of the oligonucleotide and evaporation, it may be necessary to control the humidity in the wells during preparation. In another embodiment, oligonucleotide anchors can be synthesized directly on the surface of the test region using generally accepted methods, such as activated deprotection of growing oligonucleotide chains (for example, in combination with site-directed masking) or the distribution of nanoliter drops of a deactivating compound using a pipette - dispenser. You can deprotect all growing sequences to obtain, for example, a single nucleotide, and then apply the nucleotide to the surface. In another embodiment, oligonucleotide anchors can be attached to the surface at the 3'-ends of the oligonucleotides using a conventional methodology.

Peptides, proteins, lectins, chelating agents, plastics, and other types of anchors or linkers can also be obtained by conventional methods, and anchors can be positioned on or inside surfaces using suitable available technology (see, for example, Royog c1 a1. (1996), US patent No. 5510270; Rptiid e1 a1. (1992), US patent No. 5143854; / ap / essa e1 a1. (1997), US patent No. 5643738; 1, about \\ e e1 a1. (1985), US patent No. 4562157; Meteueg e1 a1. (1994), ΝΑΚ 22, 5530-5539).

In some embodiments of the invention, the disclosed combinations are used in a wide variety of screening methods and / or to obtain information about the level, activity or structure of probes or target molecules. Such tests are called screening methods with multiple sets on tablets (MAPE) methods or analyzes, and surfaces containing sets of anchors and anchors plus probes that are used for analysis are called MAP tests or MAPE tablets.

The components of the reaction mixture, analysis or screening method can be combined in any order. For example, anchors, linkers and targets can be combined in series, or targets and linkers, in the presence or absence of reporters, can be combined in solution and then brought into contact with the anchors.

One embodiment of the invention relates to a method for detecting at least one target, comprising:

a) contacting the sample, which may include the specified target (s) with a bifunctional linker, which has a first region specific for the oligonucleotide anchor, and a second region containing a probe specific for the specified target (s) under conditions effective to obtain the first hybridization product between the specified target (s) and the specified linker;

b) contacting said first hybridization product with a combination under conditions effective to produce a second hybridization product between said first hybridization product and said combination, where said combination includes, before adding said first hybridization product:

1) a surface containing many spatially separated regions, at least two of which are basically the same, where each region includes

2) at least 8 different oligonucleotide anchors;

c) contacting said first hybridization product or said second hybridization product with a labeled detection probe, and

g) detecting said detecting probe. Each of the assays or methods described below can be carried out in a high-throughput way, in which a large number of samples (for example, approximately 864, 1036, 1536, 2025 or more, depending on the number of regions in combination) are analyzed on each plate or surface quickly and simultaneously . In addition, many tablets or surfaces can be treated at the same time. For example, when developing drugs, a large number of samples, each containing a potential drug (for example, a compound from a combinatorial chemical library, such as variants of small molecules, peptides, oligonucleotides, or other compounds) can be added to separate regions of the described combination, or you can add in biological or biochemical samples, which are then added to separate areas of the combination and incubated with sets of probes located in the areas; and tests can be performed with each of the samples. Based on the latest advances and ongoing development of high density microplates, DNA application tools and techniques such as laser technology to receive and collect data from even very dense microplates, the use of automation, advanced dispensers, sophisticated modern detection systems and data processing software , the methods of this invention can be used for screening and analysis of thousands or tens of thousands of compounds per day.

For example, in embodiments where the probes represent oligonucleotides, the analysis may be screening using a diagnostic nucleic acid or polynucleotide (e.g., in a binding assay or other) of a large number of samples for the presence of genetic variations or defects (e.g. polymorphism or specific mutations associated with diseases) such as cystic fibrosis. See, for example, 1Xa e1 a1. (1992), MoCecilia api Ce11iagr Prgoec, 505-512); pathogens (such as bacteria, viruses and protozoa that are hosted by animals, including humans, or plants) or types of mRNA transcription that are diagnostic for op

- 14 011415 certain physiological conditions or diseases. A set of nucleic acid probes, including sections of E8T (including full-size copies), can be used to assess the nature of transcription in cells from which E8T (or others) were obtained. Using nucleic acid probes, it is also possible to detect peptides, proteins or domains of proteins that specifically bind to specific nucleic acid sequences (and the like).

Similarly, in embodiments in which the probes are antigen binding molecules (e.g. antibodies), the test may be screening for variant proteins or types of expression of proteins that are diagnostic for specific physiological conditions or disease states. See, for example, FIG. 40 and 41, which show the types of molecules that can be detected.

In another embodiment, the combinations of the invention can be used to monitor biochemical reactions, for example, in the interaction of proteins, nucleic acids, small molecules and the like, for example, with respect to the effectiveness or specificity of the interaction between antigens and antibodies, or receptors (such as purified receptors or receptors associated with cell membranes) and their ligands, agonists or antagonists, or enzymes (such as proteases or kinases) and their substrates, or increase or decrease coli ETS substrate transformed into a product, as well as many other things. Some biochemical tests can be used to characterize the properties of the probe or target, or as the basis of a screening test. For example, to screen samples for the presence of certain proteases (e.g., proteases involved in blood coagulation, such as Xa protease and UIA), samples can be tested on combinations in which the probes are fluorogenic compounds specific to each protease of interest. If the target protease binds to and cleaves the substrate, then the substrate will fluoresce, usually as a result of, for example, cleavage and separation of two pairs that carry energy, and as a result, the signal can be detected. In another example, for screening samples for the presence of a particular kinase (s), (e.g., 8 Tc, tyrosine kinases or ΖΑΡ70), samples containing one or more kinases of interest can be analyzed by combinations in which the probes are peptides that can be selectively phosphorylated with the participation of one of the kinases of interest. Using well-defined conditions accepted in the art, samples can be incubated with a combination of substrates in a suitable buffer and in the presence of the necessary cofactors for an empirically selected period of time. (In some tests, for example, when conducting biochemical studies on factors that regulate the activity of kinases of interest, the concentration of each kinase can be adjusted so that each substrate is phosphorylated at the same rate). After processing (for example, washing) each reaction mixture under empirically selected conditions to remove kinases and undesired reaction components (optional), phosphorylated substrates can be detected, for example, by incubating them with detectable reagents, such as fluorescein-labeled antibodies to antiphosphothyrosine or antibodies to antiphosphoserine (for example, at a concentration of about 10 nM or higher, or lower), and as a result, a signal can be detected. In another example, you can put binding tests. For example, 8H2 domains such as SKB2 8H2 or ΖΑΡ70 8H2 can be analyzed using a combination of probes from the corresponding phosphorylated peptides; or blood serum can be screened for a combination of probes from specific receptors for immunodeficiency. In addition, using such a combination, an enzyme-linked immunosorbent assay can be performed. The combinations of the invention can also be used to detect mutant enzymes that are more or less active compared to wild-type analogues, or to screen various agents, including herbicides or pesticides.

Of course, ΜΑΡδ-tests can be used for quantitative analysis (quantification) of the active target in a sample, provided that the probe is not fully occupied, i.e., no more than about 90% of the accessible sites of the probe are connected (either reactivated or hybridized) with a target. Under these conditions, the target can be quantified, since the larger the target, the more the probe will be connected. On the other hand, in conditions where more than approximately 90% of the accessible sites of the probe are connected, then with an increase in the number of the target, there will be no increase in the number of the target associated with the probe. Thus, any of the above types of targets can be quantified. For example, Example 6 describes the quantification of oligonucleotide targets. In addition, it is shown there that even if the target is in large excess (for example, if it is in such large quantities that it saturates the amount of available probe in ΜΑΡ8 of the probes), then adding known quantities does not include the target label in the binding mixture, one can “shift the sensitivity” of the reaction in order to make it possible to quantify even such large quantities of the target.

In another embodiment, the combinations of the invention can be used to screen agents that modulate the interaction of a target and a given probe. The agent can modulate the target / probe interaction by interacting directly or indirectly with either the probe, the target, or with the complex formed by the target and the probe. Modulation can take a wide variety of forms, including but not limited to increasing or decreasing the binding affinity of the target to

- 15 011415 probe, increasing or decreasing the rate of binding of the target and the probe, competitive or non-competitive inhibition of the binding of the probe to the target or increasing or decreasing the activity of the probe or target, which, in turn, can lead to an increase or decrease in the interaction of the probe / target. Such agents can be obtained artificially, or they are natural compounds. In addition, such agents can be used unchanged, or in the form of aggregates with other compounds, and they can be combined, covalently or non-covalently, with a binding partner, either directly or using a specific binding compound. For example, to identify potential "blood thinners" or agents that interact with one of the cascades of proteases that cause blood coagulation, mixtures of proteases of interest can be analyzed to include many potential agents and then tested for activity as described above. Other examples of agents that can be used according to the invention are very diverse, and include pesticides and herbicides. Examples 16 and 17 describe high throughput assays for agents that selectively inhibit certain kinases, or for selective inhibitors of the interaction of 8H2 domains and phosphorylated peptides.

In another embodiment, the combinations of the invention can be used to screen agents that modulate the pattern of gene expression. Combinations of oligonucleotides can be used, for example, to detect mRNA species whose expression type from a group of genes correlates with a particular physiological state or developmental stages, or with a painful state (“correlative” genes, RNA, or expression types). By the terms “correlates” or “correlative” it is understood that the nature of RNA synthesis is associated with the physiological state of the cell, but it is not necessary that the expression of this RNA be responsible for or cause a certain physiological state. For example, you can identify a small subgroup of mRNAs that are expressed, their expression increases and / or decreases in cells that serve as a model for a specific disease state; this altered type of expression compared with that in normal cells in which there is no pathological phenotype can serve as an indicator of a disease state (an “indicator” of genes, RNA, or types of expression). The terms “correlative” or “indicator” can be used interchangeably. For example, cells treated with a tumor promoter, such as phorbol myristate, may have a type of gene expression that resembles that of the early stages of tumor growth. In another tumor model, in mouse insulinoma cells (for example, TCR61 cell line), when adenovirus is infected, there is an increase in expression, for example, c-1 and M1P-2, while the expression of auxiliary genes such as CAPEN and L32, mainly remains unchanged.

Agents that, after contact with a cell derived from a disease model, either directly, or indirectly, and under either ίη νίνο or ίη νίίτο conditions (for example, in tissue culture), modulate the expression index, can function as therapeutic agents or drugs on the level of the body (for example, in humans or animals, or plants) suffering from this disease. Such agents can also modulate expression by direct contact with a nucleic acid, for example, in the ίη νίίτο expression system (in vitro). In the sense in which this term is used here, “modulate” means to cause an increase or decrease in the number and / or activity of molecules or the like, which are involved in a given reaction. The combinations of the invention can be used to screen for such agents. For example, groups of cells (e.g., originating from a disease model) can be contacted with a group of agents (e.g., for a period of about 10 minutes to about 48 hours or more), and then using conventional methods generally accepted in the art (e.g. using industrially available kits) extracts of the whole fraction of RNA or mRNA are prepared. If it is desired to amplify the amount of RNA, then conventional amplification methods can be used, such as CT-PCR (see, for example, Ιηηίκ c1 a1. Ebq., (1996) RSK RgoYusoe: And Sbbe ίe Meyubk ίη Λιηρίίίχαΐίοη. ) The extracts (or products amplified from them) are contacted (for example, incubated) with a plurality of substantially identical populations that include probes for the corresponding indicator RNAs, and agents that are associated with a change in the type of indicator expression can be detected. Example 15 describes a high throughput assay for screening compounds that can alter the expression of genes that are correlative for disease states.

Similarly, agents can be identified that modulate expression types associated with specific physiological conditions or developmental stages. Such agents can be artificially produced or natural compounds, including environmental factors, such as compounds involved in embryonic development or regulation of physiological reactions, or compounds of agricultural importance, such as pesticides or herbicides. In addition, such agents can be used unchanged or in the form of aggregates with other compounds; and they can be connected, covalently or non-covalently, with a connecting member, either directly or through a specific binding compound.

- 16 011415

Another embodiment of the invention is a kit suitable for detecting at least one target in a sample, including:

a) a surface containing many spatially separated regions, at least two of which are basically the same, where each region contains at least eight different anchors (oligonucleotides, or one of the other types described here) and

b) a container comprising at least one bifunctional linker, which has a first section specific to at least one of the indicated anchor (s) and a second section that includes a probe specific to at least one of the specified target (s).

In one embodiment, a surface is provided, as indicated in paragraph a) above, and a set of instructions for connecting at least one anchor to a bifunctional linker, which has a first section specific to at least one of the indicated anchor (s) and a second section, which includes a probe specific to at least one target. Instructions may include, for example, but not limited to, a description of each anchor on the surface, a description of how many anchors are located and where they are located on the surface, and a protocol for the specific attachment (linking, linking) of linkers to anchors. For example, if the anchors are oligonucleotides, then the instructions may include the sequence of each anchor, with which the practitioner will be able to construct complementary anchor-specific groups of linkers for a specific interaction (for example, hybridization) with the anchors; if the anchors represent peptides, then the instructions may include information, for example, about antibodies that will specifically interact with peptides. The instructions may also include a protocol for binding anchors and linkers, for example, a description of conditions and reagents for hybridization (or another type of compound), such as temperature and duration of incubation, conditions and reagents for removing unbound molecules (e.g., washing solutions) and the like. In addition, instructions may include information on the design and use of any of the types of control linkers already discussed here, and on methods for performing, for example, quantification, normalization, fine tuning, or calibration with combinations. The instructions may include any of the parameters, conditions or embodiments disclosed in this application that can be performed by a person skilled in the art in the usual way using methods generally accepted in the art.

As discussed in this application, a practitioner can attach to the surface of the invention, including a given set (or sets) of anchors, a variety of types of linkers, thereby programming any of a wide range of probes. In addition, the practitioner can remove this group of linkers from the surface according to the invention and add to it another group of linkers (either the same or different from the first group), which makes it possible to reuse this surface. This flexibility and reusability are additional advantages of the invention.

In another embodiment, the combinations of the invention can be used to map E8T (expressed sequence labels). That is, MLR8 tests can be used to determine which, if any, E8T group is obtained from different (or partially overlapping) regions of the same gene (s), and which, if any, is unique. Figures 18, 19, 20, and 21 show such an analysis; in this example, a test is shown to determine which, if any, of the 16 E8Ts are “linked” to a common gene. The first stage of the test (see figure 18) is the assembly of populations in which each of the E8T to be mapped is represented by at least one oligonucleotide probe that corresponds to it. The number of aggregates equal to (or higher) the number of E8Ts to be mapped is distributed into separate areas (eg, wells) on the surface; in the presented example, the surface of the combination includes 16 holes, each of which includes a combination of 16 different EgT-specific oligonucleotides numbered 1-16. An oligonucleotide that "matches" E8T (is "E8Tspecific") is one that is sufficiently complementary to E8T so that, under the stringent hybridization conditions, the oligonucleotide will specifically hybridize with that E8T, but not with other, non-close E8T. The E8T-corresponding oligonucleotide of this type can specifically bind (under optimal conditions) to the coding or non-coding strand of cDNA synthesized from the gene from which E8T was originally obtained, or to mRNA synthesized from the gene from which E8T was originally obtained. This application has already discussed factors that should be considered when designing oligonucleotides, and hybridization parameters for their optimization in order to obtain specific hybridization. In order to collect the populations, linker molecules are prepared, each of which includes a group specific to one of the anchors of the related population plus a group comprising an oligonucleotide probe that corresponds to one of the E8Ts that are mapped; and linkers are connected to the anchors, as already discussed in this application. In the next step, an aliquot of a sample containing a mixture of nucleic acids (for example, mRNA, or single-stranded or denatured DNA), which may include sequences complementary to one or more oligonucleotide probes, is introduced into each of the regions (wells) that contains the set of probes; then the mixture is incubated in the usual

- 17 011415, defined as optimal conditions, thereby allowing the nucleic acid to contact complementary probes. If several E8T-specific probes are complementary to different regions of the same nucleic acid, then this nucleic acid will bind to each locus in the aggregate in which one of these probes is located.

In the next step, another detecting oligonucleotide (in the example presented, detectors # 1-16) is introduced into each region (well) (see Fig. 19). A detecting oligonucleotide is designed for each of the E8Ts that are being mapped. Each E8T-specific detector corresponds to a different (at least partially non-overlapping) region of the E8T than the probe oligonucleotide does so that the probe and detecting oligonucleotides do not interfere with each other. See, for example, E8T shown in FIG. 21, which correspond to E8T 1, 2, and 6 in FIG. 18-20. In FIG. Figure 21 shows that both E8T # 1 and # 2 are derived from gene X (they are “linked”), while E8T # 6 is derived from another, unrelated gene. If aliquots of the sample containing the mRNA mixture, including that obtained from gene X, are incubated with the sets of probes shown in figures 18-20, then the mRNA of gene X will, under optimal conditions, hybridize at the loci with probes 1 and 2, but not in them , in the case of probe 6. (Of course, each mRNA should be added in molar excess to the total number of probes with which it can hybridize.) If the detecting oligonucleotide 1 is introduced into the region (well) 1, then it will hybridize with the mRNA of gene X, which connected at loci 1 and 2 of aggregate z NDS, but not at locus 6. Similarly, if the detecting oligonucleotide 2 is introduced into another well, for example, well # 2, then it will also bind at loci 1 and 2, but not at locus 6. Thus, it can be determined at high throughput abilities that from E8T bind, i.e. code for sections of the same gene, and which E8Ts are unique. For the hypothetical example shown in FIG. 20, the first 3 E8Ts encode regions of the same gene, while the last 5 E8Ts encode regions of the other gene, and it turns out that the rest of the E8Ts are not connected. This application has already discussed hybridization conditions, optional washing steps, detection methods and the like with respect to other ΜΆΡδ tests. In order to confirm the binding data obtained in the ΜΆΡδ test, PCR can be performed using pairs of ЕТТ-specific oligonucleotide probes as sense and antisense primers. Each pair of bound E8T should give a PCR product. It should be noted that this PCR test by a paired basis can be performed to directly determine the connection without using the ΜΆΡδ-analysis of the connection, however, in this case, many reactions will be required and each E8T primer will need to be synthesized as sense and antisense chains. As an indicative example, 180 such reactions will be required.

In one aspect, the invention relates to a method for determining which of a plurality of E8Ts are complementary to a given nucleic acid, comprising:

a) incubating an immobilized collection of oligonucleotide probes, at least one of which corresponds to each of these E8Ts, with a test sample that may contain said given nucleic acid to obtain a hybridization product of said oligonucleotide probes and said nucleic acid;

b) incubating said hybridization product with a detecting oligonucleotide that corresponds to E8T, which corresponds to one of said oligonucleotide probes, but which is specific to a different region of E8T than said oligonucleotide probe, and

c) detecting in which oligonucleotide probe from said population there is a label made using said detecting oligonucleotide, wherein said population of oligonucleotide probes is immobilized in a region of a combination where said combination includes:

1) a surface containing a number of spatially separated, basically identical, regions, equal to the series E8T, which are examined, where each region includes

2) a number of different anchors, equal to the series E8T, which are examined, in combination with

3) a bifunctional linker, which has a first portion specific to the anchor and a second portion that includes an oligonucleotide probe corresponding to at least one of E8T.

In another aspect, the invention relates to the aforementioned method, wherein said E8T can be complementary to said nucleic acid and where each of said E8T includes two different oligonucleotide sequences, the first of which determines an oligonucleotide probe corresponding to said E8T, and the second of which determines a detecting oligonucleotide corresponding specified E8T, including:

a) contacting a sample that includes the molecules of said nucleic acid with at least one region of the combination, wherein said region comprises a plurality of oligonucleotide probes, at least one of which corresponds to each of said E8T;

b) incubating said sample with said region, thereby allowing molecules of said nucleic acid to bind to said E8T corresponding oligonucleotide probes that are complementary to regions of said nucleic acid;

c) the incubation of the specified area, including the molecules of the specified nucleic acid is connected

- 01 011415 with one or more of the indicated ΕδΤ-corresponding oligonucleotide probes with a detecting oligonucleotide, which corresponds to Ε8 которой, which corresponds to this one of the oligonucleotide probes of the specified population, thereby linking the detecting oligonucleotides to the nucleic acid molecules that are linked to the specified oligonucleotide or with other oligonucleotide probes that are complementary to said nucleic acid;

ά) detecting the presence of said detecting oligonucleotides, and thereby identifying such ΕδΤ-corresponding oligonucleotide probes of the indicated population that are complementary to the nucleic acid sites that binds to said given Inappropriate oligonucleotide probe, thereby identifying those Ε8Τ that are complementary to the specified acid the specified set of oligonucleotide probes are immobilized in the region of the combination, where the specified combination includes

1) a surface containing a series of spatially separated, basically identical regions, equal to the series Ε8Τ, which are examined, where each region includes

2) a number of different anchors equal to the Ε8Τ series that are being studied, in combination with

3) a bifunctional linker, which has a first portion specific to the anchor and a second portion that includes an oligonucleotide probe corresponding to at least one of said Ε8Τ.

The components of the Ε8Τ mapping test can be combined in any order. For example, anchors, linkers and Ε8Τ can be combined in series, or linkers and Ε8Τ in the presence or absence of reporters, can be combined in solution and then added to anchors.

In another aspect, the invention relates to a method for determining which of a plurality of Ε8Τ are complementary to a given nucleic acid, comprising:

a) incubation of a group of bifunctional oligonucleotide linkers, each of which includes a first region that represents a probe corresponding to at least one of the indicated Ε8Τ, and a second region specific for the anchor oligonucleotide, with a test sample that may contain the specified given nucleic acid, s obtaining a first hybridization product of said oligonucleotide probes and said nucleic acid;

b) incubating said first hybridization product with an immobilized set of anchor oligonucleotides, where each anchor oligonucleotide corresponds to an anchor-specific region of at least one of said linkers, to obtain a second hybridization product comprising said anchors, said oligonucleotide probes and said nucleic acid and

c) incubating either said first or said second hybridization product with a detecting oligonucleotide that corresponds to Ε8Τ, which corresponds to one of the indicated oligonucleotide probes, but which is specific to a different Ε8Τ region than the indicated oligonucleotide probe and

ά) detecting in which oligonucleotide probes of the indicated population the label was turned on using the indicated detecting oligonucleotide, where the said population of anchor oligonucleotides is immobilized in the region of the combination, where the combination includes:

1) a surface containing a series of spatially separated, basically identical regions, equal to the series Ε8Τ, which are examined, where each region includes

2) a number of different anchors, equal to the Ε8 ря series, which are being studied.

Of course, the above Ε8Τ mapping methods can be used to map test sequences (e.g., polynucleotides) in any nucleic acid of interest. For example, it can be determined whether two or more cloned DNA or cDNA fragments belong to the same genomic DNA. A similar method can help, for example, establish the structure of long complex genes. Similarly, it can be determined whether one or more spliced sequences or coding sequences belong to the same genomic DNA. A similar definition can be used, for example, in a diagnostic test in order to distinguish between normal and painful conditions, which are characterized by different types of splicing. Many other applications of the mapping method will obviously become apparent to those skilled in the art.

In another aspect, the invention relates to a method for determining which of a plurality of polynucleotides is complementary to a given nucleic acid, where one or more of said polynucleotides may be complementary to said nucleic acid, and where each of said polynucleotides comprises two different oligonucleotide sequences, the first of which determines the oligonucleotide probe corresponding to the specified polynucleotide, and the second of which determines the detecting oligonucleotide, respectively the specified polynucleotide, including

a) contacting a sample that includes the molecules of said nucleic acid with at least one region of the combination, wherein said region contains a plurality of oligonucleotide probes, at least one of which corresponds to each of said polynucleotides;

- 19 011415

b) incubating said sample with said region, thereby allowing molecules of said nucleic acid to bind to said polynucleotide-corresponding oligonucleotide probes that are complementary to regions of said nucleic acid;

c) incubating the indicated region, including the molecules of the nucleic acid associated with one or more of the indicated polynucleotide-corresponding oligonucleotide probes, with a detecting oligonucleotide that corresponds to the polynucleotide to which this one of the oligonucleotide probes of the specified population corresponds, thereby binding the detecting oligonucleotides to the molecule nucleic acids that are associated with said oligonucleotide probe or other oligonucleotide probes which e are complementary to said nucleic acid;

ά) detecting the presence of said detecting oligonucleotides, thereby identifying such polynucleotide-corresponding oligonucleotide probes of a specified population that are complementary to regions of a nucleic acid that binds to said given oligonucleotide polynucleotide-corresponding probe, thereby identifying such polynucleotides, of a given nucleic acid, which are complementary where the specified set of oligonucleotide probes are immobilized in the field of combination, where the specified combination includes

1) a surface containing a number of spatially separated, basically identical regions, equal to a series of polynucleotides that are examined, where each region includes

2) a number of different anchors, equal to a series of polynucleotides that are examined, where each anchor is associated with

3) a bifunctional linker, which has a first portion specific to the anchor and a second portion that includes an oligonucleotide probe corresponding to at least one of said polynucleotides.

In another aspect of the invention, the above methods of mapping E8T or other polynucleotides further include removing unbound portions of the sample between one or more stages.

In another embodiment of the invention, one or more target RNAs of interest (eg, mRNA or other types of RNA) are converted to cDNA by reverse transcriptase, and then the cDNA data is hybridized with a plurality of probes. This type of analysis is shown schematically in FIG. 8. RNA extracts (or purified mRNA) are prepared from cells and tissues, as described here. Then, reverse transcriptase and oligonucleotide primers specific to the RNA of interest are added to the RNA sample and, using the conditions and methods adopted in this field, which can be easily determined and optimized, the first cDNA chains are obtained. The term “specific” primer refers to one that is sufficiently complementary to the mRNA of interest to be contacted under selected stringent hybridization conditions and recognized by reverse transcription, but which does not bind to undesired nucleic acid (see above for a discussion of suitable reaction conditions to achieve specific hybridization). Residual mRNA RNA molecules that were not recognized by specific primers and / or other types of contaminating RNA molecules in an RNA extract, such as tRNA or rRNA, can be removed using any of a wide variety of ribonucleases or by chemical methods such as alkali treatment, leaving single stranded cDNA, which is then brought into contact with a set of MAP8 probes. The use of reverse transcriptase in this method minimizes the need for intensive RNA processing, which may be susceptible to degradation by the action of nucleases, and therefore causes difficulties when working with it. In addition, additional specificity resulting from specific reverse transcriptase primers introduces additional specificity into the method.

Optionally, the cDNA described above can be amplified before hybridization with a plurality of probes to increase signal intensity. The reverse transcriptase oligonucleotide primers described above may include sequences at their 5 ′ ends (which may be about 22-27 nucleotides in length), which makes initiation sites specific for RNA polymerase (e.g., T7, T3, or 8P2 polymerase and the like like that). In the example of FIG. 8, the sequence of the T7 promoter is added to the reverse transcriptase primer. The polymerase recognition site becomes included in the cDNA, and then it can play the role of a recognition site for many transcription cycles with the participation of the corresponding RNA polymerase (transcription ίη У1! Go or 1UT). Optionally, the mRNA molecules thus obtained can be further amplified using PCR and corresponding primers, or the cDNA itself can be amplified. Thus, transcription and PCR staging methods are common and well known in the art.

The flexibility of PCR allows many changes to be made to the methods of the invention. In one embodiment, one or both of the PCR primers that are used to amplify the target may include a chemical modification that results in a PCR product, specifically or non-specifically,

- 20 011415 attached to a solid substrate. Such chemical modifications include, for example, 5'amidation, which allows binding to surfaces, such as Cochag DNA binding plates (for example, modified β-oxysuccinimide ether or maleic anhydride-coated plates, such as Keac-Wb plates manufactured by P1egse, WoekGogy. 1b ) Methods for producing oligonucleotides, including similar chemical modifications, are common and generally accepted in the art. A PCR product containing such a modified primer can be attached to any desired substrate, including a solid substrate, such as the inner walls of a well on a microtiter plate, a granule (e.g., a non-magnetic or magnetic granule), or any type of surfaces described herein. Of course, the primer for PCR can be attached to the substrate before starting the PCR reaction. Several PCR cycles can be repeated without washing, but with an excess of bound primer so that the resulting PCR product remains bound to the support. Attaching an amplified target sequence to a substrate can facilitate washing (or cleaning) of the target either before it contacts (for example, hybridization) with a surface including anchors and / or linkers, or after contact with it and then release from such a surface.

In another embodiment, one or more PCR primers used to amplify a target may include one or more restriction sites, allowing the introduction of restriction sites adjacent to either end, or flanking, of the target sequence of interest. Restriction sites can be added to the amplified PCR target either before or after contact (e.g., hybridization) with a surface including anchors and / or linkers. The restriction site (s) thus introduced can, for example, facilitate cloning of the amplified target by providing cloning sites that flank the target sequence. Restriction sites can also facilitate purification of the amplified sequence. For example, one or more restriction sites can be introduced into a primer for PCR between the target specific sequence and chemical modification, which allows you to join the substrate. After amplification of the target by PCR using a modified PCR primer and binding to the substrate by chemical modification, it can be washed and then cleaved at the restriction site (s) adjacent to the target sequence, thereby releasing the washed target. See, for example, FIG. 23.

Of course, other cleavable sites than restriction sites can also be used in the methods described above, for example, a peptide can be cleaved with a specific protease, or another component that can be cleaved and / or released chemically, physically or by other means.

In another embodiment, one or both PCR primers used to amplify a target may include a sequence (which is optionally present in the target) specific to, for example, a target-specific reporter or detection linker.

Of course, the above primer modifications can be used together in any desired combination and can be added to the amplified product at any stage of the test. Examples 21 and 22 present protocols in which several of the primer modifications described above are incorporated into an amplified target.

The methods described above, in which mRNA targets are converted to cDNA by reverse transcriptase and / or PCR amplified before analysis on MARZ plates, can be used instead of the usual MARZ analysis for any of the RNA assays described above.

Nucleic acids used in the methods of the invention, for example, targets, oligonucleotides involved in target detection, or nuclease-protecting fragments (described herein) can be amplified using any of a wide variety of enzymatic methods, including PCR and ligase reactions. One of these methods is transcription-mediated amplification (see, for example, Al6 e1 a1. (1993), I. C11p. Mugo1.31, 3270-3274). See also example 32 and FIG. 36-39 and 42.

In another embodiment of the invention, one or more nucleic acid targets of interest are hybridized to specific polynucleotide protective fragments and subjected to a nuclease protection method, and these protective fragments hybridized to the target of interest (s) are analyzed on MARZ plates. Of course, such MARZ plates may include anchors that are not connected to linkers (for example, which can directly bind to a target of interest or a fragment protecting against nucleases); the benefits of nuclease protection when used in combination with any type of probe array will obviously become apparent to those skilled in the art from the present description and any of its predecessors for which an advantage is claimed. If the target of interest is RNA, and the protective fragment is DNA, then the method of protection against nucleases / MARZ (No. A-MAP§) may reduce the need for intensive processing of RNA, which may be sensitive to degradation by the action of contaminating nucleases, and thus cause difficulty working with her. Processing the sample with a nuclease protection method also makes it possible to obtain a sample with a low viscosity. Protecting the sample from nucleases also improves the sensitivity and reproducibility of the method. See, for example, example 30, which shows

- 21 011415 sensitivity and reproducibility of a typical analysis, in which the sample is processed by the method of protection against nucleases. An advantage of the invention is that the tests can be sufficiently sensitive to eliminate the need for amplification of the target (e.g., PCR) for signal detection. In the ΝΡΑ-ΜΑΡδ method, the probes in the aggregate of probes are oligonucleotides having the same number of chains as the target nucleic acids of interest, to a greater extent than complementary to them, as in the standard ΜΑΡδ test. One example of method ΝΡΑΜΑΡ8 is shown schematically in FIG. nine.

In the ΝΡΑ-ΜΑΡ8 test, the target of interest may be a nucleic acid, for example, genomic DNA, cDNA, viral DNA or RNA, rRNA, tRNA, mRNA, oligonucleotides, nucleic acid fragments, modified nucleic acids, synthetic nucleic acids, and the like. In a preferred embodiment of the invention, the method is used to analyze one or more mRNA targets that are in a tissue or cell extract of RNA. A sample that contains the target (s) of interest is first hybridized to the selected stringent conditions (see discussion above on suitable reaction conditions to achieve specific hybridization) with an excess of one or more specific protective fragment (s). The protective fragment is a polynucleotide, which may be, for example, RNA, DNA (including a PCR product), PNA, or a modified or substituted nucleic acid specific for the region of interest of the target nucleic acid. By a “specific” protective fragment is meant a polynucleotide that is sufficiently complementary to the intended binding partner to bind to it under the selected stringent conditions, but which will not bind to other nucleic acids not intended for this purpose. The protective fragment may be at least 10 nucleotides in length, preferably from 50 to about 100, or may be a full-length cDNA. In a preferred embodiment, the protective fragments are single stranded DNA oligonucleotides. Protective fragments specific for 100 or more targets may be included in a single hybridization reaction. After hybridization, the sample is treated with a mixture of one or more nucleases to destroy other nucleic acids than the protective fragment (s) hybridized to the nucleic acid (s) of interest and (optionally) the target nucleic acid region (s), hybridized and protected from action nucleases during the method of protection against nucleases (located in a duplex hybrid). For example, if the sample is a cell extract, then unwanted nucleic acids such as genomic DNA, tRNA, rRNA and mRNA, i.e. other than those of interest, can mainly be destroyed at this stage. Any of a wide variety of nucleases can be used, including, for example, pancreatic RNase, Golden Bean Nuclease, 81 Nuclease, RNase A, Ribonuclease T1, Exonuclease III, Exonuclease VII, RNase CB, RNase ΡΙινΜ. RNase I2 and the like, depending on the nature of the hybridized complexes and undesired nucleic acids in the sample. RNase H may be particularly useful for cleaving residual RNA bound to a protective DNA fragment. Reaction conditions involving these fragments are well known in the art and can be empirically optimized. In addition, you can use chemical methods, for example, alkaline hydrolysis of RNA. If necessary, then the samples can be processed using methods well known in the art to remove non-hybridized material and / or inactivate, or to remove other enzymes (for example, phenol extraction, precipitation, column filtration, etc.). The hybridization method, after cleavage by nucleases and (optionally) chemical destruction, is called a method of protection against nucleases; many different methods of protection against nucleases have been described (see, for example, Lje c1 a1. (1987), Mc111. Εηζνιηοΐ. 152, 633-648. Ζίηη c1 a1. (1983), Ce11 34, 865-879). Samples treated with protection against nucleases, followed by (optional) inactivation of nucleases, are brought into contact with the ΜΑΡδ-set of probes and carry out the usual stages of ΜΑΡδ-analysis. Bound protective fragments can be detected, for example, by hybridization with labeled target-specific reporters, as described here for standard ΜΑΡδ tests, or protective fragments can be marked on their own, covalently or non-covalently, using a detectable molecule.

If desired, one or more controls may be included to normalize method ΝΡΑΜΑΡ8. For example, one or more protective fragments corresponding to a nucleic acid that is believed to be present in each series of samples in a substantially constant amount (for example, constitutively produced mRNA, a portion of genomic DNA, tRNA or rRNA) can be used.

The ability to detect or quantify an internal control to normalize, for example, genomic DNA in a test to detect nucleic acids that are in varying amounts (e.g., mRNA), is an advantage of using protective fragments in the tests.

Since the amount of standard (s) for normalization may be lower than that of the expressed mRNA of interest, the test can be brought in such a way that the signals corresponding to the expressed genes do not drown out the signal (s) corresponding to the standard (s) of normalization. Ways to bring signals are generally accepted and, obviously, understandable for specialists in this

- 22 011415 area. For example, you can use any of the methods described here to balance the signal strength (for example, attenuating signals, fine tuning) (for example, using blocked linkers; inserting a label into a signal group designed to detect a standardization standard at a higher level than intended for detecting mRNA; locating a locus for detecting a normalization standard, a plurality of linkers specific to different regions of a nucleic acid for normalization, or protective fragments that correspond to different portions of the nucleic acid, etc.). Normalization standard (s) and target nucleic acids (e.g., mRNAs) of interest can be detected simultaneously or sequentially, for example, by any method described herein. In examples 28 and in FIG. Figure 29 shows a typical experience in which internal standards of normalization in the form of DNA are used in mRNA analysis.

In a preferred embodiment, the label is directly introduced into the protective fragment, for example, to a greater extent than when hybridized with a target-specific reporter. For example, a reporter is linked to a protective fragment via a ligand-antiligand interaction, for example, a streptavidin-enzyme complex is added to a biotinylated protective oligonucleotide. In another example, the protective fragment is chemically modified (for example, by direct conjugation with horseradish peroxidase (NCR) or fluorescent dye), and this chemical modification is detected either with or without the nucleic acid site of the protective fragment (for example, after cleavage of the modification, for example, in result of enzymatic or chemical treatment). In any of the above methods, the protective fragment can be labeled before or after hybridization with the corresponding linker molecule.

To control that the method of protection against nucleases is functioning correctly, i.e. that non-hybridized nucleic acids are cleaved, as desired, one or more protective fragments can be constructed to include protruding (non-hybridized) segments that will be cleaved by nucleases, if the method is functioning correctly. The presence or absence of protruding fragments can be determined by hybridization with a complementary, labeled, detecting probe, or protruding portions of the protective fragment can be marked on their own, covalently or non-covalently, using a detectable molecule. This control operation can be performed before the sample is brought into contact with a set of probes, or it will be an integral part of the MLR8 test itself. An example of such a control test is described in Example 15. Of course, since different labels are easily distinguished (for example, fluorescent inks with different absorption spectra), several differently labeled oligonucleotides can be included in one analysis. In addition, the standard nuclease protection test, when analyzed by gel electrophoresis, can be used during development of the assay to confirm that, as expected, the protective fragments are processed.

Those skilled in the art will obviously understand other controls for the proper cleavage of nucleases. For example, you can include in the analysis a nuclease-protective fragment that, as you know, does not have specificity for any nucleic acid in the sample (for example, in the analysis of plant nucleic acids, you can include a protective fragment specific for the animal gene, which, as you know, absent in plants).

After detecting targets, the signal of a detecting probe (e.g., NKR-labeled) can be eliminated (e.g., denature, destroy, quench, suppress, block), the plates are washed to remove any reagents, agents, or buffers that may interfere with in the subsequent step (e.g. denaturing reagent), and then the protruding fragment can be detected by another detecting probe (for example, also NKR-labeled). Signal denaturation followed by the addition of another detecting probe with the same signal group can be used at various stages of the analysis. The use of two different fluorescent probes and double color detection can be used without denaturing or blocking the signal.

As noted above, in one embodiment of the invention, an oligonucleotide probe is used to screen for a nucleic acid that includes one or more polymorphisms. In a preferred embodiment, the nucleic acid (for example, DNA, such as genomic DNA, or RNA, such as mRNA) includes one or more 8ΝΡ. To set up this method, you can use the usual operations generally accepted in this field. For example, for screening DNA that includes the known 8ΝΡ. or mRNA expressed from such DNA, the “специф-specific" protective fragment is hybridized with a sample containing nucleic acids, which may include such 8 ΝΡ. By “8-specific” protective fragment in this context is meant a protective fragment that includes an altered 8ΝΡ base or, if mRNA is analyzed, the reverse complement of this sequence. The sample is then treated with one or more appropriate nucleases that, under suitable, empirically selected conditions, cleave a non-hybridized single-stranded nucleic acid and cleave a double-stranded (duplex) nucleic acid (e.g., DNA-DNA hybrids, DNA-RNA hybrids, and the like) at the base mismatch site (for example, erroneous pairing of one base pair). Suitable nucleases include, on

- 23 011415 example, 81 or RNase N. If the sample contains nucleic acid, including 8ΝΡ, and it is hybridized with an 8№-specific protective fragment, then the protective fragment will remain intact upon digestion, and it can be subjected to MAP8 analysis and detected using a detecting probe or a detecting oligonucleotide that is specific for the sequence of the protective fragment. Nucleic acids not containing 8 содержащие will be cleaved at the base mismatch site between the ΞΝΡ-specific protective fragment and the corresponding wild-type sequence in the nucleic acid. If desired, it is possible to remove a portion of the protective fragment located distal or proximal to the cleavage site (for example, denaturation upon heating, enzymatic cleavage, etc.). The analysis can be constructed in such a way that either the split molecules (or their parts) do not bind to the linkers, or that such split molecules, even if their part binds to the linker, are not detected using an appropriately designed detection probe or a detecting oligonucleotide. In example 29 and in FIG. 30 and 31 show that when setting up the tests according to the invention, it is possible to detect erroneous pairing of one base pair in expressed 8ΝΡ. Example 32 (FIG. 41) shows an 8ΝΡ detection test, which is applicable, for example, to detect 8ΝΡ in genomic DNA.

ΝΡΑ-ΜΑΡ8 tests can be used to quantify a target in a sample. If the protective fragment is added in a sufficiently large molar excess compared to the target for carrying out the hybridization reaction to the end, then the amount of the protective fragment remaining after the stage of protection against nucleases will reflect how much target was in the sample. An example of such a quantitative reaction is shown in examples 12 and 13.

In one embodiment of the invention, various types of targets in a sample, for example, various combinations of DNA, RNA, intracellular proteins, and secreted proteins, can be analyzed using one set of probes. See FIG. 40 and 41 for examples of such tests.

ΝΡΑ-ΜΑΡΞ tests can be used to implement any of the methods described above using conventional ΜΑΡΞ tests.

In a preferred embodiment, polynucleotide protective fragments are determined on a mass spectrophotomer to a greater extent than on ΜΑΡ8 tablets. In a most preferred embodiment, none of the polynucleotides attaches to a solid surface during the hybridization or nuclease cleavage step. After hybridization, the hybridized target can be destroyed, for example, by nucleases or by chemical treatment, leaving the protective fragment in direct proportion to the amount of the fragment that has undergone hybridization with the target. Alternatively, the sample can be processed in such a way as to leave (one chain) a hybridized portion of the target or a duplex formed by the hybridized target and the protective fragment for subsequent analysis. Samples that are analyzed are separated from the rest of the mixture for hybridization and treatment with nucleases (e.g., ethanol precipitation or adsorption, or affinity chromatography, etc.), elute or solubilize, and injected into the high-throughput mass spectrophotometer through the injection loop . In a preferred embodiment, samples that are analyzed (eg, protective fragments) are adsorbed onto the surface and analyzed by laser desorption using methods well known in the art. To obtain high sensitivity, you can use Eoipeg TgaikGogt Makk 8resotogue! Gu (ETM8) (or a similar modern method), which allows to detect each protective fragment at the level of femtomoles or lower.

Protective fragments that should be detected in one (or more) samples can be constructed to produce a unique signal for the mass spectrometer used. In one embodiment, each protective fragment has a unique molecular weight after hybridization and treatment with nucleases, and their molecular weight and the characteristic type of ionization and fragmentation will be sufficient to determine their concentration. To increase the sensitivity or simplify the analysis of complex mixtures, the protective fragments can be modified (for example, derivatized) by chemical groups that give clear unique signals. For example, each protective fragment can be derivatized with various natural or unnatural amino acids linked via an amide bond to the oligonucleotide chain at one or more positions along the hybridized portion of the chain. A fragmentation of amide bonds occurs on a mass spectrometer of appropriate energy, and a characteristic amino acid ratio is released. This approach, in which medium-sized chemical groups (roughly with a molecular weight of 80-200) are used as mass spectrophotometric labels, is desirable because molecules of this size are usually easier to detect. In another example, the chemical modification is an organic molecule with a specific mass spectrophotometric signal, such as a tetraalkylammonium group, which can, for example, derivatize another molecule, such as, for example, an amino acid. In another example, positive or negative ion signals are enhanced by reaction with any of a number of agents. For example, to increase the detection of positive ions, a reaction can be carried out between a pyrilium salt (such as 2-4-dithenyl, 6-ethylpyrilium tetrafluoroborate or many others) with

- 24 011415 amine with the formation of a pyridinium salt; any other enhancing agents can be used to form other positively charged functional groups (see, for example, Ripke e! a1. (1994), Apa1u11sa1 Sjet181tu 66, 1302-1315). In a similar manner, one can react with any of the agents recognized in the art to produce negative ion enhancing compounds. Chemical modification can be detected, of course, either after cleavage from a nucleic acid, or as part of a nucleic acid. Having the ability to identify each protective fragment in a distinguishable way, it becomes possible to analyze (e.g., screen) a large number of different targets (e.g., 2, 6, 10, 16 or more different targets) in one analysis. Many of these tests can be done quickly and easily. Therefore, such a test or a group of tests can be performed with high throughput, as defined here.

Regardless of whether oligonucleotides are detected directly by their mass, or whether unique molecular labels are used, the signals for each molecule that is detected can be fully characterized with pure preparations at a known concentration. This will allow you to accurately measure the signal (determine, quantify). For any molecule that is detected by mass spectrometry, it is impossible to accurately predict the intensity and profile. The tendency of the molecule to ionize, the sensitivity of all chemical bonds within the molecule to fragmentation, the degree to which each fragment is multiply charged or is charged once, all this is too complicated to make any predictions. However, for this fixed-energy instrument and sample processing characteristics, the signal intensity and profile become very reproducible. Therefore, for each probe, a signal with pure standards can be characterized and the experimental signals can be accurately quantified.

In one aspect, the invention relates to a method for detecting one or more nucleic acids of interest, comprising processing a sample containing the nucleic acid (s) of interest, protecting against nucleases with one or more protective fragment, and detecting hybridized duplex molecules, or a protected nucleic acid , or a protective fragment, mass spectrometry.

Nucleic acid analysis methods by mass spectrometry are well known in the art. See, for example, A1reg e! a1. (1998), BS1epse 279, 2044-2045 and Coyet, US patent No. 5605798.

In addition to the diverse high-throughput methods described herein, those skilled in the art will obviously appreciate many others.

The advantage of using tests with multiple probes is the ability to include a number of “control” probes in each set of probes that are exposed to the same reaction conditions as the truly experienced probes. For example, each area in the aggregate may include positive and / or negative controls. In the sense in which this term is used here, “positive control probe” means a control probe, which, as is known, for example, mainly interacts with the target, or interacts with it in a quantitative or qualitatively known manner, thereby functioning as ( internal) standard for probe / target interaction. Such a probe, for example, may be a control of the effectiveness of hybridization. In the sense in which this term is used here, "negative control probe" means a control probe, which, as you know, basically does not interact with the target. Such a probe, for example, may be a control of the specificity of hybridization. As an example of the types of controls that can be used, consider a test in which a plurality of oligonucleotide probes are used to screen agents that modulate the expression of a group of correlated genes for a disease. For internal control of normalization of variable indicators such as the number of lysed cells in each sample, mRNA extraction, or hybridization efficiency, the set of probes may include probes specific to one or more background levels or constitutive auxiliary genes, such as structural genes (e.g., actin, tubulin or others) or DNA-binding proteins (for example, transcriptional regulatory factors or others) for which modulation of their expression by the test agents is not intended. In addition, to determine whether the test agents cause unwanted side effects, such as cell death or toxicity, the set of probes may include probes specific for genes that are known to be induced as part of apoptosis (programmed cell death), or which are induced under conditions of cell damage (e.g., heat shock proteins) or toxic effects on cells (e.g., p450 genes).

In the aggregate, you can include other control probes for "fine tuning" the sensitivity of the analysis. For example, consider a test to identify agents that modulate mRNA production associated with a particular disease state. If in previous analyzes it was indicated that one of the correlative mRNAs (say, mRNA-A) in this group is produced in such large quantities compared to the others that its signal “drowns” those coming from other mRNAs, then the linkers can be summed up for “ fine-tuning "test so as to equalize the intensity of the signals. “Blocked linkers” that include an anchor-specific oligonucleotide sequence designed for an mRNA-A target but lacking a probe

- 25 011415 specific sequence, can be added to dilute the pool of target-specific linkers and thus reduce the sensitivity of the test for this mRNA. Specialists in this field can determine the appropriate ratio of blocked and non-blocked linkers simple, generally accepted in this field methods.

The “fine-tuning” of the test for a specific target by diluting the active element with an inactive element can also be done at other stages of the analysis. For example, it can be carried out at the detection level by diluting a labeled, target-specific reporter with an “inactive" target-specific reporter, for example, one with the same target-specific group (for example, an oligonucleotide sequence), but without a signal group, or with an inactivated or inactive signal form groups. In the sense in which the term “signal group” is used here, it refers to a label, molecule or other compound that emits a detectable signal, or is capable of generating such a signal, for example, a fluorescent molecule, a luminescent enzyme, or any of the various signal groups disclosed herein ) In a particularly preferred embodiment, “fine tuning” can be carried out at the stage of contacting the target-containing complex with the detecting linker (detecting linkers are described below, for example, in the section on complex sandwich-type detection methods, example 23 and Fig. 24). You can construct a group of detecting linkers, for example, to "fine-tune" the sensitivity for the analysis of each individual target. For example, if it is known that a specific target is in a very high concentration in a sample, then the detecting linker for such a target can be diluted with an empirically selected amount of a “blocked detecting linker” including a target-specific group (for example, an oligonucleotide sequence), but in which there is no group specific to the reporter, or comprising a target-specific group and a reporter-specific group that has previously been associated with an inactive reporter. That is, instead of including a group specific to the reporter, this group may be absent, or its interaction (for example, hybridization) with the reporter is prevented (for example, blocked). In some cases, such fine tuning refers to “attenuation” of signals. In FIG. 28 presents an experience in which such signal attenuation is performed.

Samples that are tested in the assays of the invention may include any of the targets described above, or others. The assayed liquid samples may be in any volume suitable for the size of the test region, in the range of about 100 nl to about 100 μl. In a preferred embodiment, approximately 1 μl liquid droplets are added to each well of the 1536-well microtiter plate. Samples can be brought into contact with a plurality of probes in a variety of ways, suitable for analysis with high throughput, for example, using pipettes, dispensers or using replicators. Samples are incubated under conditions (for example, salt concentration, pH, temperature, incubation time, etc. - see above) effective to achieve binding or other stable interaction of the probe and target. These conditions are easily identifiable. After incubation, the samples are optionally processed (for example, washed) to remove an unbound target using conditions that are selected empirically so that specific interactions remain intact, but non-specifically bound substances are removed. For example, samples can be washed about one to ten times or more under the same or more stringent conditions compared to those used to achieve probe / target binding.

Samples containing the target RNA, for example mRNA, rRNA, tRNA, viral RNA, or the total RNA fraction, can be prepared by any various method. For example, культурыη νίίτο cell cultures from which mRNA should be extracted can be introduced into surface areas, such as individual wells of a microtiter plate. Optionally, these cells, after reaching the desired cell density, can be treated with an agent of interest, such as a stimulant or potential therapeutic agent, which can be added to the cells in any of various ways, for example, using a replicator (such as 96- or 386-needle replicators from Wesstap) , using pipettes or dispensers, and incubate with the cells for an appropriate period of time, for example, from about 15 minutes to about 48 hours, depending on the analysis. Extracts of whole fractions of RNA, mRNA, etc. from tissues or cells from the source ίη νίίτο or ίη νίνο can be obtained using conventional methods generally accepted in the art (for example, using industrially available kits).

In one embodiment, the cells are lysed (or made permeable) in the presence or absence of a nuclease-protective fragment (s) and the crude lysate is used directly (for example, in a well of a microtiter plate) without further purification, for example, of other cellular components. If cells are lysed in the absence of nuclease-protective fragments, then such protective fragments may optionally be added to the lysate.

In a preferred embodiment, for example, when nuclease-protective fragments are detected, samples are prepared by contacting cells of interest (e.g., cells on the surface of a microtiter plate well; cells in a tissue or in a sample from an organism, or the like) with an aqueous medium

- 26 011415 (lysis solution), which contains a surfactant or detergent (e.g., 8-8, at a concentration of from about 0.01 to about 0.5% w / v) and an agent (e.g., formamide (e.g. in a concentration of from about 8 to about 60%, vol./about.), guanidine HCl (for example, in a concentration of from about 0.1 to about 6M), guanidine isothiocyanate (for example, in a concentration of from about 0.05 to about 8M) or urea (for example, at a concentration of from about 40 to about 46%, w / v, or about 7M), which alone or in combination with one or more other agents may function as a chaotropic agent. The aqueous medium may be buffered with any standard buffer. In a preferred embodiment, the buffer is about 0.5-6X 88 ° C, more preferably about 3X 88 ° C. Optionally, the aqueous medium may also contain tRNA at an appropriate concentration of about 0.1 -2.0 mg / ml, preferably about 0.5 mg / ml in the aquatic environment, you can also add protection from nucleases fragments before it is added to the cells. The optimal concentration of each protective fragment can be determined empirically using conventional methods. In a preferred embodiment, the concentration of each protective moiety is from about 3 to about 300 pM, more preferably about 30 pM.

Cells are incubated in an aqueous solution until the cells become permeable and / or lysed, and DNA and / or mRNA are released from the cells into the aqueous medium. Cells are incubated in an aqueous medium for an empirically selected period of time (e.g., from about 1 to about 60 minutes), at an empirically selected optimal temperature (e.g., from about 37 to about 115 ° C, preferably from about 90 to about 115 ° C) .

For example, in one embodiment, when both DNA and RNA are released from cells in a denatured form capable of binding to a protective fragment, the cells are incubated for about 1 to about 60 minutes, preferably about 5 to about 20 minutes, in an aqueous medium at a temperature of from about 90 to about 115 ° C, preferably at about 105 ° C. If it is desirable, for example, when it is required to analyze DNA in the absence of RNA, then various commonly used ribonuclease can be included in the incubation mixture. One of skill in the art can easily select the appropriate ribonuclease and optimal cleavage conditions.

In another embodiment, mRNA can be obtained by incubating cells for about 5 to about 20 minutes, preferably about 10 minutes, in an aqueous medium at a temperature of from about 90 to about 100 ° C, preferably about 95 ° C, optionally in the presence of one or more protective fragments. In this case, the mRNA is mainly released from the cells in a denatured form capable of binding to the protective fragment, and the DNA remains mainly inside or attached to the cells, or inaccessible to the probe due to its double-stranded nature, or it is released from the cells, but in the form unable to bind to the protective fragment (e.g., undenatured). Not seeking to bind to any specific mechanism, it turned out that since the nucleic acid is released from lysed / permeable cells, it is enough to denature it to bind to the protective fragment to form a stable duplex that is resistant to the destructive action of endogenous or exogenous reagents or enzymes, and proteins within cells (e.g., nucleases) are denatured and / or inactivated.

After obtaining the nucleic acid of interest by the above method, the sample can be diluted to an appropriate volume so that the aqueous medium does not inhibit the functions of exogenous introduced proteins, such as, for example, nucleases (e.g. 81 nucleases), polymerases (e.g. polymerases necessary for PCR) or binding proteins (e.g., streptavidin). The amounts for dilution, and the identity and amounts of the components for use in the aqueous solution described above, can be determined empirically using conventional methods.

For any of the methods of the present invention, a mark (label) can be entered into the target using any of a wide variety of methods that are well known in the art and / or which are described here (for example, regarding the detection of nuclease-protective fragments). For example, chemical groups that provide a signal for detection, such as chemiluminescent molecules or enzymes that catalyze the production of chemiluminescent molecules, or fluorescent molecules, such as fluorescein or cy5, a time-resolving fluorescence molecule, can be directly or indirectly attached to target molecules such as one of the chelating metals of the lanthanide series, or a radioactive compound. Alternatively, targets can be labeled after they interact with the probe using one or more labeled target-specific reporters (eg, antibodies, oligonucleotides, as shown in FIG. 1, or any of the general types of molecules already discussed above for probes and targets) .

One type of fluorescent molecule may be “non-converting phosphorus”, i.e. A fluorescent label that absorbs and excites at far wavelengths (e.g. IR), then emits at shorter wavelengths (e.g. visible light). Since nonconvertible phosphors emit at shorter wavelengths than most potentially interfering substances typically found in a typical sample under analysis do, the use of nonconvertible phosphors makes it possible to reduce the interference caused by the substance in the sample compared to phosphors that absorb at more short wavelengths. Narrow emission spectrum of most

- 27 011415 non-convertible phosphorus also allows the simultaneous detection of a large number of different non-convertible phosphorus. Non-convertible phosphors are well known and their use is generally accepted in the art, and include, for example, rare earth metal ions such as ytterbium (Ub), erbium (Er), thulium (TT) and praseodymium (Pr), especially in the form of oxysulfide . 80 or more independently detectable non-converting phosphors are described. (See, for example, Vyuodyu1 Adep! Be1syop apy Iepysyayop, Arp1 27-30, 1999, BACRA, Vyuodyu1 HUagGeGeGeppke, BeGeppe Eaepsek OGyse.) Phosphors can optionally be attached to any surface or from a microsphere. Like other fluorescent labels, non-converting phosphors can be detected by the transfer of energy (or modulation) to the label in a fairly close linker, target, or reporter. In addition, like the other signaling groups disclosed herein, non-converting phosphors can be used to quantify the target, and can be used in any of the wide variety of methods described here, for example, to detect nuclease-protective fragments.

Of course, non-converting phosphors can also be used to detect targets that are distributed in any other way on the surface, for example, targets (including nuclease-protective fragments) that are directly attached to the surface, connected directly to a combination of different oligonucleotides on the surface, or connected via bifunctional linkers with anchors (different or basically the same) that are distributed on the surface basically evenly or in any desired organization or order. Any surface, for example a flow system or a solid surface, for example, such as granules, can be used. The granules used in any of the tests of the invention may be of any type, for example, of any material, magnetic and / or non-magnetic; and the granules used in one test may be substantially the same or different in size and / or shape.

Various more sophisticated sandwich type detection methods can be used. For example, a target can be hybridized with a bifunctional molecule containing a first group specific to the target and a second group that can be recognized by a regular (i.e., identical) reporter, for example, a labeled polynucleotide, an antibody, or the like. Bifunctional molecules can be designed so that any desired number of conventional reporters can be used in each assay.

For any of the methods of the invention, a variety of sophisticated sandwich type detection methods can be used to introduce a label into the target. For example, a target can interact, for example, hybridize with a bifunctional (or multifunctional) molecule (a “detecting linker”) containing a first group specific to the target and a second group specific to the “reporter”. The term “specific” has the meanings already defined for the interaction, for example, probes and targets. The term “reporter,” as used herein, refers to a labeled polynucleotide, antibody, or any of both types of molecules already discussed herein in connection with probes and targets. These two groups of detecting linkers can recognize (interact with or bind to) their respective partners for binding in any of the ways discussed above in connection with, for example, probes and targets. The detecting linker may also include other sequences, for example, sequences specific to the target, but differing (not overlapping) from the target-specific group of the corresponding anchor-linked linker complex. Any sequence in a detection linker can serve as a recognition sequence for a detection probe or reporter. In a preferred embodiment, the detecting linker is a polynucleotide.

Detecting linkers can be designed so that any desired number of common reporters can be used in the analysis. For example, a group of detecting linkers can be designed so that each detecting linker will be specific to different targets, but will include a binding site for the same (common), or one of a limited number of reporters. The ability to use a limited number (for example, one) of reporters for tagging different targets in a single analysis provides the advantage of low cost and lower background. Of course, detection linker / reporter combinations can be used to detect targets that are distributed in any way on the surface, for example, as described above for types of target locations that can be detected by non-converting phosphors.

In a most preferred embodiment, detecting linkers can be designed to detect nuclease-protective fragments so that the protective fragments cleaved under the action of the nuclease from the control "protruding" sequences during the method of protection against nucleases (as, for example, described in example 15), mainly were tagged. This type of detection is shown schematically in FIG. 24. In this embodiment, the detecting linker comprises a first group specific for the target and a second group specific for the common control protruding sequence, which in the preferred embodiment is found in substantially all nuclease-protective fragments at the start of the assay. If desired, the control protruding sequence can be cleaved from the nuclease-protective fragment during the reaction

- 28 011415 protection against nucleases, the target-specific group of the detecting linker will hybridize with the cleaved protective fragment, but the control protruding sequence-specific group of the detecting linker will remain unbound and available for subsequent hybridization. On the other hand, if the control protruding sequence-specific sequence is not cleaved from the protective fragment, for example, as a result of incomplete cleavage by nucleases during the reaction of protection against nucleases, then both the target-specific and control protruding sequence-specific group of the detecting linker will hybridize with the protective fragment , and they will be unavailable for subsequent hybridization. In a preferred embodiment, complexes comprising nuclease-protective fragments and linked detecting linkers are then hybridized to a reporter that includes a signal group (e.g., fluorochrome, hapten, enzyme, or any other molecule carrying a detectable signal or signal-generating group, as follows) already described) and a group (for example, an oligonucleotide) that is specific for a control protruding sequence-specific group of a detecting linker. The reporter will preferably bind and label such complexes in which the control protruding sequence of the nuclease-protective fragment is cleaved (i.e., the complex in which the control protruding sequence is a specific detection linker group is available for subsequent hybridization with the reporter).

Specialists in this field, obviously, understand the many other variations of the methods of "sandwich" detection.

The methods by which targets can be incubated with target-specific reporter (s), or target / detection linker complexes with reporters can be incubated under conditions effective to achieve binding or other stable interaction, are readily determined (see above). For example, fluorescent oligonucleotide reporters (at a concentration of about 10 nM to about 1 μM or higher, preferably about 30 nM, in a buffer such as 6X 88PE-T or others) can be incubated with bound targets for about 15 minutes to 2 hours or longer (preferably about 30 to 60 minutes) at a temperature in the range of about 15 to about 45 ° C. (preferably about room temperature). After incubation, the samples can optionally be processed (for example, washed) to remove unbound target specific reporters using conditions that are easily determined empirically, leaving specific interactions intact, but removing non-specifically bound substances. For example, samples can be washed one to ten times or more under the same or somewhat more stringent conditions than those used to achieve target / reporter binding.

Labeling with target specific reporter (s) may provide additional specificity for the initial hybridization reaction, for example, when the target specific oligonucleotide reporter binds to a different portion of the nucleic acid sequence of the target than the oligonucleotide probe, or when the probe and reporter antibodies recognize different antigen epitopes targets. In addition, tagging with target-specific reporters will allow you to "tune" the sensitivity of the reaction. For example, if a target mRNA, which is part of a correlative type of expression, is expressed at a very low concentration, then the signal level can be increased (signal amplification) by hybridization of a coupled target with several (for example, about two or about five or more) target specific oligonucleotide reporters , each of which specifically hybridizes to a different region of the target mRNA.

The ability to independently detect two types of tags allows you to have additional control in MAP8 tests. Some (for example, from about 10 to about 100%) linkers designed for a specific anchor locus (Fig. 7 shows 3 typical anchor loci, each including many basically identical anchors (A, B and C)), may have a label ( for example, a fluorescent label) associated with one end. For example, rhodamine or Cy5 fluor can be linked to the 5'-end of the linker. Such modified linkers are called “control linkers”. After linking the mixture of linkers and control linkers with the anchors and incubating the sample containing the target with the obtained set of probes, the target-specific reporter carrying another fluorescent label (for example, fluorescein or another detecting label such as chemiluminescent) can be used (or the target can be directly mark with a fluorescent label or another detecting label), and determine the ratio of the two signals. The presence of control linkers allows the calibration of the number of functional anchors (for example, capable of interacting with linkers) within and between test areas (i.e., the ability of each locus of the population to bind to a target to normalize signals), serves as a basis for quantifying the bound targets, helps with the location of anchor loci and / or provides positive control, for example, in cases where the signal is absent due to the absence of the target in the sample. In one embodiment of the invention, two different labels (eg, fluorophores) can also be used to detect two different populations of target molecules; however, the ability to recognize the presence of targets by the spatial resolution of signals allows the use of the signal type of label for various target molecules.

- 29 011415

The ability to independently detect labels (for example, using fluorescent labels that emit at different wavelengths, such as, for example, fluorescein and rhodamine, or various non-convertible phosphors), gives the methods of the invention additional flexibility. For example, each of two or more targets can be marked, directly or indirectly, using its own, uniquely detectable tag. This allows you to detect labels based on properties specific to the labels (for example, the color of the emission) in addition (or instead), for example, to identify the position of a target localized on the surface or to identify the target by the size of the granule on which it is located. In another embodiment of the invention, multiple targets can be detected independently at a single locus within a region. For example, two or more targets (for example, 2, 3, 4, 5, 6 or more) can be detected at a locus that is defined by one group of (basically identical) anchors. That is, a group of linkers can be used, each of which has an anchor-specific region specific to the same anchor plus a target-specific region specific to another target. If, for example, a group of four such linkers is used, then all four can communicate with members of the anchor group at one locus, which allows four different targets to communicate at a given locus. If each of these targets is labeled (directly or indirectly) using different, distinguishable labels, then the researcher can independently establish the presence of each of the four targets at the locus. Therefore, a collection of, for example, five anchors (groups of anchors) in a region can be used in the scenario described above to detect, for example, twenty different targets. A similar analysis is presented in Example 24 and in FIG. 25. Similarly, multiple targets, for example 80 or more, can be independently detected when one type of anchor is distributed, but not at one locus, but uniformly, or in any desired order, on a hard surface, such as a granule or flow device , and other aspects, such as the size or distribution of the granules, can be used to provide information about the identity of the targets or groups of targets.

The linking of many linkers (for example, in the range of 2 to about 50 or more) with different specificity with respect to the target, with anchors at a given locus (or a group of basically identical anchors, a “mixed locus”), in some cases, related to "Mixed linkers", forms the basis for other embodiments of the invention, which will obviously be clear to experts in this field. For example, at a given locus, anchors can be associated with a mixture of linkers that are specific for a variety of different protective fragments, each of which corresponds to (or is specific to) a different region of a nucleic acid (e.g., mRNA) of interest. The presence of such a multitude of different linkers at the locus can significantly increase the detection sensitivity of the target of interest (for example, mRNA), for example, in a small amount in the sample. Each locus can be designed so that the number of linkers corresponding to different regions of the mRNA corresponding to a given locus is inversely proportional (empirically determined) to the content of this mRNA in the sample. For example, if in a preliminary experiment it was found that one mRNA of interest is in a large excess in the sample compared to the second mRNA of interest, then the relative number of linkers corresponding to different sites of two mRNAs can be summed so that the relative intensity of the signals corresponding to each mRNA will basically be the same. That is, the signal intensity can be adjusted in such a way that the signal corresponding to the first mRNA will not overlap the signal corresponding to the second mRNA. Thus, the analysis is carried out in such a way that it is possible to simultaneously detect multiple mRNAs that are in the sample in very different quantities, balancing the intensity of the signals corresponding to each mRNA.

As noted above, in another embodiment of the invention, this locus may include linkers that are specific for a variety of unrelated or different targets, or protective fragments, which allows to detect a significantly larger number of targets or protective fragments using one set of anchors. For example, if each locus of a population of 350 anchors includes linkers specific to 10 different targets, then the population can be used to detect 3,500 targets. In practice, such an arrangement will make it possible to turn an aggregate with the help of which it is possible to determine a low density of targets into one that allows one to determine a high density of targets.

Many molecules (eg, protective fragments) linked at one locus can be detected sequentially or simultaneously, for example, using the detection methods described in this application. (See, for example, the discussion regarding “detecting linkers” and “reporters” in the section above for complex methods for detecting a “sandwich” type.) In one embodiment, a first target (eg, a protective fragment) is detected at a given locus, for example, using the first detection system (for example, a detecting linker / reporter or a detection probe specific to it); then the first detecting linker / reporter or probe is removed or inactivated using conventional methods (for example, by changing the pH to inactivate the reporter including the chemiluminescent signal generating enzyme), and a second detecting linker / reporter or detecting probe specific to the second target is used in the same loku

- 30 011415 ce, for detecting the second target; and so on, spend many cycles, if desired. In another embodiment, a first detecting linker / reporter or detecting probe is added to the combination described above, but is not removed or inactivated until a second detecting linker / reporter or detecting probe is added. In this embodiment, the number of signals corresponding to the second target can be determined by subtracting the number of signals corresponding to the first target. In another embodiment, the first and second detecting linkers / reporters or detecting probes are added to the above combination mainly simultaneously and individually detect, for example, using various detectable tags as described above. In any detection method described herein, detecting linkers may include groups specific to the same or different reporters. For example, if four targets are linked to linkers at a given locus, then each of the detecting linkers specific to each of the four targets may include a group specific to another reporter. Therefore, after hybridization, groups of all four detecting linkers with targets can be detected sequentially or simultaneously, as described above, using four different reporters. Specialists in this field, obviously, understand other methods of detection, as well as combinations of the above methods.

Of course, “mixed linkers” are also advantageous for use on surfaces containing one (non-repeating) region.

In another embodiment of the invention, the “anchors” specific to the target (s) of interest do not bind to the linkers, but more directly bind to the target (s); in turn, the target (s) may optionally interact with a detecting linker (s) or a detecting probe (s).

Targets, with or without a tag, can be detected by any of a variety of methods that are common and generally accepted in the art (see, for example, Eobog s1 a1. (1996), US patent No. 5510270; Ριιτιιιιμμ! A1. (1992), U.S. Pat. U.S. Patent No. 5,670,322; Μρδίιιιΐΐζ e1 a1. (1995), VyuTes11pk | ie5 19, 442-447; ZoyShegp (1996), Tgpbk ίη 6пеНс5 12, 110-115). Detection methods include enzyme detection, colorimetric methods, 8ΡΑ, autoradiography, mass spectrometry, electrical methods, absorption or luminescence detection (including chemiluminescence or electroluminescence) and light scattering detection, for example, from microscopic particles used as labels. In addition, it is possible to detect fluorescent labels, for example, when acquiring an image with a charge-coupled device (SSE) or using a fluorescence microscope (for example, a scanning or confocal fluorescence microscope) or by combining a scanning system with an SSC matrix or a photomultiplier, or using technology detection based on the aggregate (for example, you can detect the surface potential, each of the 10-micron part of the test area, or you can use surface plasma resonance, If you can get a high enough resolution). Alternatively, the population may contain a tag (for example, one of a pair of energy-transmitting probes such as fluorescein and rhodamine) that can be detected by the transfer of energy (or modulation by) to the tag on the linker, target, or reporter. Among the fluorescence-based detection systems are fluorescence intensity, fluorescence polarization (ΕΡ), time-resolving fluorescence, fluorescence resonance energy transfer, and time-homogeneous fluorescence (NTFE). An analysis of repeating strip-like patterns can be carried out according to the type of recognition (finding the corresponding spot or line for each specifically labeled target by its position relative to other spots or lines), followed by a quantitative determination of the intensity of the labels. Created and / or are commercially available recognition devices and software for the analysis of one- or two-dimensional populations (for example, see Wowa e1 a1. (1996), US patent No. 5545531).

Another method that can be used for detection is two-photon fluorescence, including options where the fluorescence of endogenous or conjugated fluorochromes of components associated with the surface of the population is enhanced by close binding to the surface of the population, for example, proximity to the substrate on which the population is formed, or close to other agents included in the anchor or linker, or included in the associated complex. Other fluorescence methods or applications include time fluorescence, polarization, energy transfer, etc. For example, using similar methods, it is possible to simultaneously detect or recognize multiple targets within a single locus, and in some cases it is possible to distinguish between a linked tag and an unbound tag, without the need to wash the unbound tag from the population, and this makes it easy to determine quickly reversible or weak interactions totality.

Methods for preparing or using the assemblies of the invention, including preparing surfaces or areas such as described herein, synthesizing or cleaning and attaching, or attaching compounds such as anchors, linkers, probes and detecting probes described herein and children

- 31 011415 testing and analysis of labeled or labeled compounds described herein are well-known and generally accepted technologies. In addition to the methods disclosed in the sources cited above, see, for example, patents belonging to LGHuta. AGGUTERTH, Yapodep, Rgo! Odepe, 8res! Gadep, MgShroge and Vesktap (whose products suitable for carrying out the invention are commercially available); the usual guides to molecular biology and protein science, including those cited here, and SoHen e! a1. (1991), US patent No. 5063081; 8th! Catp (1996), Siggep! Orshop ίη B1o! Eskpo1o§u 7, 85-88; More e! a1. (1996), 8c1epse 274, 610-614 and Eobog e! a1. (1993), Ya! Ige 364, 555-556.

Brief Description of the Figures

In FIG. 1 shows a design scheme for oligonucleotides in which linker 1 includes a region specific for anchor 1 and another region (probe) specific for target mRNA 1, and in which labeled detection probe 1 is specific for a target mRNA sequence that is different from sequences of a target-specific linker site.

In FIG. 2 shows a surface containing 15 test regions, each of which includes a combination of six anchor oligonucleotides.

In FIG. Figure 3 shows a linker scheme for a receptor binding assay in which the anchor-specific linker site is linked to the probe site (receptor protein) via biotin and streptavidin molecules, and in which the receptor-specific ligand is labeled with a fluorescent label. B: biotin. 8A: streptavidin. Weight: receptor protein. Ligand: natural or synthetic ligand for the receptor. *: fluorescent label associated with the ligand.

In FIG. 4 presents a surface that includes 21 test areas, each of which is additionally divided into 16 subregions (depressions, indentations).

In FIG. 5a, 5b, and 5c show three parts from which a surface such as that shown in FIG. 4. In FIG. 5a shows a well separator; in FIG. 5b is a subdivision, and FIG. 5c shows the base.

In FIG. 6 shows two test areas, each of which includes a linear set of probes (or anchors), which are located in a “strip-like” order.

In FIG. 7 schematically shows the test area, including 3 anchors (A, B and C), each of which is in multiple copies (“group”). The position of each group of anchors is called a “locus”.

In FIG. Figure 8 shows a test in which cDNA molecule (s) obtained using specific reverse transcriptase are analyzed on MAP8 plates.

In FIG. 9 shows a test that uses a method of protection against nucleases (No. A-MLP8 test). An RNA sample is obtained from cells or tissue, and it is presented as thin wavy lines. A group of polynucleotide protective fragments, presented in the form of bold, dark, and light lines, is added to the RNA sample. The dark portions of the protective fragments represent segments that are complementary to specific RNA targets, and they hybridize with these targets. Bright areas represent protruding areas: sequences adjacent to a complementary sequence but not complementary targets. Protective fragments are added in excess. After hybridization of all available targets with protective fragments, the samples are treated with an appropriate mixture of nucleases and chemical treatment is carried out, as a result of which the unwanted non-hybridized RNA and non-hybridized polynucleotide are destroyed. For example, 81-nuclease can cleave any single stranded DNA present. As a result, the excess of protective fragments is hydrolyzed as a protruding non-hybridized portion of the bound protective fragment. RNA can be hydrolyzed by the addition of ribonuclease, including ribonuclease H, or by heating samples in the base. The rest is a mixture of cleaved protective fragments, which reflects the amount of each target RNA in the sample. The remaining protective fragments are determined by the MAP8 hybridization test.

In FIG. 10 shows the specificity of hybridization in the MAP8 test.

In FIG. 11 shows the kinetics of anchor-linker binding.

In FIG. 12 shows the MAP8 test of two oligonucleotide targets.

In FIG. 13 provides a quantitative determination of the sensitivity shift.

In FIG. Figure 14 shows the determination of the melting point of four oligonucleotide linker / anchor combinations.

In FIG. 15 presents No. A-MAP8 test with mRNA.

In FIG. 16 shows a dilution curve with XPA-MAP8.

In FIG. 17 shows a test for detecting peptides containing phosphotyrosine residues.

In FIG. Figure 18 shows the first stage of the test for mapping Ε8Τ: assembling linkers corresponding to each of Ε8Τ, which are mapped onto sets of close anchors on an MAP8 tablet. Linkers comprising 16 different oligonucleotide probes located on a 4x4 matrix are attached to the surface of each of the 16 wells of the microplate. At the first locus, there is oligonucleotide 1, which is complementary to the site of the first sequence of Ε8Τ, and so on for 16 Ε8Τ, which are analyzed.

- 32 011415 cDNA or mRNA obtained from the genes from which E8T was obtained, contribute to all 16 wells and allow hybridization under appropriate conditions. As a result, any of the cDNA or mRNA that contains one of the 16 E8T sequences will bind at the locus where the probe complementary to it is located.

In FIG. Figure 19 shows the next step of the E8T mapping test: addition of detector oligonucleotides on a MAP8 plate. A detection oligonucleotide, which corresponds to one of the E8Ts that are mapped, is introduced into each well of the plate. Each detecting oligonucleotide represents an oligonucleotide associated with a molecule used to detect, for example, fluorescein, if the detection method is to obtain an image of one of the E8T using fluorescence. Each detecting oligonucleotide is complementary to one of the E8T, but differs from the E8T-specific probe so that the probe and the detecting oligonucleotide that are complementary to one E8T can both communicate simultaneously.

After washing, one detecting oligonucleotide is added to each well, as numbered in the figure. That is, a detecting oligonucleotide with sequences complementary to the first E8T is introduced into the first well, and so on.

In FIG. 20a, b show the results of the E8T mapping test of FIG. 18 and 19. After hybridization of the detecting oligonucleotides and washing under conditions of appropriate stiffness, 16 wells of the microplate are visualized using a fluorescence imaging device based on SSE. In FIG. 20a shows stylized results. It is believed that each E8T-specific detecting oligonucleotide will label mRNA or cDNA detected by an E8T-specific probe. For example, probe 5 binds to cDNA or mRNA containing the fifth E8T sequence at this locus, so the fifth detecting oligonucleotide will also hybridize with cDNA or mRNA at the same locus. This is reflected in the stylized data for each detection probe. In addition, each of the first three detecting oligonucleotides marks cDNA or mRNA held by the first three probes, thus it becomes obvious that these sequences are in the same gene. Similarly, the last five E8Ts were connected. The relationship obtained from this data is graphically represented in FIG. 20b.

In FIG. 21 shows the relationship of the probes detecting oligonucleotides and E8T # 1, 2, and 6 shown in FIG. 18-20.

In FIG. 22 shows high throughput analysis.

In FIG. 23 shows a method for preparing an amplified target.

In FIG. 24 shows a test with detecting linkers and reporters.

In FIG. 25 presents the use of multiple fluorescent labels.

In FIG. 26 shows a high throughput analysis.

In FIG. 27 shows the spatial arrangement of THP-1 cell genes along with two cell samples (taken from FIG. 26).

In FIG. Figure 28 shows a signal attenuation test.

In FIG. 29 shows a test in which genomic DNA and expressed RNA are determined in the same sample in the same well, for example, in which genomic DNA serves as a control for normalization. The left photograph shows the definition of one DNA; the right photograph shows the definition of both DNA and RNA of SLREN (defined in each corner of the population).

In FIG. 30 and 31 show the detection of expressed 8YP.

In FIG. 32-35 presents the sensitivity and reproducibility of the test.

In FIG. 36 shows some types of test configurations that are within the scope of the invention.

In FIG. 37 shows amplification of a nuclease-protective fragment by PCR.

In FIG. 38 shows the amplification of a nuclease-protective fragment using ligase.

In FIG. 39 shows the amplification of a nuclease-protective fragment using nuclease-defense.

In FIG. 40 and 41 present tests in which a protein and mRNA are simultaneously analyzed in one sample.

In FIG. 42 shows the amplification of a nuclease-protective fragment by polymerase. An application for detecting 8YP is presented.

Examples

Example 1. The specificity of hybridization (see Fig. 10)

A total MAP8 plate was prepared using an injection pipette-dispenser of the P1XI8 system (Sayyap Tespo1od1e8, 1 ps, 1 gouche, CA) to obtain the same DNA network inside each well of a microtiter plate. All oligonucleotides were produced by Vukoigse 1n! Egpa! Yupa (SatagWo, CA). On each plate, seven different oligonucleotide anchors were introduced into each well in the order shown in the form of a key (in the figure on the left). Each oligonucleotide was dispensed in the form of a droplet with a volume of 10 nl into two spots from a 2 μM solution containing 500 mM phosphate buffer with pH 8.5 and 1 mM EDTA in the wells of the DNA binding plate (Sogspd Soiag) and allowed to

- 33 011415 to whip. After attachment, the wells were blocked with 50 mM Tris buffer with pH 8, and then the oligonucleotide that did not covalently bind to the surface was washed with 0.1% 8–8 in 5x 88P buffer.

Linker oligonucleotides labeled with a fluorescent label were added to the washed plates and hybridized in 6X 88PE buffer with 0.1% Triton X-100 at room temperature for 30 minutes. This is the preferred linker attachment protocol. Linker oligonucleotides during the synthesis of Cy5-derivatized, and they were complementary in segments of 25 base pairs to specific anchor oligonucleotides. The sequences of the seven anchors and linkers were as follows (all in the direction of 5'-3 ').

# 1 Anchor *: ZEO ΙΌ: 1

TSSASSTSSASSASS0SASS6S0TSS

Linker ** 8Ef GO: 2 yyyyyyyyyyyyyyyyyyyyyyyy partyyy of the Sakha region

RNA (mimetic (mouse C-; ip); £> E <2 10: 3.

СГАТ <1АССК1АААС16ТСК1АААСОАСОАТАСТ6АОТТООАСТАААТАТССАТСТСА.ТТСА Detecting oligonucleotide * ”8EfU: 4

TOAATOAOATSOAATATTAaaTSSA # 2 Anchor * _: Zefgo: 5

SASTASOOSTOASOSSTSTSOSTOS

Linker ** 8ES) U: 6

STAaOSTOASTASTOOSGSSASASTHLHZSAOOSSSAOOTASTSSASSSaTabto

RNA mimetic (mouse ΜΙΡ-2): 8ΕΟ ©: 7

AODSTSSAStSSASA £ PTSAS <XTAS1SATASTSASL'STOAASAAAOOAAA6SSTAASTOAS

Detecting Oligonucleotide 5EfGO: 8

OTSAOOGGAOOSSTTSSSTPPTGSAO # 3 Anchor *: 8Е (2П): 9

OTSAETTAISSTGSSSTTTbTTSAO

Linker 5ES) Yu: 1O ASKHZATSTASTTOASSTSAAOAAISSOSGSSSASAASOSTSOASSOOSS

RNA mimetic (mouse CA ΡΟΗ): ΙΟ: 11

SSTGSATTSASSTSAASGASATOTTOATASTOABTAAASAAASSTOSSAAOOTATSATyAS

Detecting oligonucleotide ZEC GO: 12

OTSATSATASGGSYUSASSSPTSTSS # 4 Anchor ’: 8Εζ) 10:13 SAASSSSSTSOSSTGetTSGASASSSA

Linker ”8E <510: 14

STASSOASSAAASGSSAAATOAATGbSSGOTAOAASASOASASbbTTS

RNA mimetic (mouse protein 132): 8E (210: 15

ATTTSAT1TSSA <ZTTTOSTSS <ZTASSATAST <ZaAGSga <lTSASSAAT (X5CAASaSSa. <ESSG

Detecting oligonucleotide 8BC> 10:16

AtkhlchsathzhzhttostoASGs # 5 Anchor 8ES> 10:17 STSOT'SSSSSTSSSTSSSTSSSSa

Linker ** 5EC 10:18 STPSSSAOOSSAS6SZASASOSSOAASAO # 6 Anchor *: 8Е <r 10:19 SSKPSOSSATOOTASSASAOOTSSOS

Linker ”8E <210: 20 sazAstototAssAtahoAsse # 7 Anchor *: 5Ep>: 21 oaessossaat’AtosAtsgsgsgsgo Linker” 5E <210: 22

SOAOAOATSATAASOSOOSSZSSa • Anchors were synthesized with a C12-spayer in an amide bond at the b'-end “Linkers were synthesized with a Sub linked to the b'-end ••• Detecting oligonucleotides were synthesized with biotin bound to the b'-end

Either one linker or a mixture of linkers (as indicated in the figure) was volumetricly introduced into each well. (A mixture of all seven linkers was added to the well marked “all”.) After incubation and washing with 5x 88P buffer 3 times, the fluorescence image shown on the right side of the figure was obtained with

- 34 011415 using the Tipbga visualization device (ΣΚΙ, δΐ. SaSheppek, OgIapo). As you can see, the linkers themselves attached to the surface through specific binding to their complementary anchors.

This procedure is repeated except that eight different anchors are introduced into each well, and then linkers, preferably binding to them. Repeat the complete process with 36, 64, etc. with different anchors in each well of a 24-, 96-, 384-, 864- or 1536-well plate.

Example 2. The kinetics of binding (see Fig. 11)

The hybridization rate of the number 5 Su5-derivatized linker molecule with a complementary anchor associated with it for various linker concentrations is presented. A general MAP8 tablet was prepared as for FIG. 1, except that anchor 1 was connected at four spots per well. Incubation was carried out at room temperature in 5x88P buffer with 0.1% Tween-20, the wells were washed 3 times with 5x88P and the bound fluorescence label was determined. The fluorescence pattern on the plate was obtained using a Tipbg imaging instrument, the background was subtracted, and the total intensity of each spot inside each well was calculated using Tipbg software. The mean value and standard deviation for the total intensity values for four spots inside each of two parallel wells were calculated.

Example 3. Fluorescent Linker

A common MAP8 plate is prepared with one anchor oligonucleotide applied either to 1 spot per well (upper two rows), 4 spots per well (next four rows) or 16 spots per well (lower two rows) using the methods already discussed above. A complementary fluorescently labeled linker was attached to each well using the preferred protocol described in Example 1. After washing, the fluorescence image of the plate was reproduced using a Tipbg apparatus. The amount of functional linker available for hybridization with the target was determined by the number of fluorescent labels in each spot. The amount of signal detected on repeated spots was highly reproducible.

Example 4. Binding Curves

In the tablet obtained as described in example 3, various concentrations of the target oligonucleotide were added. The linker that joined contained a 25-dimensional sequence complementary to the target site. The target was added in 5x 88P buffer with 0.05% δΌδ in a total volume of 30 or 100 μl, the plate was coated and incubated at 50 ° С overnight. After hybridization of the target with the attached linker, the target was visualized using the preferred protocol using chemiluminescence. At a concentration of 30 nM, a biotinylated detecting oligonucleotide containing a 25-dimensional sequence complementary to a single site of the target (but not to the same site complementary to the linker) was added. A biotinylated detecting oligonucleotide can be added 30 minutes after washing off the excess of an unbound target, or it can be added together with the target for complete hybridization overnight. After the detection oligonucleotide was attached, the surface was washed twice with 5x8PP buffer, once with 1x8PP buffer containing 0.1% Tween-20 and 1% PEG (88PTP) and 250 μg / ml horseradish peroxidase was added at a dilution of 1: 50,000 conjugated with streptavidin (NKR: 8A from P1egse, Yaoskogogy, 111.) for 5 hours at room temperature. The wells were washed four times with 88PTP and washed once and then incubated with the reagent §preg §1dpa1 I11ga (P1egse). After a few minutes, a luminescent image was obtained using an SSI matrix using a Tipbg imaging device for 5 minutes. The target can be visualized in the same wells in such a small concentration as ~ 5x10 ^-13 M; as a rule, the amount of signal becomes saturated at a target concentration of ~ 10 ^-10 M. The amount of signal detected in repeating spots is highly reproducible.

Example 5. Analysis of two oligonucleotides (see Fig. 12)

A binding curve is shown showing the MAP8 hybridization test using the preferred protocol described above for two different target oligonucleotides. A general MAP8 plate was prepared with four different anchor oligonucleotides, each distributed four times in each well. For the second and fourth anchors, complementary linker oligonucleotides attached to the surface as described. Two targets were added at the indicated concentrations in a volume of 40 μl to each well, as described, and incubated at 50 ° C. overnight. The amount of each linked target was visualized by attaching a biotinylated detecting oligonucleotide specific to each target, followed by NKR: 8A, and a chemiluminescent image was obtained as described. At the bottom of the figure is a quantitative determination of the intensity of the image. We scanned the intensity of the images along the lines between the arrows, as shown in the upper photograph, using software that is part of the Tipbg system. At the lowest target concentration, 1.1 pcM, Gaussian peaks in each spot are clearly visible in the scanned images, while in the leftmost sample at the 0-th target concentration, there are no distinguishable background peaks.

- 35 011415

Example 6. The shift of sensitivity (see Fig. 13)

The MARZ hybridization test can also be used to determine the concentration of a group of oligonucleotides when they bind to the surface and label them. These methods are suitable for oligonucleotides that are in medium or low concentration. In this case, two samples can be distinguished, because the more oligonucleotide is contained in one sample, the more it will bind. On the other hand, if the concentration of the target oligonucleotide is saturated for the surface (i.e., if it is high enough to occupy all binding sites), then with an increase in the concentration of binding, an increase in binding cannot occur and, therefore, it is impossible to determine the amount . However, the target binding curve can be shifted by the addition of an unlabeled competitive ligand.

Binding curves were obtained for four different oligonucleotide targets, which all led to surface saturation (i.e., reached maximum binding) roughly at 3 nM. By adding unlabeled competitive targets to all wells, the binding of the labeled oligonucleotide was shifted in order to achieve binding at a lower concentration and at which the saturation level is shifted. You can add competitive oligonucleotides, for example, for targets 1 and 3, but not 2 and 4. This leads to a shift in the sensitivity of the test only for targets 1 and 3. Thus, within one test well, you can define many oligonucleotide targets with different concentrations if the relative amount of oligonucleotide is allegedly known.

As already explained above, data can be processed quantitatively to bind one of the oligonucleotide targets. In FIG. 13 quantitatively shows that the inclusion of a competitive oligonucleotide in the test shifts the binding curve used to analyze this target at higher concentrations.

Example 7. The melting temperature of the four probes (see Fig. 14)

We plotted the concentration of four different fluorescently labeled linker oligonucleotides that specifically hybridize with anchor oligonucleotides in the MARZ test versus temperature increase. Initially, four oligonucleotides were hybridized at 50 ° C for 1 h at a concentration of 300 nM. Then the wells were washed with GLC without probes and the bound amount was determined as described above by fluorescence (point 50 ° C). Then, the surface was incubated at 55 ° C for 30 min, and the associated fluorescent label was determined, and so on, at all temperature values shown.

Example 8. Methods of detection

Two detection methods can be directly compared with each other. To the MARZ plate with four attached oligonucleotide anchors, each at four spots per well, two oligonucleotides were added to each well, both included a covalently bound Cy5 group or both contained biotin. Epi-fluorescence was determined as described in relation to visualization and determination of the fluorescence linker. The determination of chemiluminescence was performed as described for the MARZ test, followed by the addition of NKR: 3A and a chemiluminescent substrate. The generated signals were approximately the same value. However, due to the geometric properties of microplates that have walls separating each well, and random liquid bubbles or liquid miniscus, the resulting reflections in epi-fluorescence images may interfere with the interpretation of the data.

Example 9. Chemiluminescence Products

Two commercially available products can be compared as chemiluminescent substrates for horseradish peroxidase for detection in the MARZ test. MARZ plates were prepared as in Example 8 and incubated with biotinylated linker oligonucleotides. Then, either alkaline phosphatase conjugated with streptavidin (L1Cryo8: ZL) or NKR: ZA was added, followed by washing and SEP-Z1ag ((τορίχ) into wells with A1kP1y5: ZA or ECb-P1i8 into wells with NKP: Labeling with ZA-derivatized enzymes and substrates was carried out according to the manufacturer's instructions regarding labeling for Western blotting. These two substrates (as well as other commercially available substrates) can be used to evaluate the hybridization of oligonucleotides with MARZ plates.

Example 10. Resolution at 0.6 mm

The resolution of this system for the MARZ test was evaluated when preparing MARZ tablets with four different oligonucleotide anchors for each well, each four times added to the well, with a module (distance from center to center) of 0.6 mm. Then either su5 derivatized linkers or biotinylated linkers were hybridized, detection and scanning were performed as described above. To determine the epi-fluorescence, the resolution was higher (and the module can probably be reduced). When detecting chemiluminescence, adjacent spots were not completely separated, but still, even at this distance, individual peaks could be resolved using a computer.

Example 11. Protocol test protection against nucleases

In the test for the analysis of optimal conditions for hybridization and treatment with nucleases in the protocol for

- 36 011415 nuclease shields used a nuclease protection test kit manufactured by Αιηόίοη (ΛυδΙιη, Texac) to provide conditions, buffers and enzymes. Eight samples were prepared in one of the three buffers. Nu Vie £$ 1 represents a 100% hybridization buffer (ΑιηΝοη); NuBi £$ 2 represents 75% of the hybridization buffer and 25% of the dilution buffer for hybridization (Bt ^ oi), and NuBi £ 3 contains 50% each. A 70-dimensional oligonucleotide was synthesized, containing 60 residues complementary to the mRNA tested (B1O8Oigse Shtayoyaf SatagSho, CΑ) and labeled with Psoralen-fluorescein (Sсееесіёге and 8сійе11, Кссис, ΝΗ) followed by the conclusion of the protocol proposed for. Briefly, the protective fragments were diluted to a concentration of 50 μg / ml in 20 μl of TE buffer (10 mM Tris buffer, 1 mM EDTA, pH 8), boiled for 10 min and quickly cooled on ice. Added 4 μl of a solution of Psoralen-fluorescein in DMF with a concentration of 130 μg / ml and the sample was illuminated for 45 min at 40 ° C with a manual source of long-wave ultraviolet waves. Free psoralen-fluorescein was removed by extraction with saturated butanol. The mRNA used was the antisense mΡΗΚ-CΛΡ ^ Η obtained from the antisense plasmid (ρΤΡΙ-ΟΑΡΌΗ mouse antisense control matrix from ЬΑЬ ^οο)) using the T7 promoter and the Max1-8sp1 set (ΑΑΑ ^οοи). The short protective fragment represented a 60-dimensional complementary region, synthesized separately and labeled in a similar manner. The sequences of the protective fragments are as follows:

Full-size protective fragment; 5Εζ} U: 23

SSAOAAATATOL <> ASTSASTAAAOATTOTSASSAATOSAGSST <ZSASSASSAASTOSTTySGGOT STEEL Short protective fragment: 5ES> P>: 24

SSAOAAATATOASAASTSASTSAAOATGOTSAOSAATOSATSSTOSASSASSAASTOS'GT

Hybridization is carried out by mixing protective fragments at a concentration of 20 nM and mRNA-CΛΡ ^ Η at a concentration of 60 nM in 10 μl of the final volume for two hours at 22 and 37 ° C. After hybridization, 200 μl of the nuclease mixture was added according to the manufacturer's instructions (a nucleotide protection kit manufactured by Ltb, nuclease mixture diluted 1: 200) and re-incubated at the same temperature for 30 minutes. The hydrolysis is stopped with a hybridization inhibition buffer (^ 11 ^ 1011), the oligonucleotides are precipitated by centrifugation and washed with ethanol. Add 10 μl of 1x gel sample buffer (ΑιηΝοη) and the oligonucleotides are separated in 15% TBE-urea gel. The gel is washed in a buffer for 30 minutes, placed on a plastic plate and visualized using TiPh using fluorescein filters at selected excitation and emission wavelengths. The image is accumulated on a set of SSI for 2 minutes. The best results are obtained when the samples are incubated in NaBiBi £$ 2 at 37 ° C or in LiuBi £ £ 3 at 22 ° C. No detectable full-sized protective fragments were present in these samples, and significant amounts of a portion of the full-sized protective fragment of about the same size as short protective fragments could be detected.

Example 12. Test determination of mRNA with ΝΡΑ-ΜΑΡδ (see Fig. 15)

The complete ΝΡΑ-ΜΑΡδ protocol was used with hybridization and nuclease treatment conditions similar to those described in Example 11. Ten samples were examined in the test. All samples contained the same amount of 70-dimensional oligonucleotide protective fragments and different amounts of mRNA-CΛΡ ^ Η. Hybridization samples in an amount of 10 μl in a mixture of 50% hybridization buffer and 50% dilution buffer containing 0.08 mg / ml yeast RNA (ΑιΜοη) were heated at 90 ° C for 6 min, quickly centrifuged, heated at 70 ° C for 5 min, allowed to cool to 19 ° C and incubated for 19 hours. Then, 20 μl of the nuclease mixture was added to each sample for 30 min at 19 ° C. 60 μl were taken from each sample for the ΜΑΡδ test. 2 μl of 10N No. 1OH and 2 μl of 0.5 M EDTA were added and the sample was heated at 90 ° C for 15 minutes, at 37 ° C for 15 minutes and allowed to cool to room temperature over 20 minutes. Then the samples were neutralized with 2 μl of 10 M HCl and 12 μl of 20x δδ ^ buffer containing 2 M No. of δ with pH 7.5 and 200 nM biotinylated detecting oligonucleotides specific for the protective fragment, as well as 1 μl of 10% δΌδ were added. The samples were mixed, heated at 80 ° C for 5 min, and two aliquots of each sample with a volume of 35 μl were pipetted into two wells of a ΜΑΡδ-tablet (each sample was divided into two and parallel analysis was performed on ΜΑΡδ-tablets). The tablet was prepared according to the usual protocol for ΜΑΡδ with the addition of a Su5-derivatized linker specific to the already attached protective fragment. ΜΑΡδ plates were coated and incubated at 50 ° C. overnight, detection and luminescent analysis were performed as described. Nucleases were not added to the last sample during the test and were visualized as a control to evaluate how one protective fragment would be detected in the ΜΑΡδ test. At the bottom of the figure, an intensity scan is presented (analysis using a visualization tool) for the top row of holes. The amount of mRNA-CΛΡ ^ Η present in the sample (i.e., the amount in each parallel well after applying an aliquot to the ΜΑΡδ plate) is shown in the figure.

The oligonucleotides used for the ΜΛΡδ tablets were as follows:

- 37 011415

Anchor *: ZEF ΙΟ: 25

S6sss5ts5asssttstsosazssss

Linker ** ZEY 10:26

STTYAYTBASTTSTSATATTTSTSSSSATASTSASSSSSSTSSASAASSSTSeASS boss

Protective fragment (complementary antisense mRNA for

SCROY)

ZE (2 U: 27 SOaAAAATATSASAASTSASSTSaAOATTSTSASSAATEATSATSTOSTASSASSASSAASTOSTTSST TSTSTAA

Detecting oligonucleotide *** - labeled with biotin at the 5'-end £ Ες GO: 28

AASSASTTSSTSSTSSAOOYATSSAT * Anchors were synthesized with a C12 spacer at the amide link at the 5'-end ** Linkers were synthesized with Cy5 linked to the 5'-end *** Detecting oligonucleotides were synthesized with biotin linked to the 5'-end

Example 13. The dilution curve, ΝΡΑ-ΜΑΡ8 (see Fig. 16)

The data discussed in Example 12 and shown in FIG. 15, were quantified and plotted as a dilution curve. The mean and standard deviation were calculated for all eight spots in two parallel wells for each mRNA concentration. The binding curve is presented as:

Bound fraction = maximally bound * 1 / (1 + 1C ₅₀ / b), where maximally bound represents maximum binding upon saturation, the bound fraction represents the bound amount at ligand concentration, b, and 1C ₅₀ is the ligand concentration at which the bound fraction is half from the maximum related. The curve is represented as dots and dashes in the figure with a value of 1C50 = 4.2.

Example 14. Test ΝΡΑ-ΜΑΡ8 determination of mRNA-ΟΑΡΌΗ in the whole extract of the total fraction of RNA from mouse liver

The extract of the total fraction of murine RNA is analyzed for the content of mRNA-ΟΑΡΌΗ in test ΝΡΑΜΑΡ8 and a dilution curve is constructed. The total RNA fraction from mouse liver was prepared using the Οίαβοη kit. RNA is precipitated with 70% of UNN with 0.5 M acetate and resuspended in 10 μl of 5x88C buffer with 0.05% 8–8 with 1.8 nM protective fragment. A protective fragment is added to the oligonucleotide of 70 base pairs, of which 60 bases were complementary to mouse ΟΑΡΌΗ. Either a fragment complementary to mouse mRNA-ΟΑΡΌΗ (a “protective fragment”) or a complementary sequence used as a negative control (an “antisense fragment”) is used.

Samples of RNA with protective fragments are heated at 90 ° C for 5 minutes and hybridization is carried out by heating the samples to 70 ° C and then they are slowly cooled to room temperature overnight. Nuclease 81 (Gutes ^ a) was added at a dilution of 1:10 in 30 μl of Nuclease 81 (Gute ^ a) buffer for 30 min at 19 ° C and stopped by the addition of 1.6 μl 10n Ν αΟΗ and 2.7 μl 0.5 M EDTA. Samples are heated at 90 ° C for 15 min and then at 37 ° C for 15 min to denature and destroy RNA, neutralize 1.6 μl of 10 M HCl and incubate on 8-plates overnight in 5x 88C buffer with 0, 5% 8–8 with the addition of 200 mM ΗEΡE8 with a pH of 7.5, to which 30 nM biotinylated detecting oligonucleotide is added. Rinsing and imaging with 8Α-ΗΚΡ is carried out as already described. The amount of signal decreases in parallel with a decrease in the amount of mouse RNA (samples contain 500, 170, 50, 5, or 0.5 μg of total mouse RNA). Two control samples are included in which nuclease 81 is not added. The signal is detected only for a complementary protective fragment.

Used oligonucleotides:

- 38 011415

Anchor *: 5BC> 10:25

SOSYUEOTT ^ OSSTTSTSSSOAOOS

Linker * 8Εζ) TO: 26

SP'OAOOTOOPOTOKTATTGSTSOOATASYUAOOTSSOSGSSSS ^

Protective fragment (complementary to mouse antisense mRNA for SZARRN) ZET? ©: 27 SOAOAAATATOASAASTSASTAAAOATTOTSabSAATSSATSTASASSASSASSAASSTSTST OSTGOTSTAA.

Detecting oligonucleotide *** 8Εζ) W: 28

AAOOSAOOTTOOTOSTSSAOOATOSAT

For samples with semantic mRNA ΘΑΡΡΗ.

Anchor *: 8FFS: 25 SassootsalosattotoooAasos Linker ’* 8FU: 29

ATOSATSSTOSASKHAOSMSTOSTGS ^^^

Protective fragment (complementary to mouse mRNA for ΘΑΡΡΗ) 8Е (? ©: 30

AAS5CASSTOSTGOSTOSASaATOSASTOASAATATOASGOAOTTSTSATA1TTSTSO OSGTOTSTAA

Detecting oligonucleotide *** 5Е <Э ©: 31 SOAOAAATATALSAASTSLSTSAAO • Anchors were synthesized with a C12 speyer in an amide bond at NabConce ·· Linkers were synthesized with Sub linked to bConce “• Probes were synthesized with biotin linked to b’-end

Example 15. The N8 nuclease protection test with mRNA controls is extracted from the mouse liver and the nuclease protection is mainly carried out as described in Example 14, except that the ΟΑΡΌΗ-specific protective fragment contains 60 nucleotides that are complementary to the ΟΑΡΌΗ mouse, followed by 15 “protruding” nucleotides at the 3'-end of the fragment that are not complementary to the target. After hybridization and nuclease digestion, the remaining protective fragment is hybridized with a ΜΑΡ δ plate as described in Example 14, except that two different oligonucleotide detecting fragments are used to detect the immobilized protective fragment. One detecting fragment is complementary to the ΟΑΡΌΗ-specific portion of the protective fragment, and the other, the control one, is complementary to the protruding portion of the protective fragment of 15 bases. Each detecting fragment is used in different reproduced samples (i.e., different wells) so that both detecting fragments can be labeled with a single detecting molecule. In the present example, both fragments label NKR. Without nuclease addition, signals come from both detecting fragments, while after nuclease digestion, a signal corresponding to ΟΑΡΌΗ sequences can be detected. The value of the ΟΑΡΌΗ-specific signal decreases relative to that observed in the absence of nuclease cleavage, since the protective fragment is added in excess with respect to the amount of mRNA-ΟΑΡΌΗ present. This makes it possible to limit the amount of mRNAΟΑΡΌΗ by protective hybridization so that the amount of a double-stranded hybrid formed (and, therefore, the amount of a protective fragment that is protected from the action of nuclease) reflects the amount of mRNA. When mRNA is not included in the reaction mixture, no signal is detected when nucleases are added. The above established facts show that the stages of hybridization and cleavage in the analysis proceed as desired.

When protective fragments corresponding to different targets are included in this test, each of the protective fragments may contain the same protruding portion of 15 bases. This allows the use of a single detecting fragment to test the remaining protruding portion in all samples.

Example 16. A transcription test for screening compounds that can alter the expression of genes that are correlative for disease states

A cell line derived from a human tumor is used. It was found that 30 genes are expressed in it at a higher level than in normal cells (that is, these 30 genes were used to a greater extent than normal cells for the production of mRNA and then for the production of proteins that are encoded by these genes. Analysis transcription allows you to determine how many genes are used to determine how much mRNA is present for each gene). Using the nuclease protection test on ΜΑΡδ-plates (ΝΡΑ-ΜΑΡ8), 8800 chemical compounds are tested to determine whether expression of some of the 30 correlative genes in cultured cells in the presence of compounds decreases without affecting the expression of six normal genes (constitutive, auxiliary genes) . Any compound having an effect could be suitable in the future development of drugs for the treatment of this type of tumor.

Approximately 10,000-100,000 cells are added to each well of 100 96-well polystyrene plates and the cells are cultured for 2 days until they cover the surface of each well. AT 8

- 39 011415 wells of each tablet, cells grow without the addition of test compounds. In the remaining 88 wells of each plate, various chemical compounds are added so that the effect of each of them can be estimated. On 100 tablets, 8800 compounds can be tested or screened simultaneously. Cells were cultured for 24 hours in the presence of compounds, and then the cells were harvested for analysis. The cells in each plate are treated according to the instructions for obtaining RNA in samples from 96-well plates (for example, attached to the O1adep KNeaku 96 kits). After receiving RNA, 36 different types of mRNA were quantified by the ΝΡΑ-ΜΑΡ8 method, including 30 correlative genes and 6 normal auxiliary genes. 36 oligonucleotide DNA protective fragments, each corresponding to one of the genes of interest, were introduced into each well and hybridized under selected stringent conditions with their target mRNA sequences. Nuclease 81 was then added to destroy the hybridized DNA, and the samples were also chemically treated to destroy the RNA. The rest represents oligonucleotide protective fragments for each of the 36 genes in proportion to the amount of mRNA present in the treated cells in each sample.

One thousand 96-well plates were prepared, each containing a plurality of 36 different anchor oligonucleotides in each well, by adding 36 different linker oligonucleotides to each well. Linkers attach to the surface of each well, converting common plates into MAP8 plates containing specific probes for each of the 36 oligonucleotide protective fragments. Each linker has a site specific to one of the 36 anchors, and a site specific to the segment of one of the 36 protective oligonucleotides. A sample of oligonucleotides from each well of 100 sample plates was added to the corresponding well of 100 MAP8 plates. After hybridization under selected stringent conditions, a detecting oligonucleotide was added for each target with a conjugated chemiluminescent enzyme so that each specific spot in each well glowed in proportion to the amount of mRNA in the sample. All wells in which reduced amounts of correlative genes were observed without affecting 6 auxiliary genes are of interest. Perhaps the compounds added to the cells of these samples represent the starting points in the development of antitumor agents.

Example 17. Expression of induced and constitutive genes

RNA was obtained mainly, as described in example 14, from the liver of mice, either uninfected ("control"), or one hour after infection ("infected") with adenovirus. 60 μg of hepatic RNA was used for each sample, and samples were prepared in parallel. Each well for analysis contained three groups of parallel loci corresponding to the three genes described above. Each locus contained an anchor connected to a linker including a probe, which was complementary to the protective fragment corresponding to one of the three genes. The MAP8 nuclease protection test was performed mainly as shown in FIG. 12, and images were collected and scanned as already described. Presented by collected raw visualized data and intensity scans for parallel wells for each of the three mRNA targets. The numbers along the scan lines represent the integrated intensity values and standard deviations for each condition (n = 4). Auxiliary gene ΟΑΡΌΗ, the change of which was not expected, showed a slight increase of 1.3 times in the infected sample, which was statistically unreliable. The transcription of M1P-2 and c-cnc increased 4 and 6 times, respectively. These established facts indicate that there are two genes, M1P2 and c-alk. show increased expression in response to infection with adenovirus compared to the control constitutively expressed by the gene - SAREN.

Example 18. Enzymatic method of screening compounds selectively inhibiting tyrosine or serine kinase (see Fig. 17)

Kinases are enzymes by which phosphate binds to proteins. It has been shown that many of them stimulate the growth of normal or malignant cells. Therefore, compounds that inhibit specific kinases (but not all kinases) can be used to analyze the participation of kinases in the development of pathology and, if so, whether they serve as a starting point for the development of pharmaceuticals. For example, the five tyrosine kinases that are involved in stimulating cell growth or regulating the inflammatory response are kgf, 1sk, Hup, Ζηρ70, and uek. Each kinase has substrates that are partially established such as short peptides containing tyrosine. The specificity of some kinases overlaps so that different kinases can equally phosphorylate some peptides, but others are only preferred. For five kinases, 36 peptide substrates were selected that show a spectrum of specific and overlapping specificity.

Use one thousand 96-well plates; each well contains 36 common oligonucleotide anchors. Get 36 linkers for converting the total set of oligonucleotides (only with anchors) in the aggregate, including peptide substrates. 36 peptide substrates are synthesized and each is covalently linked via an amide bond, for example, with an oligonucleotide containing a 5'-amino group. Oligonucleotides contain sequences that hybridize specifically with anchors. Peptide / oligonucleotide linkers themselves attach to the surface when they are added to all wells on MAP8 plates.

- 40 011415

For screening, five kinases at appropriate concentrations (so that the phosphorylation rates of the substrates are as balanced as possible) are added to each well along with one of 8800 different compounds for testing. Compounds are tested for their ability to directly inhibit isolated enzymes. The degree of phosphorylation of each individual peptide is detected by the addition of labeled antibodies, which bind only to tyrosine phosphorylated peptides. Of interest is any well in which there is a decrease in the fluorescence of phosphotyrosine spots, but not all of the wells. Compounds that are added to these wells can be tested additionally as possible selective inhibitors of some of the tested kinases.

An analysis chart is presented at the top of FIG. 17. A chimeric linker molecule is obtained in which an oligonucleotide of 25 base pairs complementary to one of the anchors is crosslinked with a tyrosine phosphokinase peptide substrate. The chimeric oligonucleotide-peptide substrate itself binds to a set of oligonucleotide anchors, the kinase is used to phosphorylate the peptide region of the chimera, and after the enzymatic reaction, the degree of phosphorylation of the peptide is determined using antibodies to antiphosphothyrosine and antiphosphoserine with conjugated detecting fluorochrome or.

The test results are presented at the bottom of the figure. A homobifunctional cross-linker linker, Ό88 (Pegsé), was used to link the 5'-amino group of the oligonucleotide linker to the конц-end of the synthesized peptide with phosphorylated tyrosine. The peptide sequence, represented in the form of a code from the initial letters, represented представля8ΕΡ0ρΥ0Ρ6ΕΝΕ (8Е0 GO: 32), where ρΥ is phosphotyrosine. The chimera was used either directly or initially the pH was adjusted to 14 within 60 minutes to partially hydrolyze the phosphate group in tyrosine. Phosphorylated or partially dephosphorylated chimeric molecules themselves bind to complementary anchor molecules on plate ΜΑΡ8 in concentrations presented for one hour. After washing and blocking the wells of 0.3% Β8Α in 88ΡΤΡ, anti-phosphotyrosine antibodies conjugated with ΗΒΡ (antibody 4610 manufactured by irk! A! E Vu1ec1to1odu. Lake Ρ ^ ίά, ΝΥ) were added at a dilution of 1: 3000 to 88ΡΤΡ for one hour, and we determined the number of antibodies bound to a chemiluminescent substrate, 8 and 8 bp1 B1ax. The presented image was accumulated on the SSE aggregate for 1 min. As expected, differences were observed in the amount of phosphate binding to the oligonucleotide peptide. These differences are the basis for the subsequent analysis of what will be the activity of kinases when treated with various possible inhibitors.

Example 19. A binding test for the detection of selective inhibitors of the interaction of 8-2 domains and phosphorylated peptides

8H2 domains serve as cutting subunits for some growth-regulating proteins. Domains bind to low specificity proteins or peptides containing phosphotyrosine. That is, some peptides containing phosphotyrosine specifically bind to one or more 8H2 proteins, while others to many 8H2 proteins.

For this test, linkers are phosphorylated peptides covalently linked to oligonucleotides. Peptide groups are selected for their ability to bind to a group of selected 8H2 proteins. Linkers convert general-type plates into ΜΑΡ8-plates with ligands specific for the group of proteins containing 8H2. 100 968 96-well plates carrying ligands are prepared. Proteins are isolated and labeled, for example, with a Cy5 fluorescent molecule.

To screen inhibitors of 8H2-domain-phosphopeptide interaction, a group of 8H2 labeled proteins are added to each well of 100 96-well plates and various test compounds are added to each well. Therefore, the effect of each compound on the interaction of 8H2 proteins with their phosphopeptide ligands is individually tested. The test allows you to determine the fluorescence of the bound 3H2 protein connected to each peptide linker linked to the surface. For any well in which reduced fluorescence is observed in some spots, but not all spots, the introduced compound can be tested further as a putative selective 3H2-cutting domain inhibitor.

Example 20. High throughput screening (see Fig. 22)

Presented is a ΜΑΡ8-tablet with high throughput for detecting a signal from 96 wells in one experiment. The hybridization of one oligonucleotide with 16 replicates in 80 wells was determined. As shown, the reproducibility of 1280 hybridization tests was very high. The leftmost and rightmost columns serve as controls for signal standardization at various oligonucleotide concentrations.

Similarly, in each well, 16 different oligonucleotides can be tested and the test repeated in 80 different wells on a plate. Of course, even more different oligonucleotides or other probes (e.g., 100 nucleotide probes) can be tested in each well, and many plates (e.g., 100, such as a 96 well microtiter plate) can be analyzed at the same time. A large number of analyzes that can be set with each sample (for example, in the latter case, approximately 100 different analyzes) and a large number of samples,

- 41 011415 which can be analyzed simultaneously (for example, in the latter case, approximately 96x100 or 9600 different samples) provides an analytical system with high throughput.

Example 21. Preparation of an amplified target (see Fig. 23)

A PCR primer (one primer) is attached to a solid support (e.g., a granule or reaction vessel) by chemical modification, which is introduced at the 5'-end of the primer oligonucleotide. The primer includes a chemical modification in the 5′-3 ′ direction, a restriction site, and a sequence complementary to the target of interest (for example, a cDNA copy of the mRNA of interest). The target is amplified by PCR, using one plus two as a primer for the PCR primer, which includes in the 5'-3 'direction, a sequence specific for the detecting oligonucleotide, and a sequence complementary to a different region of the target than that in primer one. After PCR amplification, the amplified DNA target is washed to remove excess reaction material and released from the solid support by restriction enzyme specific site restriction in primer one. Thus, the amplified primer is released into the liquid phase. Thermal and / or chemical treatment can be used to deactivate the restriction enzyme and denature the double-stranded DNA product. The released single stranded target DNA can then be contacted with a surface containing anchors and / or linkers, and the target can be detected using detecting oligonucleotides complementary to the detector-specific primer two sequences.

Example 22. Preparation of an amplified target

The PCR primer (primer one) is attached to a solid support (for example, a bead and a reaction vessel) by chemical modification, which is introduced at the 5'-end of the primer oligonucleotide. The primer includes a chemical modification in the 5'-3 'direction, a peptide sequence that can be cleaved with a protease, and a sequence complementary to the target of interest (for example, a cDNA copy of the mRNA of interest). Instead of the peptide, any other element that can be specifically cleaved can also be used. After PCR amplification, as described, for example, in Example 21, the PCR product, still attached to the solid support, is denatured and (optionally) washed, leaving a single chain molecule attached to the support. Then, the washed, attached molecule can be cleaved and released (for example, by treatment with an appropriate protease) and contacted with a surface containing anchors and / or linkers. Alternatively, the chain of the amplified target that is released after denaturation may contact a surface containing anchors and / or linkers. In this case, only one chain of the amplified target (for example, hybridized) is in contact with the linker so that there is no competition from the opposite side of the amplified target with respect to hybridization, and the background is reduced. Linkers can be designed to be specific to one or both amplified target chains.

Example 23. Test with detecting linkers and reporters (see Fig. 24)

A sample containing the mRNA of interest is subjected to a nuclease protection method using an oligonucleotide as a protective fragment, which includes a specific target group and a control protruding group that is not complementary to the mRNA. After cleavage with nuclease, the control protruding group can be cleaved as shown in the figure to the right. The obtained nuclease-protective fragments hybridize with a detecting linker that includes a target-specific group and a control protruding sequence-specific group. In the test shown in the figure to the left, the control protruding group of the detecting linker remains unhybridized; in contrast, in the test shown in the figure to the right, the control protruding group of the detecting linker hybridizes with the remaining control protruding sequence of the protective fragment. In a subsequent analysis step, a reporter comprising a group that can interact with a control protruding-specific group of a detecting linker interacts with the complexes. In the test shown in the figure to the left, the reporter hybridizes with a control protruding sequence by a specific group of a detecting linker that remains available for hybridization, and the complex can be detected by a signal group in the reporter. In contrast, in the test shown in the figure to the right, the reporter cannot bind to the complex, since complementary sequences are not available for hybridization, so the signal associated with the complex is absent.

In many of the tests of the present invention, the reporter can interact with any sequence present in the detection linker, not limited to the sequence specific to the control protruding group.

Example 24. Multiple fluorescent labels (see Fig. 25)

An area including five loci AE is shown in FIG. 25. Each locus includes a different group of basically the same anchors, anchors AE. Four different types of linkers hybridize with anchors at locus A, each of which includes a group specific to anchor A. However, each of the anchors included a different target-specific group: for targets 1, 2, 3, or 4. Therefore, after hybridization of targets with complexes anchor / linker, all targets 1, 2, 3 and 4 are localized in

- 42 011415 locus A. Similarly, four different types of linkers can be hybridized with locus B. Each linker contains a group specific for anchor B, and target-specific groups are specific for targets 5, 6, 7 and 8. Similarly, target 9-12 can contact locus C, targets 13-16 - with locus Ό and targets 17-20 - with locus E. If each of these targets is labeled, either directly or indirectly, with a different, independently detectable fluorescent label, for example, as non-convertible phosphors, it is possible to independently detect all 20 targets at five indicated loci.

Example 25. Analysis in high performance form

In this example, the transcription analysis of the invention is used to detect and quantify changes in the nature of gene expression, in the form of high-throughput screening. All stages of the analysis are performed automatically. Typical washing steps are described schematically. All reactions are carried out using conventional methods that are known in the art and / or are described herein.

Human THP-1 monocytes were cultured in 96-well V-bottom microtiter plates at a titer of 50,000 or 150,000 cells / well. Cells are either not treated or differentiated with phorbol-12-myristate-13-acetate (PMA) for 48 hours, followed by activation by lipopolysaccharide (L8) for four hours. After treatment, the cells are lysed with guanidine isothiocyanate and frozen until use. mRNA is prepared using streptavidin paramagnetic particles to which biotin-polyBT is bound. Alternatively, the total RNA fraction is obtained by extraction with a trireagent (8CTa C11C1cca1 Co., Lox, MO). Samples containing either mRNA or the total RNA fraction are treated with nuclease protection using a mixture of thirteen 60-dimensional single-stranded oligonucleotides as DNA protective fragments, each of which includes in the 5'-3 'direction a 25-dimensional specific to one of thirteen targets of interest (ΟΆΡΌΗ, 1L-1, TIR-a, cathepsin C, ox-2, cyclin-2, vimentin, ΕΌ78-β, NMS-17, osteopontin, (β-thromboglobin, angiotensin or actin); 10-dimensional spacer; 25-dimensional specific oligonucleotide detection probe specific and 15-dimensional general control the protruding sequence. In this case, mRNA is converted into a stoichiometric amount of the “corresponding DNA protective fragment", which is detected in the test. In control experiments in which the corresponding DNA protective fragments are incubated with a probe specific to the control protruding sequence, it was shown that mainly the corresponding protective fragments are only sequences specific for target mRNAs of interest, which was expected in the case if the cleavage of nuclease occurred on the desired path.

Surfaces are prepared according to the methods of the invention. A set of sixteen different 25-dimensional oligonucleotide anchors is placed in each well of a 96-well DNA binding plate. Fourteen different types of anchors are used. One kind of anchors is used in three of the four corners of the population, and 13 different types of anchors are used, one each in the remaining positions of the population. Then the aggregates hybridize, in a certain orthogonal order, with 60-dimensional oligonucleotide linkers, each of which includes in the 5'-3 'direction a 25-dimensional, corresponding to one of the target of interest, a 10-dimensional spacer and a 25-dimensional, specific to one of the anchors. Thus, in each multiple sequence of 16 spots, each of the thirteen target-specific linkers is located in a certain position (locus) in the population. See FIG. 18 showing such an orthogonal population. Linkers corresponding to SAREN. the constitutively expressed “housekeeping” gene, which serves as an internal control of normalization, is represented at three loci within each population. In control experiments, it was shown that linkers, as well as protective fragments and detecting oligonucleotides used in the experiment, exhibit the desired specificity.

Samples, including mixtures of the corresponding protective fragments, prepared as described above, hybridize with the anchor / linker aggregates. Samples obtained from untreated or induced cultures are used. Then, the presence is detected and the number of hybridized protective fragments at each locus is determined by hybridization with labeled detecting oligonucleotides. In order to normalize the signal at each locus, the detecting oligonucleotides are diluted with the appropriate amounts of blocked oligomers, as has already been described. The magnitude of the signal at each locus is processed and normalized to the signals of the control SAREN. The data obtained are reproduced in eight parallel samples, as well as in samples obtained in three independent experiments, delivered on different days. The total data on the relative content of thirteen transcripts in one experiment are presented in the table below.

- 43 011415

Relative intensity (10 * “lvgok / well)

Average

Sarra fifth ΤΝΡ OARON Cathepsin 0 SOX-2 N-2 cycles Vimentin Ts378 NM6-17 Osteopontin Tronbotobupin SAGON Angiotensin Actin (the control)

Induced

SU {p-10

Ratio

Example 26. Computer algorithm for quantifying data on a tablet with multiple sets

Using the preferred algorithm, you can find the position of all the spots on the MAP8-tablet and automatically calculate the value of the signal amplitude for each data point. Preferably, the algorithm is carried out using a computer program.

1. Select a small part of the image data, a 40x40 square, containing the intensity value of each pixel (picture element) of the image, which includes the first well for analysis.

2. Define a function by which it would be possible to calculate the estimated intensity at each pixel position using 16 unknowns. Unknown are:

the amplitudes of each of 13 different spots of micro-aggregates (that is, the brightness of real signals in each position of the DNA aggregate). There are 13 such 4x4 spots (= 16) inside each well, since some of the 16 spots are parallel to the same target;

x offset and y offset determine the exact position of the 4x4 spots within a given well;

background intensity of the image inside the well.

Using the function for each pixel position, the distance between the pixel and each spot is calculated and the contribution of each spot to the intensity determined in the pixel is added by multiplying the spot amplitude by the pulse response function for a given distance. For images, the used response function of the momentum is determined by the sum of the Saikyap and LogepShap corresponding (constant) radii.

3. Start lighting this well, quickly determining the parameter values. Having done this, calculate the average image intensity for 16 areas of the image where spots are supposed to be. Subtract the bias from these 16 averages and scale the difference using a constant factor. The displacement and scale constant are determined empirically. Regroup the data to get 16 spots with 13 amplitudes. For background and offset use any small numbers.

4. Optimize the value (for 16 unknowns) along the curve. In particular, use the nonlinear smallest area algorithm using the Magdiai method! to linearize functions with 16 unknowns to 40x40 = 1600 equations (although, of course, not all equations are linearly independent).

5. Use the x, y offset for this well to establish with increased accuracy where the grid of the next well will be on the microplate. It is believed that the distance will be 9 mm with respect to the next neighboring hole (turn the number of pixels into a distance using the magnification factor of the image system). Since the distance between the wells is small relative to the size of the tablet, using local position values is

- 44 011415 the most accurate.

6. With a more precise positioning, limit the smaller square of the image to the next well by moving to a square of 30x30 pixels. This makes the definition faster.

Return to stage 2 and repeat for each well.

Example 27. High throughput screening (see Fig. 26 and 27)

In FIG. 26 shows raw visualization data for a test using detecting linkers and one reporter. The test analyzes the expression of 13 naturally occurring mRNA species from 96 different cell samples. Each well of a 96-well plate contains 10 ⁵ untreated THP-1 cells (in the figure on the left) or induced to monocytes with PMA and LRA (in the figure on the right).

The nature of expression changed sequentially. Inductions are subjected to 1b-1, ΤΝΡ, COX-2, vimentin, bb78, osteopontin and beta-thromboglobin. The expression of cathepsin-C, cyclin-b, NMS-17 and angiotensin is reduced. Expression of SARBN and actin does not change.

In FIG. 27 shows the spatial arrangement of the THP-1 cell genes along with the wells for two samples (selected from FIG. 26).

The oligonucleotides used in these experiments are listed below. For some targets, the signal intensity decreases when the detecting linker is diluted with an incomplete detecting linker oligonucleotide containing 25 bases complementary to the protective fragment, but not containing a sequence complementary to the reporter. These incomplete oligonucleotides are referred to as “attenuation factors”.

In the table. 1 shows the quantitative processing of the data presented above. This screening test is performed with high throughput. Cells are cultured and processed in 96 well plates with an average titer of 10 ⁵ cells / well, with this titer the cells are easily treated on microplates. Determine the expression pattern of 13 genes by high throughput in small cell samples. The data obtained are presented in table. 1 using a high throughput method. Using the method, less than one copy per cell can be detected. The literature reflects observations collected for various closely related cell types, such as I-937 cells. Noticeable significant differences between the control and induced conditions are the result of very low background signals for determinations made by the applicants. The use of detecting linkers with only one type of reporter helps reduce the background for analysis. This is possible due to the fact that the total concentration of the NKR-containing reporter is significantly reduced.

Table 1. The relative content (RNA molecules per cell) in the format MAPE 96-16, 10 ⁵ cells / well

Gene The control Induced Literature SARRN 30 ± * 7% 30 ± 14% Without changes 1b-1-beta * + Not detectable 684 ± * 14% Increase ΤΝΕ 3.0 ± 40% 214 ± 23% Increase Cathepsin-s 53 + 8% Not detected Decline SOX-2 Not detected 8.3 ± 23% Increase Dicklin-2 2, 8 ± 11% 0.5 ± 46% Without changes Vimentin Not detected 37 ± 33% Increase Bc78-b Not detected 3360 ± 28% Increase NMS-17 33b ± 5% 33 ± 23% Decline Osteopontin Not detected 18 ± 23% No messages Thromboglobulin Not detected 66 ± 15% Increase Angiotensin 0.5 ± 18% 0.1 ± 66% No messages Actin 7 9 + 7% 43 ± 21% Without changes

+ Established values, assuming that SARBN is 30 / cell *% SU (standard deviation / mean value as%) (n = 48)

The oligonucleotides used in example 27 with detecting linkers and one probe are:

- 45 011415

Fixed probe: SASSTSSAAASabtbaAAOOSASASSA (sogfida Day ίο NKR) (5Е <2 Ιϋ: 33)

TargetM; S: M178516AR0N (572-513)

Anchor: -length = 25

SSSSSSTSSASSSTTST6S6ASS6S (ZEO U: 34)

Target: -length = 60

TSASAASTATSASAAASASSSTSAASATSATSabSAATBUUTSTSTSSASSASS

AASTSSTT (ЗЕ <2 Ιϋ: 35)

Linker: -length = 60

ATSSSTSSSTSSASSASSASAASTPSTTSATASTASSetSSSSSTSSSASAASSSTSS

ASSSSSS (S <3 10:36)

Protective fragment: - length = 75

AASSASTTSSTSSTOSASSASSASSATTSSTSATeATSTTteASOSTSTTSTSTSAT

ASTSTSTSAOOSTGSTSTAAOTSTO (8E0YU: 37)

Detecting linker: long

TOSTSTSSTGSASTOTSSAOOTSbATASTOAOOTTOAOAAETATOASAASA

Θ

SSTSAA6 (8VrGO: 38)

Attenuation Factor: Length = 25

TOAOAA0TATOASAASAOOSSTSAOO (3Ε <? ΠΞ>: 39)

MtJ; GO: M15840 PL-Yya (43924333)

Anchor: length = 25

TSSASSTOAOASASSSOASOOSTSTSS (8E (} GO: 40)

Target: Dpiner)

SOASASATOS (ITAAS (CJSCSSPATSTOSASSATSSASSTSTASSATSASTS

AASTOSAS (5E () GO: 41)

Linker: dpina = 60

SASSTOTASSZSATSASTAASASTOSASOASTOAOOTSSASOSSSTSSbOTSST

SASSTOSA (5E <5GO: 42)

Protective fragment length = 75

STOSAS1DSAOOTSATSSTASA (ZOT (K) ATSOT (YuASATAAZHSTS (NTATSSA

TSTOTSSOSSGTOTSTAASTSTO (BEb'P): 43)

Detecting linker: length = 60

TOYHZTSSTGSASTbTTTbOASOTSSATASTOABTSbASASATSZOOATAASO

BUT

OSSTTAT (8TH: 44)

Attenuation Factor: Length = 25

SOASASATSOSATAAbAeSTSTAT (8E <? GO: 45)

Target # 3; HOMO98UYUT (780-721)

Anchor: length = 25

SASTASbOSTSSASSASbTSSSSSTOS (8ES! GO: 46)

Target: Length = 60

SbOAASSSAAOOSSTAOAASTTGAAbsAASAAOAASSASSASTSTSOAAASSTOS

SAGGSASS (WITHOUT GO; 47)

Linker: dpina = 60

ASSASTTSSAAASSTSS (LATTSASYUSATAST (CJSC (ZSA (lS

SSOTAOTO (8E (} GO: 48)

Protective fragment: length = 75

- 46 011415 “LHZAATSSSA

SSTTSSSSSTGOTSTAASTSTA (5EfP> 49)

Detecting linker: length = 60

TOSGSTS (^ GSAST01TG (X5AO (N ’(YuATASTSAOOTSOSAASSSAASSTTASA SGGGAAO (8BC5 PE: 50)

Attenuation Factor: Length -25

S (YuAASSSSAASSGGASAASTTTAAO (8EOP> 51)

Target # 4; SHMG78510ARON (572-513) (the same as target # 1)

Target # 5 _; GO: MK11TKetepsin- (3 (373-314)

Anchor: length = 25

OAASSOSTSHYUOTSTTSTASASZSSSSA (ZefGO: 52)

Michelin = 60

SSASASSATSSASAATSASATSATSTTATTOSA6STAOOSAOAAA6ASTSASA SSOAATSS (£ Е (2ПЗ: 53)

Linkerdpina = 60

TSABSASAASAOTSAOASSSSAATSSATASTSAOOTTOSSTSTAOAASASbS 6ASSSSTTS (5E0 GO: 54)

Protective fragment: length-75

SKhTAZTSSSOTSTSASSTGTSSTSSTSabSTSSAATAAASATOTSZTSATTSTSTOOAT

OSTSSSSOSTOSTTSTAAOTSTO (8TH: 55)

Detecting linker: dpina = 60 ta

SATSATS (WES2GO: 56)

Attenuation factor; dpina = 25

OSOOASSATSSAOAATSASATSATO (Zef GO: 57)

Target # 6; ПЖ9010000Х-2 (240-181)

Anchor: dpina-25

STSTSTSSSSOTSSOTHEOSTOSSAO (8E () PE: 58)

Mi1ienpina = 60 csoAashiAtstatoASshztasAttto ^

SOOASAOO (ZEOU: 59)

Pinker: Length = 60

- 47 011415

ATAAST6S6ATTETASSASSSSSASASS <ZASTASTOA (ZTSTSKgSA <ОССАС6 <ДАССС 6ОААССАО (5В0 ГО: 60)

Protective fragment: length = 75

SSGOTSSSYUOTAS. \ ATCO € ASTGATASTSOTSAAATSSSASASTSATASATAS

ASSTOOSKZSTGOTSTAASTSTS (5E (} GO: 61)

Detecting linker: length = 60

TSSTSTSSTTSASGOTTTOOA <ЗСгГСОАТАСОА <ЗТСС0А (ЭОТ (ЭТАТАТАТАА (} ТОТОООА (ЗЕРГО: 62)

Attenuation Factor: Length = 25

ССОАЗЗТЗТАТЗТАТЗАОТСЗТбСЗА (3Εζ ГО: 63)

Target # _7; Go: M74091cycline (932-873)

Anchor: dpina ^with 25

SOOTSOOOSATOATASSASAOOTSSOS (8EfGO: 64)

Target: Length = 60

SASSTSSAAASA6TOAAOZASZASSASKYUTSSAAATSSAASTSAOAAS7TSTA OSTASASSS (ZefSh: 65)

Linker: Length = 60

SZAAOTSAOAASTSTAZSTASAOSSOATASTASTASTSSSOASTSTSHlGASSAT SSSOASSO (8ER Go: 66)

Protective fragment: dpina = 75

C9 <ZSGSTA (YUGAOA & TTSTSASTT (HATTTSSASSSTASTSTSTSS1TSASTOTTT

OZABOTSOSTTOTSTAAOTSTO (ZefGO: 67)

Detecting linker dpin = B0

TOSGSTSSTTSAST31TGOZAS <71KHYUATASgaAOOTSASSTSSAAASAOOTOAO

OASAOSA (5EfGO: 68)

Attenuation Factor: dpina = 25

SASSTSSAAASAOOTOAASOAASSA (5Ef GO: 69)

Target # 8; PCM14144vimeitif1338-1279)

Anchor: dpina = 25

SSOS (XC (X: 31TATSSATSTSTGSZ (ZETSGO: 70)

Target: length = 60 sp's <tAtSkhSssttaaaa6saassaatsaotssstsS’Aas6SaaoatszsstsaaaaAOAOO (Zef GO: 71)

Pinker: dpina = 60

ASSSSAOATOSOTAAATSKHZAASAZOATASTZASTSSAAOAOATySATAAAS

- 48 011415

OSSSSSSOSOS (5TH: 72)

Protective fragment: dpina-75 _____

STSTTSSSATGTSASbSATSTOOSOTTSSAOobOASTSATTSYUTTSSTTTAAOSS SATSSASOSTSTOTSTAASTSTS (ЕО ©: 73)

Detecting linker: length = 60

TOSTSTSS1TSAST (LTG (YuAOOTSOATASTOAOOTOT (YuATOSSSTTAAASSA

ASSAATO (5ECI: 74)

Attenuation Factor: Length = 25

OTSUATSSSSTTAAASHEAAASSAAT (8EC 10:75)

Target Yu; Ts9O145 Sh78-b (2049-1990)

Yayurydpina = 25

SGGAOOSATASOTOTSASSASASSASSO (8Е <31П: 7б)

Target: Length = 60

SASSTSSSSASASATTSSASASAATTGSATAOOSTOASGASTTGSAOASOAOOS

AOSASTO (8E (2GO: 77)

Linker: dpina = 60

ASTASTTTOASASSASSA <lSSASTSSATASTOA (ZTSSO <dTSTSST6ASASOT

ATSSTAAS (8ETs1O: 78)

Protective fragment: length = 75

SASTOSST (H7GSOTSTSAAAOTASTS <ESTAT (LAAATTST <lT (l <}

OSASATOOSGTSTSTAAOOTSTO (5VOP>: 79)

Detecting linker: length = 60

TSSTSTSSTTSAST6TTTOSAOOTSX1 \ TASTOAOTSASSTSSSSASAOATTSS ASASAAT (SOS: 80)

Attenuation Factor: dpina = 25

SASSTSSSOASASATTSSASAOAAT (5E0GO: §1)

Target #! ^ C> _: X13546NMS-17 M12b23-: m-RNA (191-132)

Anchor: length = 25

S0TSASTSSSTSSSSSSA6STSTTSS (5E $ 10:82)

Target: Length = 60

SAAASYUTSAAOOASAAASSASASAOAASATSSSOAOOSTTSTSTSTAAAS SGSSTOSTS (8Е <210: 83)

Linker: length = 60 ’

AOS1TOTST (HYAAA4SS7G <KTSSTSSATASTAOAOTOAAAOOSGa <ZSSOAS <E SASSSASS (ЗЕ4} 1О: М)

- 49 011415

Protective fragment: length = 75

CASOASYUASYUTAOOSAOASAASSTSSYOOOOSTSTTSTSTSTSOTTSatSSTGS

ASSTTGSSSTTSTSGAAOOTSTO (8EOYU.-85)

Detecting linker: length ·· ®}

TOSGSTSSTTSASGOTGTSSASOTOOATASTOAOOTSAAAOOTOAASSASSOAA

SSASAOAO (8EOSH: 8b)

Attenuation Factor: Length = 25

SAAAOOOOTSAAOOSASSAASSASAOAO (8Ef10: 87)

Target#! one; S: X1369FK ^{; theopon} ™ ⁿ (783-724)

Anchor: length = 25

ATSSASTGAASASSASATSSGASTASS (5ErP>: 88)

Target: dpnna = 60

SSSTOSSAAObASAOOTTATOAAASSAOOTSAbSTSSATbASSAOASTOSTA AASSSSASAO (8E0 GO: 89)

Linker: Dliva = 60

ATSASSAOAbTOSTOAAASOSASA (3 <ZATASTA (ZT <l <ZTASTA (ESAT (ZT60TT AASTOAT (8Βς GO: 90)

Protective fragment: dpina = 75

STSTbGESTTTSAbSASTSTOOTSATSSAbSTOASTSSTGTSATAAASTOTSSTT

S SSASKHESbSTTaTSTAAOOTSTS (8ES> GO: 91)

Detecting linker: dpina = 60

TSSTSTSSTTSASTOTTTSSASYUTSSATASTSASSTSSOTOSOAAOOOOSAOTTA TOAAASO (8Er GO: 92)

Attenuation Factor: dpina = 25

SSSTOSSAASOASAOGTATSAASO (5E (2GO: 93)

TTAbSOGGOZSSSAOeTTSATAOOSS (5Ε (} Π> 94)

Target. Dpina = 60

STSTAAASATSASTGSSAASSGOSYUSSTbOSTSTSTSTSSSSAOOSSTTSSTOATG

TSTOSAS (8ERGO: 95)

Linker: length = 60 ttssg ^ susttskhtgssattgstssassata ^^ ASSSTAA (8ΕςΠλ96)

Protective fragment: dpins = 75

- 50 011415

STSKZAOAAATSASYUAASSSTOSSAASKHASZAOOSSASASSOSSASSTSTOSAASSTS TSPTASASOSTTOTSTAAOTSTO (ZERGO: 97)

Deterfing linker: ₽60 length

TSSTSTSSTTSASTSTTTOOASOTSSATASTSabTSTOTAAASATSASTTSSA

AOSTOOS (8EOGO: 98) Attenuation factor: length = 25 OTOTAAASATOASTTSSAASSTSSS (ZER GO: 99)

Targets # 13; GO: M1785YuARPN (572-513) (same as target # 1)

Mission 4; W; K02215 ^en ™ ° ^{tens h} (805-74b)

Anchor: dpina = 25

SATGASSASTOSATTSOSATSAASR (ZVR GO: 100)

Target: length = 60 CASOSGSTSTOOASTTSASAOAAASTSSATOTTASTOSTOASAASATTSASAO STGSATSS (5E <2 GO: 101)

Linker: length = 60.

STOASAAOATTSASA (KGTTSATOSSATASTAAOTSSTTOATOS (1AATOSAS TSOTAATO (8ERGO: 102) Protective fragment: dpina = 75 osatoaasststraatstststsaosaosaasatssaststststaastssao

A SAOSOTSKYUTTSTSTAASTSGS (ZEO GO: 103).

Detecting linker: dpina-60

T (YUGSLGS1TSAST <ZTTTsZSASKZTSSATAST (1ASTSASSSTSTSG (E <ZASTTSA SASAAST (ZERGO: 104)

Blinding factor: length = 25 SASSSTSTOSTOTASGTSASAOAAST (ZERGO: 105)

Target # 15; GOA0277rctin (2627-2568)

Anchor: dpina = 25

АТСА1ХИАА <ЖТГШЗТСО <ЭТС6С (ZERGO: 106)

Target: dpina = 60 CA <31ShGST <YUSATSSAS (hAAASTASSTGSAASG <HATSATSZAA6TOT0ASS T

SOACATS. (5E () GO: 107)

TACATOAT (ZEPh: 108) Protective fragment: length = 75

OATetSSASOTSASASTGSATOOOOOOTTOAAOOTASTTTSbTOSATSSSA

S AOOASTSbSTTOTSTAAOTSTO (ZefSh: 109)

Detecting linker: dpina = 60

TSSTSTSSTTSASTaTTTSOASbTSRATASTOARTOAOTSSTBT & OSATSSAS OAAASTA (ZER GO: 110)

Attenuation Factor: Length = 25 OASTSSTSTSSSSATSSASSAAASTA (ZERGO: 111)

Target # 16; (YOU εροί ίε ηοΐ hoarfrost)

Target # 16; (this stain was not applied).

Example 28. Simultaneous determination of DNA and RNA (see Fig. 29)

Human THP-1 monocytes were cultured in 96 well U-bottom microtiter plates at a titer of 30,000-150,000 cells / well. Control cells were used that did not differentiate using PMA and did not activate using LB8, since RNA for some genes (e.g., L2-2, Cox-2, LO78, osteopontin and thromboglobulin) is absent in these cells and, therefore, the test determines only DNA, while for SAREN are present, and therefore determine both DNA and RNA. Cells are heated to 105 ° C in an aqueous medium (lysis buffer) and nuclease-protective fragments are added to the lysates for targets of interest. Upon lysis at elevated temperature, DNA is released in a detectable form as well as RNA. To determine the DNA, nuclease-protective fragments are added as such for 1b-1, Cox-2, bO78, osteopontin and

- 51 011415 thromboglobulin. For the simultaneous determination of DNA and RNA, the previous nuclease-protective fragments, as well as specific for SAREN, are added. Nuclease protection reactions are carried out in the wells described in the application.

Combinations are obtained and hybridization of the protective fragments is carried out mainly as described in Example 27. Detection of DNA against DNA + RNA is carried out by serial hybridization of the detecting linkers. In this case, serial hybridization is carried out in order to balance the signals from the RNA and DNA targets (as discussed below); serial hybridization, of course, is not a prerequisite for tests in which both DNA and RNA targets are detected. At the first stage, detecting linkers for 1b-2, Cox-2, 778, osteopontin and thromboglobulin are introduced. In a second step, a detecting linker for SAREN is added. Serial hybridization is carried out in order to visualize a DNA signal, which is relatively much weaker than the RNA signal, due to a significantly smaller number of copies per sample, over a longer period of time in order to accumulate a higher signal intensity.

The results are presented in FIG. 29. The figure on the left shows that when one genomic DNA is examined, the tested genomic sequences — 1L-1, Cox-2, ΕΌ78, osteopontin and thromboglobulin — can all be detected at the corresponding loci, and they are found in approximately equal amounts. The results of the determination of this genomic DNA are presented in table 1, from the data of which it follows that in these control cells, RNA for these genes is not detected. The figure on the right shows that when both DNA and RNA were detected, visualization was carried out for a significantly shorter period of time, since the DNA signal is much weaker than, as shown on the left, and it was found that when DNA and RNA are detected together, the control genomic sequences can be detected before, as internal standards for normalization, and the expressed SAREN gene is in a much larger amount than in the control. By quantifying the relative amounts of signal in the control and expressed SAREN mRNA, the amount of expressed mRNA per cell can be calculated.

Example 29. Detection of expressed 8IR (see Fig. 30 and 31)

In FIG. 30 schematically shows one type of analysis of expressed 8IR. In this case, the nuclease protective fragment is designed to hybridize with the RNA region containing 8IR, so that when the corresponding enzyme is introduced (for example, RNase H), the nuclease protective fragment hybridizes with RNA for which there is an erroneously paired base (in In this case, 8IR), the enzyme will cleave the nuclease-protective fragment. In this case, the obtained cleaved fragment cannot hybridize with the aggregate (for example, due to hybridization conditions such as the temperature used). In other embodiments, hybridization together can occur, but the detecting linker cannot bind to the cleaved protective fragment (for example, due to hybridization conditions such as the temperature used); or the cleavage proceeds in such a way that the cleaved protective fragment can hybridize together and with a detecting linker.

In FIG. 31 shows the results of such a test carried out by conventional methods, as described herein. Wild type actin was used as an internal control, and the protective fragment corresponding to the wild-type SAREN containing the constructed 8IR was different from the protective fragment corresponding to wild-type SAREN. In FIG. Figure 31 shows the analysis of a plurality of samples containing either wild-type SAREN or wild-type actin (left column and left image in high magnification), or containing 8IR SAREN and wild-type actin (right column and right image in high magnification). The central image in large magnification shows the scheme of populations.

Example 30. High throughput screening (see Fig. 32-35)

The transcription analysis is carried out mainly as described in example 28, except that, for example, anchors are placed more on irradiated plates than on DNA binding plates. Use the same anchors described in example 27, but change some targets in the aggregate, namely, they use only one anchor to determine SAREN and add tubulin, actin and LNE. Other analyzed targets are 1L-1, TIR-a, cathepsin-C, Cox-2, C-C8E, CM-C8E, C8T-P11, NMS-17 cyclophilin, β-thromboglobulin, T1MP-1, MMP-9. The combination is shown in FIG. 32, both the linker and the sequences of the nuclease-protective fragments are presented below. Approximately 30,000 cells per well are used (not treated for prolonged proliferation of control cells during treatment with PMA and BEN-treated cells). Sequences:

Target # 1 SAREN (the same as target # 1 in example 27)

Target # 2 1b-1b (same as target # 2 in example 27)

Target # 3 TIR-α (same as target # 3 in example 27)

- 52 011415

Target # 4 tubulin (ARN1347) anchor: dpina = 25

TAABSOTSTSGASYUAAOOSASKHZTSO (8E <5GO: 112)

Target: Length = 60

SASOTOOGTSSSAAAOATOTSAATOSTSSSATTOSSASSASSATSAAOASSAASS 6TASSATS (8EC GO: 113)

Linker: Length = 60

SASSATSAAOASSAAOOSaTASSATSSATASTSASTSSASSTSSSTGSSGASA SASSgSTTA (5E <2 LE: 114)

Protective fragment: length = 75satsotas <zstt <s <n <7ph7atsyutsyusaatsiyuask / ATT <tasatsttt (E {KG AASSASOTSSSGTOTSTAASTSTO (5E <GO: 113) linker Detektaruyuschy: length = 60 TSSTSTSSTLDASTOTGTSSASSTOSATASTSASTOASSTbSGGSSSAAAOAT "ZTSAATS (2E <2nd: 116)

Attenuation Factor: Length = 25

SASStKHGGGSSSAAASATOTSAATS (ZERSH: CT)

Target # 5 cathepsin-6 (the same as target # 5 in example 27)

Target # 6 Sox-2 (the same as target # 6 e example 27)

Target # 7 (ЗС5Г (ё01219)

Anchor: length = 25

SSYUTOOSATSSTASSASASAOOTSSSS (F) TO: 118)

Target: dpina = 60

OAOOOSabSASASAOOSAOOSAATSATSTSAOOSSSTSTOTTOAAAOOOOASSTS SASTOTSAS (Zeo GO: 119)

Linker: Length = 60

STSTSAAAS6AA0STSSASASTSTASAS0ATAST0A0TaS0ASASTets (PASSAT

SSSSSASSO (Zh2 GO: 120)

Protective fragment: length = 75

STaASASTASAOSTOSTSSGGTSASASASASSSSSTSASATSATTSSTSTSTSTSTS

STSSSTSOSTTETSTAASTSTO (8ETSH: 121)

Detecting linker: length = 60

TOSTSTSSPSSASGotteAOOOTOOATASTSAOOTOOOOOASSOASABSAaATSATO / 2EO I): 122)

- 53 011415

Target # 8 (MS8G (E02975)

Anchor: length = 25 (ЗСССССС6С (ЛТАТаСАТСТСТГСа (8РёУ: 124)

Target; lengths.

SASTASAASSASSASSTSSSSSTSSAASSSSSSAAAASTTSSTOTOSASSSSAOA

TTATSASS (8E () GO: 125)

Linker: Length = 50

TTSSTbTbSAASSSabATTATSASSbATASTSASSTSSAASASATOSATAAASS

S (ZSSSSSSSS (8E () GO: 126)

Protective fragment: length = 75

OSTSATAAATSTBSSTSTSSASASHAZAASTPSSSSSOSTGOSASSYUSAOTSSTSS

POTAOOTOSSPSSTAAOOTSTS (ZVr GO: 127)

Detecting linker: length-60 TOSTSTSSPSSASTbTTGOOAOOSTOSATASTSAOOTSASTASAASSAZHASTOS SSTSSAA (ZEOU: 128)

Attenuation Factor: Length = 25

SASTASAAOASASSASTSSSSSTSSAA (5Εζ) GO: 129)

Target#? 68Τ-ΡΙ1 X06547

Anchor: length = 25

OTGAOOSATASSTOTSASSASASASSOO (8Е0 Yus 130)

Target: Length = 60

SASHKhKAOOSSAASASSTTSATTOTBOSADASSASATSTSSTTSbSTSASTASA

ASST6ST0 (^ GO: 131)

Linker: Length = 60

STSSPSSSSTOASTASMSSTSSTSYUA ^^^

TOSTAAS (8YER: 132)

Protective fragment: length = 75.

SAOOS6OPOTADTSA6S (1AA (YuAOATSTOSTSTSSSSASAATSA6STSTT (}

SSTSSSTSOSTTSTSTMOTSTS (8E () GO: 133)

Detecting linker: length = 60 t “dstsssssASt <ttoo ^^

ΑΤΤΟΤΟϋ (3ΕςΐΏ: 134)

Attenuation Factor: Length-25

SAOSDAOOOOAAOASSTGSATTbTOO (5ES> GO: 135)

- 54 011415

Target # 10 ΗΜ <3 · 17 (the same as target # 10 in example 27)

Target # 11 Cycle ofel in X52851

Anchor: length = 25

ATSSA0TTAAASSASAT6STASTASS (ZER GO: 136)

Target: dpina = 60 6

Linker: Length = 60

TAATOK: ATSTSUTSZSSAAOOTSSATSSATASTSAOOTS <ETASTAOOSAT <7GOO'GG AASTSSAT (F) GO: 138)

Protective fragment; length-75

SATSSASGGOSSASSASSAOOTSSA'P'ATSSSOTOTOAabTSASSASSSSSTSASAS

ATAAASSSOSGTSTSTAAOOTS (8Е <2 GO: 139)

Detecting linker: dpina = 60

TOSTSTSSTTSASTStSSASbTOSATASTBAOOTOBOTTGATOTSTSAOaOT SUTSAST (3Εζ> ΙΒ; 140)

Attenuation factor: length = 25 (ZbKLTTATOTSTSTSAOobbTSOTSAST (5Е <2 GO: 141)

Target # 12 b-thromboglobulin (same as target # 12 in example 27)

Target # 131GON X02152

Anchor: length = 25

TSTSOSTSTOSAASSSSSSSSOSAAST (ZER GO: 142)

Target: length = 60 bOToETTOAOAOOTOSTATATOAASOTOATSAAASTAAAOOSGASASATSSTOS SSGATGSS (WEU> GO: 143)

Linker; length = 60

AASYUSTASA (> ТССТ6ОССТА1Т (ЗС0АТАССАеТА0ТГ0СС0СССОТГССА

SAOOSOASA (8ERGO: 144)

Protective fragment: length = 75

SSAATAOOSSSASYATSTSTAOSSPTbAOOTTTSATSASSTSATAAASSASTSTS AASSASSASS5STTOTSTAASTSTS (8E (2GO: 145)

Detecting linker: dpina = 60

TSSGSTSSTTSASTSTP'SSASHZTOOATASTASAOTStSgTbSTGSASASTOSTTA TSASOTO (8EOGO: 146)

Attenuation Factor: dpina = 25

OOTOSGGOAOA6TSSGGAT <ZAOOTO (8ert: 147)

Miyen # 14TMR-1 X03124

- 55 011415

Anchor: length = 25

SATTASOAOOTSSATTSOSATSAAAOO (ZER GO: 148)

Target: Length = 60

SASSAAOASSTASASTASTTTAOSTOASASSAATOSASASTSTTGSSSTOTTTAT

SSATSSS (ZERGO: 149)

Linker: dpina = 60

SAOTAGGTSSSTOTTTATSSATSSSOATASTSAOOTSSGGOATbSaAATbSAST

STAAT6 (5ES) GO: 150)

Protective fragment: length = 75

OOSATSOATAAAAAOOOOAAASASTOTSSATGSSGSASASSASAASABTSTAOO

TSTTOOTbOSTNETSTAASSTO (ZEST GO: 151)

Detecting linker: length = 60

TOSTSTSSTTSASTOTTTASAOOSTOOATASTOAOOTSASSAAOASSTASASTOT

TSSSTOT (8ERGO: 152)

Attenuation Factor: Length = 25

SASSAAOASSTASASTETTOOTOST (ZER GO: 153)

Target # 15 MMR-9 GO5070

Anchor: dpina * 25

ATSATSTAAASTTSTTSTSHOTSSSTOSS (ZER GO: 154)

Target: long = 60

OSAASSTOAASATSTTSOASSSSATSSSSSSAOATTOOAAASASZSTSTATGG (ZTTSAASO (ZERSh: 155)

Linker: Length = 60

OS <ZAASSA <ZSTOTATTTSTSTSGAA6SSATASTSA6TASSASS (} ASSOAAOAST TASATSAT (ZER GO: 156)

Protective fragment: length = 75

SSTTSAASAAATASAOOSGSOTTSSSAATSTSSSSOATOOC6TSOAAOATOTT

SASOTGOSSSTTOTSTAAOOTSTS (5EP GO; 157)

Detecting linker: dpina = 60

TSSTSTSSTTSASTSTTTOSASSTOBATASTSAOTOSAASBTSAASATSGGSb

ASSSSAT (ZER GO: 158)

Attenuation factor; length = 25 aАААСТААААТАТТССССАССАТ (ЗРП): 159)

Target # 16 Actin M10277

Anchor: dpina = 25

STSAOOTSSTSSSSGSZSSGASSTOOS (ZER GO: 160)

Target: Length = 60

OAOTSSTOTAssATSSASAAAASTASSTTSAASTSSATSATOAASTSTbASO

TOOASATS (ЗВрГО: 161)

Linker: dlia = 60

SATSATSAAOOTBTAASSTOOASATSSATASTOAOOTOSSASOTAOOSSASSOA 6SASTS AO (ZEO P): 162)

The protective fragment of dpin = 75 satotssasotsasaspattsatsatooootstsotaota gtsotssatsssa SAOSASSTOSGTOTSTAAOOTSTO (5EP ΙΟ: 163)

Detecting linker: length = 60

TOSTSTSSTTSASTOGGTSYUOSTOSATASTSAATOAASP-SSTSTSEOSATSSAS OAAASTA (8E () U: 164) Attenuation factor: length = 25

OAOTSSTOTOOSATSSASOAAASTA (ZERSh; 165)

In FIG. 33 shows that the reproducibility of the assay is high, providing% SU in the range of about 3 to 13% when 30,000 cells are analyzed.

In FIG. 34 shows that the sensitivity of the assay is high, for example, the RNA target for ΟΑΡΌΗ can be detected when the RNA comes from a sample obtained from 1000 analyzed cells or less.

In FIG. 35 presents an analysis mainly carried out according to the protocol of Example 25 and using the aggregates and targets of Example 27, in which it was shown that test mRNA targets can be detected even when RNA comes from a sample obtained from 1000 cells or less, and all targets , even expressed in low concentration, can be detected when RNA is derived from 1000 cells or less.

Example 31. The choice of oligonucleotide reagents (see Fig. 36)

In FIG. 36 schematically illustrates several types of oligonucleotide reagents that can be used in the methods of the invention. The figure shows the analysis scheme in which oligonucleotide anchors are attached to the surface either through their 5'- or their 3'-ends ("inventor

- 56 011415 “), and in which the oligonucleotides have two recognition sites that are either adjacent to each other (“ shortened ”) or separated by nucleic acid spacers. Each square represents 5 nucleotides.

Example 32. Methods of amplification of a nuclease-protective fragment (see Figs. 37-39 and 42)

In FIG. 37 shows amplification of a nuclease-protective fragment by PCR.

In FIG. 38 shows the amplification of a nuclease-protective fragment using ligase. When choosing a 'in the form of a sequence of 12 bases out of 25 bases that bind to the aggregate in ligated a'a, one can choose hybridization conditions under which it becomes possible to bind only a ligated sequence of 25 bases of a'a molecules. Recognition can be improved by using modified nucleotide (s) in each region of the linker-binding region of sequences a 'and a. If dissociation cycles destroy the ligase under the influence of heat, it can be added to the cycle again.

In FIG. 39 shows the amplification of a nuclease-protective fragment using nuclease-defense. A strand (DNA) complementary to the (nuclease-protective fragment b) may include modified bases that hybridize at a lower temperature than (RNA), or (RNA) can be destroyed before the addition of (DNA). Similarly, (linker a) for (DNA) may contain modified nucleotides that hybridize at a lower temperature than (a protective fragment from nucleases b) even if the hybrid (linker a) / (DNA) contains 25 bases, and (DNA) / (a protective fragment from nucleases b) is a hybrid of 50 base pairs, especially taking into account the experimental data that the DNA chains in solution are diluted and pass through a high, mostly uniform concentration (linker a). The flow device can be replaced with a tablet containing a grip assembly. A linear population can be replaced by a 2-Ό or 3-Ό population.

In FIG. 42 shows the amplification of a nuclease-protective fragment by polymerase. After completion of the reaction to protect against nucleases and dissociation of the nuclease-protective fragment from RNA, add a primer (a '), which contains a double-stranded promoter for RNA polymerase (e.g., T7 polymerase) and a primer for building up a fragment that is protective against nucleases (e.g., using reverse transcriptase (JT) for RNA or DNA replication, or Tad polymerase for DNA replication) so that after binding of the nuclease-protective fragment (for example, JT or Tad polymerase) it will be used as a matrix for images a double-stranded DNA complex with a double-stranded promoter located at the end of the sequence of the nuclease-protective fragment. Addition of a ligase will ligate the second promoter chain with a chain of a nuclease-protective fragment, with the exception of the first base, which is an erroneously paired δΝΡ and, therefore, during the reaction of protection against nucleases, δ1 will cut an unprotected base. The ligase will not ligate the promoter with the nuclease-protective fragment due to the excised base. In the case of ligation, the L chain is converted to an extended L chain including the polymerase promoter, and amplification can be continued using the polymerase, and continue until the JT primer b 'is added. The promoter / extended end of the nuclease-protective fragment is used to bind to the population or a detection linker or detection probe. To detect δΝΡ, this hybridization is carried out in such a way that the δΝΡ site is located approximately in the middle of the sequence used for hybridization with the population (a detecting linker or a detecting probe, etc.). The totality (detecting linker or detecting probe) of the hybridization region of the extended nuclease protective probe does not include all bases at the end (some of the bases at the end of the extended nuclease protective probe may protrude without a sequence complementary to hybridization with the linker, etc.). In variations that are not shown, there is no need for ligation before using a polymerase (for example, T7), and therefore, δΝΡ detection is carried out by choosing a sequence positioning δΝΡ in the middle of the sequence hybridized with a 'and using δΝΡ detection as described above . There is no need to grow the a 'chain, but instead, an RNA polymerase can use a promoter hybridized with a single-chain nuclease-protective fragment (skipping the indicated extension using JT or an alternative stage of extension using Tad polymerase) to obtain RNA.

From the above description, specialists in this field will obviously understand the main features of the present invention and, without departing from its essence and scope, it is possible to make changes and modifications of the invention to adapt it for various applications and conditions.

Without further development, it is believed that those skilled in the art will be able to make full use of the present invention using the foregoing description. Therefore, the foregoing preferred specific embodiments are provided for the purpose of illustration only and in no way limit the invention.

The full disclosure of all uses, patents and publications cited above and presented in the figures, incorporated herein by reference.

Claims (15)

CLAIM 1. A solution for cell lysis or permeability, containing from about 8 to about 60% (v / v) formamide, from about 0.01 to about 0.5% (v / v) sodium dodecyl sulfate (EBU ) and 0.5-6X EEC as a buffer. 2. The solution according to claim 1, containing from about 10 to about 30% (vol./about.) Formamide. 3. The solution according to claim 1, containing from about 0.03 to about 0.10% (vol./about.) EBU. 4. The solution according to claim 1, containing approximately 2X-4X EEC. 5. The solution according to claim 2, containing from about 0.03 to about 0.10% (vol./about.) EBU. 6. The solution according to claim 5, containing approximately 2X-4X EEC. 7. The solution according to claim 1, containing 3X EEC. 8. The solution according to claim 6, containing 3X EEC. 9. A method for detecting at least one nucleic acid target, comprising: a) ensuring contact of the sample, which may include the specified target (s) with a fragment (s) protecting from nucleases (s), which (s) are specific (s) to the target (s), and binds (s) to the specified target (s); b) exposing the sample to a nuclease effective to cleave a single-stranded nucleic acid and to ensure that the resulting sample is in contact with a device that, before adding the specified sample, has a surface including many spatially separated regions, at least two of which are essentially the same, where each the region includes at least two different loci of oligonucleotide anchors, where each anchor is associated with a bifunctional linker, which has a first olig specific site a nucleotide anchor, and a second region that includes a probe specific to the indicated nuclease protecting fragment (s), under conditions effective to bind said protecting fragment (s) to said device, the anchors, located in at least one locus, are associated with about 2-4 different bifunctional linkers with different specificities, and where the specified sample is exposed to a solution according to any one of claims 1 to 8. 10. A method for detecting at least one nucleic acid target, comprising: a) ensuring contact of the sample, which may include the specified (s) target (s) with a fragment (s) protecting from nucleases (s), which (s) are specific (s) to the target (s) and binds (s) to the specified target (yami); b) exposing the sample to a nuclease effective to cleave a single-stranded nucleic acid and to ensure that the resulting sample is in contact with a device that, before adding said sample, has a surface including many spatially separated regions, at least two of which are essentially the same, where each the region includes at least two different loci of oligonucleotide anchors, where each anchor is associated with a bifunctional linker, which has a first olig specific site a nucleotide anchor, and a second portion that includes a probe specific to the indicated nuclease protecting fragment (s), under conditions effective to bind said protecting fragment (s) to said device, wherein said the sample is exposed to a solution according to any one of claims 1 to 8. 11. A method for detecting at least one nucleic acid target, comprising a) ensuring contacting of the sample, which may include the specified target (s) with the fragment (s) protecting from nucleases (s), which (e) is specific (s) to the target (s) and binds (s) to the specified target (yami); b) exposing the sample to a nuclease effective to cleave a single stranded nucleic acid, and C) ensuring contact of the obtained sample with a surface having a probe specific to the specified fragment (s) protecting from nucleases (s), under conditions effective to bind said protecting fragment (s) to said device, the specified sample is exposed to the solution according to claim 1. 12. A method for detecting at least one nucleic acid target, comprising: a) ensuring contact of the sample, which may include the specified target (s) with a fragment (s) protecting from nucleases (s), which (s) are specific (s) to the target (s), and binds to (s) target (s); b) exposing the sample to a nuclease effective to cleave a single-stranded nucleic acid and to ensure that the resulting sample is in contact with a device that, before adding the specified sample, has a surface including many spatially separated regions, at least two of which are essentially the same, where each the region includes at least two different loci of oligonucleotide anchors, where each anchor is associated with a bifunctional - 58 011415 by a single linker, which has a first region specific for the oligonucleotide anchor and a second region which includes a probe specific for the nucleus protecting fragment (s), under conditions effective to bind said protecting ( their) fragment (s) with the specified device, where the specified sample is exposed to a solution according to any one of claims 1 to 8 at a temperature of about 105 ° C or lower, in which essentially only mRNA, but not DNA, is released in the form, in which she binds to the specified protective (s) f fragment (s), and / or is exposed to the specified solution at a temperature of about 105 ° C or higher, in which both mRNA and DNA are released in the form in which they bind to the specified protective (s) fragment (s). 13. The method according to item 12, where the anchors located in at least one locus are associated with about 2-4 different bifunctional linkers with different specificities. 14. A method for detecting at least one nucleic acid target, comprising: a) ensuring contact of the sample, which may include the specified target (s) with a fragment (s) protecting from nucleases (s), which (s) are specific (s) to the target (s), and binds (s) to the specified target (s); b) exposing the sample to a nuclease effective to cleave a single-stranded nucleic acid and to ensure contact of the obtained sample with a surface, including a probe specific for the fragment (s) protecting against nucleases, under conditions effective to bind said protecting ( their) fragment (s) with the specified surface, where the specified sample is exposed to a solution according to any one of claims 1 to 8 at a temperature of about 105 ° C or lower, in which essentially only mRNA, but not DNA, is released in the form, in which it binds to the specified protecting (s) fragment (s), and / or is exposed to the specified solution at a temperature of about 105 ° C or higher, in which both mRNA and DNA are released in the form in which they bind to the specified protecting ( mi) fragment (s). 15. The method according to any one of claims 12-14, wherein a solution of said sample containing a solution according to any one of claims 1 to 8 is diluted so that said solution does not inhibit the functions of said nuclease with respect to cleavage of a single stranded nucleic acid.