JP2005315844A - Substrate, and manufacturing method therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To inspect mistakes in an order and a direction of a container with a selective coupling substance stored alignedly, in a short time, and to use a substrate after the inspection. <P>SOLUTION: A confirmation probe for confirming the arrangement of the selective coupling substance is arranged on the substrate, in the substrate immobilized with the selective coupling substance. A dedicated reagent is not required thereby. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a DNA chip substrate designed to prevent changes in product components due to misplacement.

  Recent advances in analysis and analysis technology have made it possible to identify macromolecules in many biological species. In particular, for humans, information on all genes, all proteins encoded by them, and various in vivo macromolecules such as secondary sugar chains and lipids are rapidly accumulated. Although it was possible to proceed with identification and functional analysis of these in vivo macromolecules from conventional technologies, biochip technologies represented by DNA chips can simultaneously detect much more types of interactions than conventional methods. There is a big feature in the point.

  Analytical methods or methodologies such as the DNA chip method or DNA microarray method, and the macroarray method can be performed on a solid surface made of silicon, surface-modified glass, polypropylene, activated polyacrylamide, or the like called an array or chip. Diagnosis of genetic diseases and cancer, detection of mutants, and development of new drugs by interacting labeled samples derived from in vivo RNAs called targets with a substrate on which selective binding substances consisting of all kinds of known DNAs are placed It is a technology or methodology used as a sensor for microorganisms and environmental pollution.

  As a method of arranging various kinds of selective binding substances on the substrate at high density, the selective binding substances are stored in a container divided into 96 to 384 small sections, and are picked up and arranged on the substrate in a certain order. Or there is a method of synthesis on a substrate. However, all the selective binding substances have the same color and odor and cannot be distinguished visually, so it is difficult to detect a mistake in the order and orientation of the containers.

  As a method for confirming the arrangement state of the selective binding substance, a method of detecting by a laser scanner that a salt contained in a DNA probe solution deposited on a glass surface of Brown et al. Although this method can be inspected immediately after placement, the salt is washed away after the next immobilization treatment, and there is a problem that confirmation after production cannot be performed. Further, since salt precipitation occurs with respect to all the selective binding substances on the substrate, it is not possible to detect a mistake in the order and orientation of the containers only by salt precipitation.

  As another method, there is a method of staining DNA on a glass surface with a compound having a high affinity for single-strand DNA described in Non-Patent Document 1. This compound is SYBRGreen II, which is a fluorescent substance, and DNA on the substrate after staining is detected by a laser scanner. Although there is an advantage that it can be confirmed after production as compared with the method of Brown et al., A complicated staining operation is required, and the residual fluorescent substance may affect the interaction. Further, since a fluorescence signal is detected for all the selective binding substances on the substrate, it is impossible to detect a mistake in the order and orientation of the containers only with the fluorescence signal.

  In addition, there is a quality control method in which a DNA solution containing a first fluorescent substance excited / released at a specific wavelength described in Patent Document 1 is placed on a substrate, and fluorescence emitted from the placed DNA spot is detected. Although this method has an advantage that there is no staining operation, the point that the fluorescent substance is mixed with all the selective binding substances arranged on the substrate is the same as in Non-Patent Document 1, and the total selective binding substance on the substrate is Since a fluorescent signal is detected, it is impossible to detect a mistake in the order and orientation of the containers only with the fluorescent signal.

  Further, there is a method of specifying a DNA chip by arranging a dye on a substrate described in Patent Document 2, and converting the position information of the arranged dye into index information. Although this method uses the same dye as the position information on the substrate, it can distinguish individual substrates, but it cannot detect mistakes in the order and orientation of containers in which selective binding substances are arranged. .

In order to confirm that containers with selective binding substances arranged and stored at present are used in the correct order and orientation, it is necessary to interact with selective binding substances and to clarify the degree of such substances, for example, There is a method in which a target sample synthesized and labeled from a clear RNA interacts with a selective binding substance on a substrate, and the result is evaluated. In this method, the result varies depending on the quality of RNA used, the test procedure, the execution environment, etc., and the test requires a dedicated reagent, and it takes time to obtain the result. In addition, there are disadvantages that the inspected substrates cannot be reused and that all the substrates cannot be inspected.
Battaglia C, Salani G, Consolandi C, Bernardi LR, De Bellis G, Analysis of・ Dynenue microarrays by non-destructive fluorescent staging using SYBR green II, “Biotechniques” 2000 Year 29, p. 78-81 Japanese Patent Laid-Open No. 15-279576 JP-A-12-338110

  The present invention solves the problems of such conventional products, and can inspect in a short time the order and orientation of containers in which selective binding substances are aligned and stored. Is intended to be provided in a usable state.

  The process for confirming the alignment and storage state of the selective binding substance in the container from the molecular weight or the gene sequence in the case of DNA, and the type, order and orientation of the container can be uniquely determined from the optical characteristics. Providing the substrate produced from the step of storing a probe and the step of aligning the selective binding material and the verification probe on the substrate.

That is, the present invention has the following configuration.
(1) A substrate on which a selective binding substance is immobilized, wherein a confirmation probe for confirming the arrangement of the selective binding substance is disposed on the substrate.
(2) The confirmation probe is disposed at a specific location on the substrate that is adjacent to the selective binding substance but does not overlap with the arrangement of the selective binding substance. Board.
(3) The confirmation probe is adjacent to the selective binding substance, but is arranged at a specific location on the substrate that does not overlap with the arrangement of the selective binding substance, and the position information of the confirmation probe is The substrate according to (1), which shows an arrangement state of the selective binding substance.
(4) The confirmation probe is adjacent to the selective binding substance, but is arranged at a specific location on the substrate that does not overlap with the arrangement of the selective binding substance, and the position information of the confirmation probe is The substrate according to (1), wherein the substrate has a configuration of a selective binding substance.
(5) The method for manufacturing a substrate according to (1), wherein the selective binding substance and the confirmation probe are aligned and stored in a container, the aligned storage state in the container is confirmed, A method of manufacturing a substrate comprising the step of aligning and arranging the selective binding substance and the confirmation probe at a predetermined position on the substrate by using containers in a predetermined order and orientation.
(6) In the method according to (5), the container is used in the correct order and orientation based on the step of detecting the positional relationship of the confirmation probe and the positional relationship of the confirmation probe on the substrate. And a step of detecting the substrate.

  The confirmation by the conventional method is invasive to the manufactured DNA chip, and the confirmation makes the DNA chip unusable. Therefore, all the manufactured substrates could not be confirmed, and there was no choice but to check them. However, this detection operation is non-invasive to the selective binding substance and can be 100% tested.

  The effect of the invention according to claim 1 is that the dedicated reagent is used because the optical property of the confirmation probe is used to confirm the arrangement state of the selective binding substance and the confirmation probe on the substrate. This is a substrate that can be reused because it is unnecessary, has a short inspection time, and is non-invasive to the selective binding substance.

  The effect of the invention described in claim 2 is that the confirmation probe is arranged at a specific location on the substrate that is adjacent to the selective binding substance but does not overlap with the arrangement of the selective binding substance. That is, the substrate does not affect the interaction between the active substance and the target sample.

  The effect of the invention of claim 3 is that the selective binding substance and the confirmation probe have a specific positional relationship in the arrangement of the selective binding substance and the confirmation probe on the substrate. The substrate can detect a difference in arrangement of the selective binding substances due to a difference in order or orientation of the containers as a difference in arrangement of the confirmation probes.

  The effect of the invention described in claim 4 is that the confirmation probe is arranged at a position on the substrate where the container can be uniquely determined when there are many kinds of the container. This is a substrate that can detect the misunderstanding as the misplacement of the confirmation probes.

  The effect of the invention described in claim 5 is that, in the substrate manufacturing method of the type in which the selective binding substance is picked up from the container in which the selective binding substance is aligned and stored and arranged on the substrate in a specific order, the selective binding substance and The substrate having the effects described in claims 1, 2, and 3 can be manufactured by manufacturing the substrate using the container in which the alignment storage state of the confirmation probe is confirmed.

  The effect of the invention described in claim 6 is that the substrate is manufactured by the manufacturing method described in claim 5 so that the difference in arrangement of the selective binding substance on the substrate can be determined from the arrangement state of the confirmation probe. Since the substrate that can be inspected and used for the inspection can be reused, it is a manufacturing control method that can inspect all the manufactured substrates.

  In the present invention, the “substrate” refers to a support on which a selective binding substance can be arranged on the surface, for example, a plate shape, a film shape, a cylindrical shape, or a thread shape, for example, glass, Or nylon, or nitrocellulose, or polystyrene, or polymethyl methacrylate, or polyethyl methacrylate, or polypropyl methacrylate, or polycarbonate, or various polymer materials such as polypropylene, or platinum, aluminum, or magnesium oxide Made of metals and alloys. At this time, the portion where the substrate surface and the selective binding substance are in contact with each other may be subjected to physical treatment for strengthening the binding of the selective binding substance to the substrate surface or stabilizing the binding. For example, coating, eg etching, eg masking, eg molding. Further, at this time, the portion where the selective binding substance is in contact with the substrate surface may be subjected to chemical treatment for strengthening or stabilizing the binding of the selective binding substance to the substrate surface. Well, for example, a treatment such as functional group application, for example, polar application, for example, functional catalyst application. Furthermore, at this time, the biological contact treatment for strengthening the binding of the selective binding substance to the substrate surface or stabilizing the binding is applied to the portion where the selective binding substance contacts the substrate surface. For example, it refers to the application of virus particles, for example, the application of microorganisms, such as the application of in vivo macromolecules such as transcription factors, ribosome molecules, albumin proteins, and the like.

  In the present invention, the “selective binding substance” refers to a substance that specifically interacts with a macromolecular compound in vivo, and indicates a nucleic acid, protein, sugar chain, lipid, or low molecular weight compound. At this time, the portion where the substrate surface and the selective binding substance are in contact with each other may be subjected to chemical treatment for strengthening or stabilizing the binding of the selective binding substance to the substrate surface. For example, it refers to a treatment such as functional group application, for example, polar application, for example, functional catalyst application. Furthermore, at this time, the biological contact treatment for strengthening the binding of the selective binding substance to the substrate surface or stabilizing the binding is applied to the portion where the selective binding substance contacts the substrate surface. In other words, it refers to a treatment in which a selective binding substance is localized, for example, a virus particle, for example, a microorganism such as a eukaryotic cell is fixed to a substrate surface. In this case, “interaction” means that two or more substances are specifically bound by the structure or property of each other. This “bond” is achieved by physicochemical bonding between all molecules such as covalent bonds, ionic bonds, hydrophobic bonds, hydrogen bonds, metal bonds, etc., even if the recognized partner substance is in the contaminant. But it is done specifically.

  In the present invention, the “target sample” refers to a biopolymer or a substance obtained by changing a biopolymer to be detected to a detectable state. In this case, “in vivo polymer” refers to a substance synthesized in a cell composed of, for example, a nucleic acid, such as a protein, such as a sugar chain, such as a lipid. A detectable state is a state in which a selective binding substance and a target sample interact with each other, and each target sample has at least one optical property of fluorescence, luminescence, absorption, polarization, color development, and radiation dose. In a state that can be specifically detected even in contaminants, for example, RNA molecules present in cells by reverse transcription using reverse transcriptase, dATP, dCTP, dGTP, dTTP and Biotin-dATP, biotin is obtained by a reverse transcription reaction using a reverse transcriptase and an RNA polymerase from a target having cDNA as a constituent molecule, which is a complementary sequence of an RNA molecule composed of Cy3-dCTP, such as an RNA molecule present in a cell. -Details composed of dCTP, biotin-dGTP, biotin-dTTP Refers to a target that the cRNA molecule complementary sequences of RNA molecules of the inner and constituent molecules.

    In the present invention, the “confirmation probe” has at least one optical property of fluorescence, luminescence, absorption, polarization, color development, and radiation dose, a selective binding substance, a target sample, and a selective binding property. A substance designed so that the substance and the target sample do not interact with the interacted substance, for example Cy3, eg Cy5, eg Alexa Fluor 546, eg Alexa Fluor647, eg R-Physyrin Streptavidin (SAPE), eg green fluorescent protein (GFP), eg gold colloids, eg latex, eg Coomassie Brilliant Blue (CBB), eg high molecular compounds such as [γ-32P] -dCTP, eg Cy3, eg Cy5, eg A exa Fluor 546, such as Alexa Fluor 647, such as R-Phycoerythrin Streptavidin (SAPE), such as green fluorescent protein (GFP), such as gold colloid, such as latex, such as Coomassie Brilliant Blue (CBB), such as [γ-32P] -dCTP It refers to an in vivo polymer bonded to at least one polymer compound. At this time, the portion where the substrate surface is in contact with the confirmation probe may be subjected to a chemical treatment for strengthening the binding of the selective binding substance to the substrate surface or stabilizing the binding, For example, it refers to a treatment such as functional group application, for example, polar application, for example, functional catalyst application. Further, at this time, even if the portion where the substrate surface and the confirmation probe are in contact with each other is subjected to biological treatment for strengthening the binding of the selective binding substance to the substrate surface or stabilizing the binding. For example, it refers to a treatment such as immobilizing an organism in which a selective binding substance is localized in a virus particle, for example, a microorganism, such as a eukaryotic cell, to the substrate surface.

    In the present invention, the action of “confirming the arrangement of the selective binding substance” means that when each substance constituting the selective binding substance is placed on the substrate, each of the selective binding substances is After each of the selective binding substances stored in the container is disposed on the substrate, each of the selective binding substances in which the selective binding substance has been stored, Refers to uniquely associating each component of the arranged selective binding substance and has traceability between the container in which the selective binding substance is stored and the selective binding substance arranged It is characterized by.

    For example, 96 different selective binding substances are not mixed in each accommodating part of a 96-well microtiter braid having accommodating parts of 8 columns and 12 rows and whose row number and column number are uniquely determined. Each selected binding substance is picked up in the ascending order of 1 column, 1 row, 1 column, 2 rows, 1 column, 3 rows, 1 column, 12 rows, then 2 columns, 1 row, and 1 row. When the direction increases to 12 columns, the column numbers are picked up in ascending order, the column numbers picked up in the column direction are increased, and the selective binding substances are picked up again from the smallest row number in the row direction in ascending order. In the case where the selective binding substances picked up in the picking time are arranged on the surface of the selective binding substance in an arrangement state of 96 rows and 1 column, 95 kinds of the selective binding substances are respectively P1, P2 to P95 and P Select the binding substance, its right side Each of the components of the selective binding substance and the selective binding substance arranged in 96 columns in one column on the substrate, wherein the added numbers are the numbers that uniquely associate the 95 kinds of the selective binding substances. Is uniquely associated with each component of the selective binding substance stored in the container in which is stored.

    In the present invention, “a specific location on the substrate that is adjacent to the selective binding substance but does not overlap with the arrangement of the selective binding substance” means each of the selective binding substance and the confirmation probe. When the substance is disposed on the substrate, the selective binding substance and the confirmation probe do not mix with each other on the substrate, and each component is not the selective binding substance or the This means that the probe can be confirmed as a confirmation probe.

    For example, each of the 96-well microtiter braids having 8 columns and 12 rows of containing sites and having the row number and the column number uniquely determined includes 95 different selective binding substances and one type of The confirmation probes are stored without being mixed, and the confirmation probes are stored in one column and one row, and each of the selective binding substances or confirmation probes is stored once in one column. If the column direction increases from the first column to the second row, the first column to the third row, and the first column to the third row, then the second column to the first row and the row direction to 12 columns, the column direction increases to the first column. The column number picked up is increased, and the selective binding substance is picked up again in ascending order in the row direction from the smallest row number, and the selective binding substance picked up in each picking time is put on the surface of the selective binding substance. 1 row, 96 rows In this case, the selective binding substances and the confirmation probes arranged in one column and 96 rows on the substrate are arranged in an independently distinguishable manner, and the confirmation probes are arranged. Is in a state where the selected binding probe is not located.

    In the present invention, “the selective binding substance and the confirmation probe are aligned and stored in a container” means that the selective binding substance and each of the substances constituting the confirmation probe are each of the selective binding substances. And each confirmation probe are not mixed with each other, and each component is in a state where it can be distinguished whether it is the selective binding substance or the component of the confirmation probe. Point to.

    For example, each of the 96-well microtiter braids having 8 columns and 12 rows of containing sites and having the row number and the column number uniquely determined includes 95 different selective binding substances and one type of When storing the confirmation probe, each of the selective binding substance and the confirmation probe is uniquely accommodated in each accommodation site, and each of the selective binding substance and the confirmation probe is stored in each of the accommodation parts. The state which can be matched by recording the order uniquely accommodated in the accommodating part is pointed out.

    In the present invention, “detecting the positional relationship of the confirmation probe” means that the selective binding substance and each of the substances constituting the confirmation probe are arranged on the substrate. After the substance and each confirmation probe are placed on the substrate from the container in which each of the selective binding substance and the confirmation probe is stored, the fluorescence, light emission, absorption, polarization, color development that the confirmation probe has , Refers to confirming where the confirmation probe is arranged on the substrate by detecting at least one optical characteristic in the radiation dose.

    For example, each of the 96-well microtiter braids having 8 columns and 12 rows of containing sites and having the row number and the column number uniquely determined includes 95 different selective binding substances and one type of The confirmation probes are stored without mixing, the confirmation probes are stored in one column and one row, and 95 types of the selective binding substances are stored in the remaining 95 portions. Each of the selective binding substances or one time is picked up in ascending order of 1 column, 1 row, 1 column, 2 rows, 1 column, 3 rows, 1 column, 12 rows, then 2 columns, 1 row, and the row direction. When the number increases to 12 columns, it is picked up in the column direction in ascending order, the column number picked up in the column direction is increased, and the selective binding substance is picked up again in ascending order from the smallest row number in the row direction. The selected results taken up in When the verification substance and the confirmation probe are arranged on the base surface in the arrangement state of one row and 96 rows, the selective binding substance of the first row and 96 rows in which the confirmation probe is arranged on the substrate, and the This means that it is possible to uniquely determine which of the confirmation probe arrangement states is the confirmation probe.

    In the present invention, “the positional information of the confirmation probe indicates the arrangement state of the selective binding substance” means that the confirmation probe is mounted on the substrate by detecting the positional relationship on the substrate. Further, it means that the arrangement of the selective binding substance and the confirmation probe on the substrate can be determined.

    For example, each of the 96-well microtiter braids having 8 columns and 12 rows of containing sites and having the row number and the column number uniquely determined includes 95 different selective binding substances and one type of The confirmation probes are stored without mixing, the confirmation probes are stored in one column and one row, and 95 types of the selective binding substances are stored in the remaining 95 portions. Each of the selective binding substances or one time is picked up in ascending order of 1 column, 1 row, 1 column, 2 rows, 1 column, 3 rows, 1 column, 12 rows, then 2 columns, 1 row, and the row direction. When the number increases to 12 columns, it is picked up in the column direction in ascending order, the column number picked up in the column direction is increased, and the selective binding substance is picked up again in ascending order from the smallest row number in the row direction. The selected results taken up in In the case where the luminescent substance and the confirmation probe are arranged on the substrate surface in an arrangement state of one column and 96 rows, the confirmation probe is M, and 95 types of the selective binding substances are selectively bonded to the selective binding substances. P1, P2-P95 and P are selective binding substances in the order of picking up from the container in which the chemical substances are stored, respectively, and the numbers added on the right side are numbers that uniquely associate the 95 types of the selective binding substances. In this case, at least one optical characteristic of fluorescence, light emission, absorption, polarization, color development, and radiation dose included in the confirmation probe is detected, and the confirmation probe is arranged in one row and 96 columns on the substrate. By determining that the selective binding substance is mounted at the position of 1 row 1 column in the arrangement state of 1 row 1 column 1 in the positional relationship from 1 row 1 column to 1 row 96 column To 1 row 96 columns are the selective binding substances And the selective binding substances from P1 to P95 uniquely determine each selective binding substance in the order in which the numbers appended to the right side of P are arranged in ascending order. It means that it is possible to determine that it is mounted at a position from the column to 1 row and 96 columns.

    In the present invention, “the positional information of the confirmation probe indicates the configuration of the selective binding substance” means that the confirmation probe is mounted on the substrate by detecting the positional relationship on the substrate. It means that the combination of each component of the selective binding substance and the confirmation probe can be determined.

    For example, each of the 96-well microtiter braids having 8 columns and 12 rows of containing sites and having the row number and the column number uniquely determined includes 95 different selective binding substances and one type of The confirmation probe is stored without intermingling, and there are a plurality of types of containers in which the selective binding substance and the confirmation probe are stored in this way, and uniquely with a specific “A1” There is a container in which the selective binding substance to be expressed and the confirmation probe are stored, A1 stores the confirmation probe in a site of one column and one row, and 95 types of the selective binding substances are left There is a container in which the selective binding substance uniquely expressed as “A2” and the probe for confirmation are stored in the state where the part is stored in 95 parts. The confirmation probe is stored In a state where 95 types of the selective binding substances are stored in the remaining 95 sites, each of the selective binding substances or each time, 1 column 1 row to 1 column 2 rows, 1 column 3 rows If the column direction increases to 12 columns, then 2 columns and 1 row, then the column number increases in the column direction, the column number picked up in the column direction increases, and the row minimum again The selective binding substances are picked up in ascending order in the row direction from the row numbers of the lines, and the selective binding substances and the confirmation probes picked up at each pick-up time are arranged in a state of arrangement of 96 rows in one column on the base surface. In this case, the 95 kinds of the selective binding substances stored in “A1” are picked up in the order of picking up the selective binding substances from the container in which the selective binding substances are stored, respectively, P1, P2 to P95 and P Selective binding substance, added on the right side When the number is expressed as a number that uniquely associates 95 kinds of the selective binding substances, at least one of the fluorescence, emission, absorption, polarization, color development, and radiation dose of the confirmation probe is provided. By detecting the characteristics and determining that the confirmation probe is mounted at the position of 1 row and 1 column of the array state of 1 row and 96 columns on the substrate, the selective binding substance and the The selective binding substance in which the type of the container in which the confirmation probe is stored is uniquely expressed as “A2” and the selection in which the confirmation probe is uniquely expressed as “A1” instead of the container in which the confirmation probe is stored. It means that it can be determined that the container contains the binding substance and the confirmation probe.

    In the present invention, “detecting that the containers have been used in the correct order and orientation” means that the selective binding substance and the confirmation probe are detected by detecting the positional relationship of the confirmation probe on the substrate. The selective binding substance stored in the container storing the gas, the confirmation probe, the correspondence between the selective binding substance mounted on the substrate and the confirmation probe can be uniquely determined, and the selective binding It means that it can be determined that the container storing the sex substance and the confirmation probe has been used with a unique direction.

    For example, each of the 96-well microtiter braids having 8 columns and 12 rows of containing sites and having the row number and the column number uniquely determined includes 95 different selective binding substances and one type of The confirmation probes are stored without mixing, the confirmation probes are stored in one column and one row, and 95 types of the selective binding substances are stored in the remaining 95 portions. Each of the selective binding substances or one time is picked up in ascending order of 1 column, 1 row, 1 column, 2 rows, 1 column, 3 rows, 1 column, 12 rows, then 2 columns, 1 row, and the row direction. When the number increases to 12 columns, it is picked up in the column direction in ascending order, the column number picked up in the column direction is increased, and the selective binding substance is picked up again in ascending order from the smallest row number in the row direction. The selected results taken up in The selective binding substance or the confirmation probe picked up at the first floor in the first row in the arrangement state of the first row of 96 substances and the confirmation probe on the substrate surface in the first row and the first row in the second floor in the selection state In the case where the binding substances or the confirmation probes are mounted in one column by increasing the row number in ascending order up to 1 row and 96 rows in the order picked up in 1 row and 2 columns, 95 types of the selective binding materials are selected. In order of picking up the binding substances from the container in which the selective binding substances are stored, P1, P2-P95 and P are the selective binding substances, respectively, and the numbers attached to the right side are the 95 types of the selective binding substances. Is expressed by a numerically associated number, the detection probe detects at least one optical characteristic of fluorescence, luminescence, absorption, polarization, color development, and radiation dose, and the confirmation probe 1 row on the board 9 By determining that it is mounted at the position of 1 row and 1 column in the array state of the column, the confirmation probe is stored in the site of 1 column and 1 row, and 95 types of the selective binding substances remain. The container stored in 95 sites of the above is picked up on the confirmation probe in the first row and in the first column, and the second time, the selective binding substance of P1, and then from P2 to P95 is added to the right side of P. It means that it is determined that the numbers have been picked up to P95 in an increasing order.

    In the present invention, “the selective binding substance is immobilized on the substrate, and the confirmation probe for confirming the arrangement of the selective binding substance is disposed on the substrate. The method for realizing “the substrate to be performed” has, for example, 8 columns and 12 rows of accommodating parts, and row numbers A, B, C, D, E, F, G, and H, and 1, 2, 3 respectively. 4,5,6,7,8,9,10,11,12 column numbers are added to uniquely identify each accommodation site, and each accommodation site of a 96-well microtiter plate (cornig) On the other hand, a confirmation probe made of Cy3-dUTP (GE Healthcare) is accommodated as a confirmation probe in the section where the accommodation part number in one column and one row is A1, and Human Genome for the remaining 95 sections. Sample Oligo Se Each selective binding substance consisting of a solution adjusted to 40 μM with Solution-I (Takara Bio) in which all oligo DNAs except for the A1 section of Version 2.0 (Operon) were diluted 4-fold with ultrapure water was used in different sections. When using the GMS417 Alayer (Affymetrix), a 96-well microtiter plate A1 section is placed on the left side when viewed from the front of the GMS417 Alayer on a substrate made of Hubble slide glass (Takara Bio). Of the four vertices of the Hubble slide glass, the rectangular notch part is placed in the back left as viewed from the front of the GMS417 Alayer, for confirmation that it is housed in a 96-well microtiter blade The probe and each selective binding substance When mounted in a positional relationship of 8 columns and 12 rows above, the confirmation probe is mounted so that 1 row from the left end of the Hubble slide glass is located 5 cm from the left end of the GBS 417 arrayer, and 1 row and 1 column is located. The selective binding substances stored in 8 rows and 12 columns excluding the first row and the first column are arranged in a square lattice form at intervals of 0.5 mm at the remaining positions, that is, 96 holes As column numbers uniquely determined by the microtiter plate, 1 column 1 row to 1 column 12 rows are A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns. 1 row to 2 columns 12 rows are B1, B3, B5, B7, B9, B11, B2, B4, B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7 , C9, C11, C2, C4, C6, C8, C10, C12, 4 columns 1 row to 4 columns 12 rows are D1, D3, D5, D7, D9, D11, D2, D4, D6, D8, D10, D12. 5 columns 1 row to 5 columns 12 rows are E1, E3, E5, E7, E9, E11, E2, E4, E6, E8, E10, E12, 6 columns 1 row to 6 columns 12 rows are F1, F3, F5 , F7, F9, F11, F2, F4, F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10. , G12, 8 columns 1 row to 8 columns 12 rows are loaded with confirmation probes and selective binding substances corresponding to H1, H3, H5, H7, H9, H11, H2, H4, H6, H8, H10, H12. Refers to the foundation that is.

  In the present invention, the above-mentioned (1), wherein the confirmation probe is arranged at a specific location on the substrate that is adjacent to the selective binding substance but does not overlap with the arrangement of the selective binding substance. For example, a method for realizing the “substrate described in FIG. 1” has a receiving portion of 8 columns and 12 rows, and the row numbers A, B, C, D, E, F, G, H, and 1, 2, respectively. , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 for each receiving part of the 96-well microtiter braid to which each receiving part is uniquely determined. Thus, a confirmation probe made of Cy3-dUTP is accommodated as a confirmation probe in the section whose accommodation part number is A1 in one column and one row, and Human Genome Sample Oligo Set Version 2.0 is A1 for the remaining 95 sections. of When each selective binding substance consisting of a solution adjusted to 40 μM with Solution-I obtained by diluting all oligo DNAs 4 times with ultrapure water was stored in different compartments, onto a substrate made of a Hubble glass slide. Using the GMS417 Alayer, the 96-hole microtiter plate's A1 section is located in the back left as viewed from the front of the GMS417 Alayer. Of the four vertices of the Hubble glass slide, the rectangular corners are notched. With the GMS417 arrayer placed in the back left as viewed from the front, the confirmation probe and each selective binding substance contained in the 96-well microtiter blade are placed in a positional relationship of 8 columns and 12 rows on the Hubble slide glass. When installed, the confirmation probe is GMS417 array. 1mm from the left end of the Hubble slide glass when viewed from the front of the front of the jar, so that 1 row and 1 column are located on the 1st row and 1st column, the selective combination stored in 8 rows and 12 columns excluding the 1st row and 1st column As a section number uniquely determined by a 96-well microtiter blade, that is, a selective binding substance is arranged in a square lattice at intervals of 0.5 mm in the remaining positions, that is, from column 1 row 1 to 1 Column 12 rows are A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns 1 row to 2 columns 12 rows are B1, B3, B5, B7, B9, B11, B2, B4, B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7, C9, C11, C2, C4, C6, C8, C10, C12, 4 columns 1 row 4 columns and 12 rows from D 1, D3, D5, D7, D9, D11, D2, D4, D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 rows are E1, E3, E5, E7, E9, E11, E2, E4 E6, E8, E10, E12, 6 columns 1 row to 6 columns 12 rows are F1, F3, F5, F7, F9, F11, F2, F4, F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 The rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10, G12, 8 columns 1 row to 8 columns 12 rows are H1, H3, H5, H7, H9, H11, H2, Confirmation probes corresponding to H4, H6, H8, H10, and H12, and a substrate on which a selective binding substance is mounted, and a confirmation probe in which a GMS417 arrayer is accommodated in a 96-well microtiter plate and each selection Binding material Are mounted in a positional relationship of 8 columns and 12 rows on the Hubble slide glass, the occupied area on the circular Hubble slide glass formed by the probe for confirmation and each selective binding substance on the Hubble slide glass is as follows: Arrangement of 8 columns and 12 rows without touching the area occupied on the circular Hubble slide glass formed by the other selective probe mounted on the slide glass and each selective binding substance on the Hubble slide glass There is a base that is mounted with.

  In the present invention, “the confirmation probe is in the arrangement state described in the above (2), and the positional information of the confirmation probe indicates the arrangement state of the selective binding substance. As a method for realizing the “described substrate”, for example, there are 8 columns and 12 rows of accommodating parts, and the row numbers A, B, C, D, E, F, G, and H are 1, 2, For each accommodation site of the 96-well microtiter braid, in which the column numbers 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 are added and each accommodation site is uniquely determined A confirmation probe made of Cy3-dUTP is accommodated as a confirmation probe in a section where the accommodation site number in one column and one row is A1, and for the remaining 95 sections, A Gen of Human Genome Sample Oligo Set Version 2.0 When each selective binding substance consisting of a solution adjusted to 40 μM with Solution-I obtained by diluting all oligo DNAs 4 times with ultrapure water was stored in different compartments, onto a substrate made of a Hubble glass slide. Using the GMS417 Alayer, the 96-hole microtiter plate's A1 section is located in the back left as viewed from the front of the GMS417 Alayer. Of the four vertices of the Hubble glass slide, the rectangular corners are notched. With the GMS417 arrayer placed in the back left as viewed from the front, the confirmation probe and each selective binding substance contained in the 96-well microtiter blade are placed in a positional relationship of 8 columns and 12 rows on the Hubble slide glass. When installed, the confirmation probe is GMS417 array. 1mm from the left end of the Hubble slide glass when viewed from the front of the front of the jar, so that 1 row and 1 column are located on the 1st row and 1st column, the selective combination stored in 8 rows and 12 columns excluding the 1st row and 1st column As a section number uniquely determined by a 96-well microtiter blade, that is, a selective binding substance is arranged in a square lattice at intervals of 0.5 mm in the remaining positions, that is, from column 1 row 1 to 1 Column 12 rows are A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns 1 row to 2 columns 12 rows are B1, B3, B5, B7, B9, B11, B2, B4, B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7, C9, C11, C2, C4, C6, C8, C10, C12, 4 columns 1 row 4 columns and 12 rows from D 1, D3, D5, D7, D9, D11, D2, D4, D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 rows are E1, E3, E5, E7, E9, E11, E2, E4 E6, E8, E10, E12, 6 columns 1 row to 6 columns 12 rows are F1, F3, F5, F7, F9, F11, F2, F4, F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 The rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10, G12, 8 columns 1 row to 8 columns 12 rows are H1, H3, H5, H7, H9, H11, H2, Confirmation probes corresponding to H4, H6, H8, H10, and H12, and a substrate on which a selective binding substance is mounted, and a confirmation probe in which a GMS417 arrayer is accommodated in a 96-well microtiter plate and each selection Binding material Are mounted in a positional relationship of 8 columns and 12 rows on the Hubble slide glass, the occupied area on the circular Hubble slide glass formed by the probe for confirmation and each selective binding substance on the Hubble slide glass is as follows: Arrangement of 8 columns and 12 rows without touching the area occupied on the circular Hubble slide glass formed by the other selective probe mounted on the slide glass and each selective binding substance on the Hubble slide glass On a Hubble glass slide on which a probe for confirmation contained in a 96-well microtiter plate and each selective binding substance are mounted using a GMS417 arrayer for 18 hours. DN after holding at room temperature Using a probe laser scanner GenePix 4000B (Axon Instruments), a laser output of 100%, a photomultiplier voltage setting of 500 and a confirmation probe contained in a 96-well microtiter plate and each selective binding substance The GBS417-mounted Hubble slide glass with GenePix4000B seen from the front front and the right apex of the Hubble slide glass has a notch part with a rectangular corner and a 96-hole microtiter The probe for confirmation contained in the braid and the surface on which each selective binding substance is mounted are set downward with the gravitational direction downward, and the Hubble slide glass is configured to detect the excitation light of Cy3. When the excitation data of Cy3 can be confirmed on the lower left side with the longitudinal direction as the longitudinal axis, the 96-well microtiter plate's A1 section is placed in the back left as viewed from the front of the GMS417 layerer. Of the four vertices of the Hubble glass slide, the rectangular notch is located in the back left when viewed from the front of the GMS417 layerer. Probe and each selective binding substance are mounted on the Hubble slide glass in a positional relationship of 8 columns and 12 rows on the Hubble slide glass and determined by the GMS417 arrayer, that is, unique to the 96-well microtiter plate. As a partition number to be determined 1 column 12 rows are A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns 1 row to 2 columns 12 rows are B1, B3, B5, B7, B9, B11 , B2, B4, B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7, C9, C11, C2, C4, C6, C8, C10, C12, 4 columns 1 4 columns and 12 rows are D1, D3, D5, D7, D9, D11, D2, D4, D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 rows are E1, E3, E5, E7, E9 , E11, E2, E4, E6, E8, E10, E12, 6 columns 1 row to 6 columns 12 rows are F1, F3, F5, F7, F9, F11, F2, F4, F6, F8, F10, F12, 7 Column 1 row to 7 columns 12 rows are G1, G3, G5, G7, G9, G11, G2. , G4, G6, G8, G10, G12, 8 columns and 1 row to 8 columns and 12 rows are H1, H3, H5, H7, H9, H11, H2, H4, H6, H8, H10, H12 on the Hubble slide glass. There is a base that can be determined to be installed.

    In the present invention, “the confirmation probe is in the arrangement state described in the above (2), and the positional information of the confirmation probe indicates the configuration of the selective binding substance,” described in the above (1) As a method for realizing the “substrate”, for example, there are 8 columns and 12 rows of accommodating portions, and the row numbers A, B, C, D, E, F, G, and H are respectively 1, 2, 3 4,5,6,7,8,9,10,11,12 column numbers are added to uniquely identify each accommodation site for each accommodation site of the 96-well microtiter braid. A confirmation probe made of Cy3-dUTP is accommodated as a confirmation probe in a section whose accommodation part number is A1 in one column and one row, and for the remaining 95 sections, a section of A1 of Human Genome Sample Oligo Set Version 2.0 When each selective binding substance consisting of a solution adjusted to 40 μM with Solution-I diluted 4-fold with ultrapure water was stored in different compartments, onto a substrate made of a Hubble slide glass, Using the GMS417 array, the A1 section of the 96-well microtiter blade is placed in the left back as viewed from the front of the GMS417 arrayer, and the notch part with the rectangular corners of the four vertices of the Hubble slide glass is the GMS417. The probe for confirmation contained in the 96-well microtiter blade and each selective binding substance are mounted in a positional relationship of 8 columns and 12 rows on a Hubble slide glass in a state of being arranged in the back left as viewed from the front side of the arrayer. The base that was made was Chip1, and the accommodating part of 8 columns and 12 rows And row numbers A, B, C, D, E, F, G, and H and column numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 respectively. Cy3-dUTP is used as a confirmation probe for each storage site of the 96-hole microtiter braid that is appended and uniquely determined for each storage site in which the storage site number in one column and one row is H12. Solution-I was prepared by diluting all oligo DNAs except the A1 section of Mouse Genome Sample Oligo Set Version 2.0 (Operon) 4 times with ultrapure water for the remaining 95 sections. When each selective binding substance consisting of a solution adjusted to 40 μM is housed in different compartments, a GMS417 array is placed on a substrate made of a Hubble slide glass. Using the ear, the A1 section of the 96-well microtiter blade is placed in the back left as viewed from the front of the GMS417 arrayer, and the notch part with the rectangular corners of the four vertices of the Hubble glass slide is the GMS417 arrayer. The probe for confirmation accommodated in the 96-well microtiter blade and each selective binding substance are mounted in a positional relationship of 8 columns and 12 rows on a Hubble slide glass in a state of being arranged in the back left as viewed from the front. When the base is Chip2, the substance stored in A1 of the 96-well microtiter plate is 1 cm from the left end of the Hubble slide glass as viewed from the front side of the GMS417 arrayer, so that 1 row and 1 column are located. It is mounted in 8 rows and 12 columns excluding the first row and the first column. As the section number uniquely determined by the 96-well microtiter blade, that is, the selective binding substance is placed in a square lattice at 0.5 mm intervals in the remaining positions. Column 1, row 1, column 12, row A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, column 1, row 2, column 2, row B1, B3, B5, B7 , B9, B11, B2, B4, B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7, C9, C11, C2, C4, C6, C8, C10, C12. 4 columns 1 row to 4 columns 12 rows are D1, D3, D5, D7, D9, D11, D2, D4, D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 rows are E1, E3, E5 , E7, E9, E11, E2, E4 6, E8, E10, E12, 6 columns 1 row to 6 columns 12 rows are F1, F3, F5, F7, F9, F11, F2, F4, F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 The rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10, G12, 8 columns 1 row to 8 columns 12 rows are H1, H3, H5, H7, H9, H11, H2, Confirmation probes corresponding to H4, H6, H8, H10, and H12, and a substrate on which a selective binding substance is mounted, and a confirmation probe in which a GMS417 arrayer is accommodated in a 96-well microtiter plate and each selection When the binding substance is mounted in a positional relationship of 8 columns and 12 rows on the Hubble slide glass, the confirmation probe and each selective binding substance are placed on the Hubble slide glass. The area occupied on the circular Hubble slide formed is another circular probe that is formed on the Hubble slide by another confirmation probe mounted on the Hubble slide and each selective binding substance. A confirmation probe and each selective binding substance contained in a 96-well microtiter blade, which are mounted on a substrate having an array of 8 columns and 12 rows, without contacting the occupied area on the glass, are GMS417. A glass slide mounted on an arrayer is held on a glass slide for 18 hours under light-shielding conditions and at room temperature, and then a DNA scanner laser scanner GenePix4000B is used to set the laser output to 100% and the photomultiplier voltage to 500. Hole microtiter The Hubble slide glass on which the probe for confirmation contained in the Blate and each selective binding substance is mounted using the GMS417 arrayer is viewed from the front side of the GenePix4000B. There is a notch portion with a corner, and the surface on which the probe for confirmation accommodated in the 96-well microtiter blade and each selective binding substance is mounted is set downward with the direction of gravity as downward, When the image data obtained when measuring a Hubble slide glass with a setting capable of detecting the excitation light of Cy3 can confirm the excitation light of Cy3 on the lower left side with the long axis direction vertical, the A1 section of the 96-well microtiter plate Placed in the back left as viewed from the front of GMS417 Of the four vertices of the Hubble slide glass, the rectangular notch part is placed in the back left as viewed from the front of the GMS417 Alayer, and is housed in a 96-hole microtiter blade, which is Chip1. The probe for confirmation and each selective binding substance are mounted on the Hubble slide glass in a positional relationship of 8 columns and 12 rows on the Hubble slide glass and determined by the GMS417 arrayer, that is, a 96-well microtiter As partition numbers uniquely determined by Brate, 1 column 1 row to 1 column 12 rows are A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns 1 row 2 columns 12 rows from B1, B3, B5, B7, B9, B11, B2, B4, B6, B 8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7, C11, C2, C4, C6, C8, C10, C12, 4 columns 1 row to 4 columns 12 rows D1, D3, D5, D7, D9, D11, D2, D4, D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 rows are E1, E3, E5, E7, E9, E11, E2, E4 E6, E8, E10, E12, 6 columns 1 row to 6 columns 12 rows are F1, F3, F5, F7, F9, F11, F2, F4, F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 The rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10, G12, 8 columns 1 row to 8 columns 12 rows are H1, H3, H5, H7, H9, H11, H2, H4, H6, H8, H10, H12 as Hubble slide glass It is mounting, and the like that this check probe and selective binding substance is a foundation that dictated be derived from a substance which is stored in 96-well microtiter plates Chip1.

    In the present invention, “the method for manufacturing the substrate according to the above (1), wherein the selective binding substance and the confirmation probe are aligned and stored in a container, and the aligned storage state in the container is confirmed. And the step of aligning and arranging the selective binding substance and the confirmation probe at a predetermined position on the substrate by using the containers in a predetermined order and orientation. As a method for realizing the “substrate manufacturing method”, for example, there are 8 columns and 12 rows of storage parts, and A, B, C, D, E, F, G, and H are respectively assigned row numbers 1 and 2. , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 for each receiving part of the 96-well microtiter braid to which each receiving part is uniquely determined. Cy as a probe for confirmation in a section where the storage site number in one column and one row is A1 -A confirmation probe consisting of dUTP is accommodated, and the remaining 95 compartments are Solution-I obtained by diluting all oligo DNAs except for the A1 section of Human Genome Sample Oligo Set Version 2.0 with ultrapure water 4 times. When each selective binding substance consisting of a solution adjusted to 40 μM is accommodated in different compartments, the A1 compartment of a 96-well microtiter plate is placed in front of the GMS417 array on the substrate made of a Hubble glass slide using a GMS417 arrayer. A 96-well microtiter with the rectangular corners of the four vertices of the Hubble glass slide located at the back left as viewed from the front of the GMS417 Alayer. Contained in the Blate The material stored in A1 of the 96-well microtiter plate on the base on which the confirmation probe and each selective binding substance are mounted in a positional relationship of 8 columns and 12 rows on the Hubble glass slide are viewed from the front side of the GMS417 layerer. It is mounted so that the position 1 cm from the left end of the Hubble glass slide is 5 mm from the left so that 1 row and 1 column are located, and the selective binding substances stored in 8 rows and 12 columns excluding the first row and the first column are the remaining positions. The selective binding substances are arranged in the form of a square lattice at intervals of 0.5 mm, that is, as the partition numbers uniquely determined by the 96-well microtiter blade, 1 column 1 row to 1 column 12 rows are A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns 1 row to 2 columns 12 rows are B1, B 3, B5, B7, B9, B11, B2, B4, B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7, C9, C11, C2, C4, C6 C8, C10, C12, 4 columns 1 row to 4 columns 12 rows are D1, D3, D5, D7, D9, D11, D2, D4, D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 rows E1, E3, E5, E7, E9, E11, E2, E4, E6, E8, E10, E12, 6 columns 1 row to 6 columns 12 rows are F1, F3, F5, F7, F9, F11, F2, F4 F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10, G12, 8 columns 1 row to 8 columns 12 The rows are H1, H3, H5, H7, H9, H11, H2, H4, H6, H8, 10. Confirmation probe corresponding to H12 and selective binding substance are mounted on the base, and GMS417 arrayer is accommodated in a 96-well microtiter plate. Each selective binding substance is a Hubble slide. When mounted in a positional relationship of 8 columns and 12 rows on the glass, the occupied area on the circular Hubble slide glass formed by the probe for confirmation and each selective binding substance on the Hubble slide glass is the same as that on the Hubble slide glass. Another array of confirmation probes mounted on the and each selective binding substance has an array of 8 columns and 12 rows without touching the area occupied by the circular hub slide formed on the hub slide. It is a mounted base and 96 holes This is a laser scanner for DNA chip after holding the probe for confirmation contained in the icrotator blade and each selective binding substance on a Hubble slide glass equipped with GMS417 arrayer for 18 hours under light-shielding condition and room temperature condition. A Hubble slide glass on which a probe for confirmation and each selective binding substance contained in a 96-well microtiter blade are mounted using a GMS417 layerer with a GenePix 4000B laser output of 100%, a photomultiplier voltage setting of 500, and a selective binding substance. The GenePix4000B is seen from the front, and the right corner has a notch with a rectangular corner out of the four vertices of the Hubble glass slide, and is housed in a 96-well microtiter blade. Image data obtained when the surface on which the probe for confirmation and each selective binding substance are mounted is set downward with the gravitational direction downward, and the Hubble slide glass is measured with a setting capable of detecting the excitation light of Cy3 When the excitation light of Cy3 can be confirmed on the lower left side with the longitudinal direction as the vertical axis, the A1 section of the 96-hole microtiter blade is placed in the back left as viewed from the front of the GMS417 layerer, and the four apexes of the Hubble slide glass Among the above, the confirmation probe and the respective selective binding substances accommodated in the 96-well microtiter blade in a state in which the notch portion having a rectangular corner is arranged in the left rear as viewed from the front side of the GMS417 arrayer On a Hubble glass slide with a positional relationship of 8 columns and 12 rows and a GMS417 array As a section number uniquely determined by the 96-hole microtiter blade, 1 column 1 row to 1 column 12 rows are A1, A3, A5. A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns 1 row to 2 columns 12 rows are B1, B3, B5, B7, B9, B11, B2, B4, B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7, C9, C11, C2, C4, C6, C8, C10, C12, 4 columns 1 row to 4 columns 12 rows are D1, D3 D5, D7, D9, D11, D2, D4, D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 rows are E1, E3, E5, E7, E9, E11, E2, E4, E6, E8, E10, E12, 6 rows 1 6 columns and 12 rows are F1, F3, F5, F7, F9, F11, F2, F4, F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10, G12, 8 columns 1 row to 8 columns 12 rows are H1, H3, H5, H7, H9, H11, H2, H4, H6, H8, H10, H12, Hubble In producing a substrate that can be determined to be derived from a 96-well microtiter plate mounted on a slide glass, it has 8 columns and 12 rows of storage parts, and each of A, B, C, D, The row numbers E, F, G, and H and the column numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 are added to uniquely determine each accommodation site. Each of the 96-well microtiter blades attached With respect to the content area, a confirmation probe made of Cy3-dUTP is accommodated as a confirmation probe in a section where the accommodation part number in one column and one row is A1, and Human Genome Sample Oligo Set Version 2 for the remaining 95 sections. The selective binding properties so that each selective binding substance consisting of a solution adjusted to 40 μM with Solution-I obtained by diluting all oligo DNAs except for 0.0 A1 compartment with ultrapure water 4 times is contained in different compartments. A step of aligning and storing the substance and the probe for confirmation in a container; and having a receiving portion of 8 columns and 12 rows, and row numbers A, B, C, D, E, F, G, and H, respectively , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 column numbers of 96-well microtiter plates are uniquely assigned to each receiving site. For each housing part, a confirmation probe made of Cy3-dUTP is accommodated as a confirmation probe in a section where the accommodation part number in one column and one row is A1, and for the remaining 95 sections, Human Genome Sample Oligo The selection so that each selective binding substance consisting of a solution adjusted to 40 μM with Solution-I obtained by diluting all oligo DNAs except for the A1 section of Set Version 2.0 with ultrapure water 4 times is contained in different sections. In the container, the binding substance and the confirmation probe are aligned and stored in the container, and only the section A1 visually confirms the color that is one of the optical properties of Cy3 of the confirmation probe. A step of confirming the aligned storage state, and a row number of A, B, C, D, E, F, G, and H, each having an accommodating portion of 8 columns and 12 rows; Accommodation of each of 96-well microtiter plates in which 1,2,3,4,5,6,7,8,9,10,11,12 column numbers are added to uniquely determine each accommodation site For the part, a confirmation probe made of Cy3-dUTP is accommodated as a confirmation probe in a section where the accommodation part number in one column and one row is A1, and for the remaining 95 sections, Human Genome Sample Oligo Set Version2. This selective binding substance which contains each selective binding substance which consists of the solution adjusted to 40 micromol with Solution-I which diluted all oligo DNAs 4 times with ultrapure water except the section of 0 A1 in a different division, respectively That the confirmation probe is aligned and stored in the container, only the section A1 is one of the optical characteristics of the confirmation probe Cy3. A 96-well microtiter plate in which a confirmation probe and a selective binding substance in which the alignment storage state in the container is confirmed so as to visually confirm a certain color is stored on a substrate made of a Hubble glass slide, GMS417 arrayer The A1 section of the 96-hole microtiter blade is placed in the back left as viewed from the front of the GMS417 arrayer, and the four corners of the Hubble glass slide are notched with rectangular corners on the front of the GMS417 arrayer. When the confirmation probe and each selective binding substance housed in a 96-well microtiter blade are mounted in a positional relationship of 8 columns and 12 rows on the substrate in a state of being arranged at the left rear as viewed from the front , The probe for confirmation is Hubbles as seen from the front of GMS417 1 mm from the left end of the id glass is mounted so that 1 row and 1 column is located, and the selective binding substance stored in 8 rows and 12 columns excluding the first row and first column is 0 in the remaining positions. The selective binding substances are arranged in a square lattice at intervals of 5 mm, that is, as the partition numbers uniquely determined by the 96-well microtiter plate, 1 column 1 row to 1 column 12 rows are A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns 1 row to 2 columns 12 rows are B1, B3, B5, B7, B9, B11, B2, B4, B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7, C9, C11, C2, C4, C6, C8, C10, C12, 4 columns 1 row to 4 columns 12 rows are D1, D3, D5, D7, D9, D11 D2, D4, D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 rows are E1, E3, E5, E7, E9, E11, E2, E4, E6, E8, E10, E12, 6 columns 1 row 6 columns and 12 rows are F1, F3, F5, F7, F9, F11, F2, F4, F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10, G12, 8 columns 1 row to 8 columns 12 rows correspond to H1, H3, H5, H7, H9, H11, H2, H4, H6, H8, H10, H12. By using the container for producing a base on which the confirmation probe and the selective binding substance are mounted in a predetermined order and orientation, the selective binding substance and the confirmation probe are predetermined on the substrate. The base including the step of aligning at a position of It may be a manufacturing method.

In the present invention, the step of detecting the positional relationship of the confirmation probe on the substrate manufactured by the method described in (5) above and the positional relationship of the verification probe on the substrate In order to realize the “manufacturing management method of the substrate including the step of detecting that the substrates are used in the correct order and orientation”, for example, each of A, B, C, and D has an accommodating portion of 8 columns and 12 rows. , E, F, G, and H and column numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 are added to uniquely identify each receiving portion. For each of the 96-hole microtiter braids to be determined, a confirmation probe made of Cy3-dUTP is accommodated as a confirmation probe in a section where the accommodation part number in one column and one row is A1, and the remaining 95 For the parcels, Human Geno Each selective binding substance consisting of a solution adjusted to 40 μM with Solution-I in which all oligo DNAs except the A1 section of the me Sample Oligo Set Version 2.0 were diluted 4-fold with ultrapure water was accommodated in different sections. A step of aligning and storing the selective binding substance and the probe for confirmation in a container, and 8 rows and 12 rows of receiving parts, and rows A, B, C, D, E, F, G, and H, respectively. Each of the 96-well microtiter plates is numbered and numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 to uniquely identify each receiving site. For the remaining 95 sections, a confirmation probe made of Cy3-dUTP is accommodated as a confirmation probe in the section where the accommodation section number in one column and one row is A1, and for the remaining 95 sections, Human Ge Each selective binding substance consisting of a solution adjusted to 40 μM with Solution-I obtained by diluting all oligo DNAs with ultrapure water 4-fold diluted with ultrapure water except for the A1 section of nome Sample Oligo Set Version 2.0 is accommodated in different sections. The selective binding substance and the confirmation probe are aligned and stored in the container, and only the section A1 confirms the color that is one of the optical characteristics of Cy3 of the confirmation probe by visual observation. A substrate made of a Hubble slide glass using a 96-well microtiter plate in which the positional relationship of the confirmation probe is confirmed from the stage of confirming the alignment storage state in the container and the confirmation probe and the selective binding substance are stored. Up, using the GMS417 arrayer, the A1 section of the 96-well microtiter plate is GMS In the state where the part of the notch that has a rectangular corner out of the four vertices of the Hubble slide glass is arranged in the back left side when viewed from the front side of the GMS 417 layer, The probe for confirmation accommodated in the 96-well microtiter plate and each selective binding substance are stored in the A1 of the 96-well microtiter plate on the base plate on which 8 columns and 12 rows are mounted on the Hubble glass slide. The substance is mounted so that the position 1 cm from the left end of the Hubble slide glass as viewed from the front side of the GMS 417 arrayer is located 1 row and 1 column, and is stored in 8 rows and 12 columns excluding the first row and the first column. The selective binding substances are arranged in a square lattice at 0.5 mm intervals at the remaining positions. As a section number uniquely determined by the 96-hole microtiter blade, A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns 1 row to 2 columns 12 rows are B1, B3, B5, B7, B9, B11, B2, B4, B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows C1, C3, C5, C7, C9, C11, C2, C4, C6, C8, C10, C12, 4 columns 1 row to 4 columns 12 rows are D1, D3, D5, D7, D9, D11, D2, D4 D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 rows are E1, E3, E5, E7, E9, E11, E2, E4, E6, E8, E10, E12, 6 columns 1 row to 6 columns 12 The rows are F1, F3, F5, F7, F9, F11, F 2, F4, F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10, G12, 8 columns 1 row 8 columns and 12 rows are bases on which confirmation probes and selective binding substances corresponding to H1, H3, H5, H7, H9, H11, H2, H4, H6, H8, H10, H12 are mounted, and When the confirmation probe and each selective binding substance accommodated in the 96-well microtiter blade are mounted in a positional relationship of 8 rows and 12 rows on the Hubble glass slide, the confirmation probe and each selective binding Occupied area on the circular glass slide formed by the active substance on the glass slide, Mounted with an array of 8 columns and 12 rows without touching the area occupied on the circular Hubble slide formed by the other confirmation probes and other selective binding substances formed on the Hubble slide. On a Hubble glass slide equipped with a GMS417 arrayer and a confirmation probe housed in a 96-well microtiter blade and each selective binding substance using a GMS417 layerer under light-shielding conditions and room temperature conditions After holding, using the GenePix 4000B which is a laser scanner for DNA chip, the laser output is 100%, the voltage setting of the photomultiplier is 500, and the confirmation probe and each selective binding substance contained in the 96-well microtiter blade GMS417 Arrayer Using the Hubble slide glass mounted on it, the GenePix 4000B is seen from the front front, and the right apex of the Hubble slide glass has a notch part with a rectangular corner, and is housed in a 96-well microtiter blade. Image obtained when measuring the Hubble glass slide with a setting capable of detecting the excitation light of Cy3 with the surface on which the probe for confirmation and each selective binding substance are mounted set downward with the direction of gravity facing downward By confirming that the excitation light of Cy3 is detected on the lower left side with the longitudinal direction of the long axis, the A1 section of the 96-well microtiter plate is placed in the back left as viewed from the front of the GMS417 arrayer, and the Hubble slide Of the four vertices of the glass, the knot that has a rectangular corner In the state in which the part of H is arranged in the back left as viewed from the front side of the GMS417 arrayer, the confirmation probe and each selective binding substance accommodated in the 96-well microtiter blade are placed in 8 rows and 12 rows on the Hubble slide glass. As a section number uniquely determined by the 96-hole microtiter blade, it is mounted on the Hubble slide glass in the arrangement state determined by the GMS417 arrayer. A1, A3, A5, A7, A9, A11, A2, A4, A6, A8, A10, A12, 2 columns 1 row to 2 columns 12 rows are B1, B3, B5, B7, B9, B11, B2, B4 B6, B8, B10, B12, 3 columns 1 row to 3 columns 12 rows are C1, C3, C5, C7, C9, C11, C2, C4 C6, C8, C10, C12, 4 columns 1 row to 4 columns 12 rows are D1, D3, D5, D7, D9, D11, D2, D4, D6, D8, D10, D12, 5 columns 1 row to 5 columns 12 The rows are E1, E3, E5, E7, E9, E11, E2, E4, E6, E8, E10, E12, 6 columns 1 row to 6 columns 12 rows are F1, F3, F5, F7, F9, F11, F2, F4, F6, F8, F10, F12, 7 columns 1 row to 7 columns 12 rows are G1, G3, G5, G7, G9, G11, G2, G4, G6, G8, G10, G12, 8 columns 1 row to 8 The substrate of the confirmation probe for confirming that the column 12 rows are mounted on the Hubble slide glass as H1, H3, H5, H7, H9, H11, H2, H4, H6, H8, H10, H12. Correct the container from the position above Order, there is a production control method to include the step of detecting that used in orientation.

  Hereinafter, a DNA chip will be taken up as a more specific example of an embodiment of the present invention. However, the present invention is not limited to this. The reagents used in the examples were Nacalai Tesque products unless otherwise specified.

  The selective binding substance was mouse kidney-derived cDNA. First, a mouse kidney-derived cDNA library clone was prepared in order to prepare mouse kidney-derived cDNA. That is, total RNA was extracted from mouse kidney using TRIzol test (Invitrogen), and polyadenine-added RNA present in this total RNA was purified using mRNA Purification Kit (GE Healthcare GE Healthcare). The purification procedure followed the manual attached to this kit. Furthermore, this polyadenine-added RNA was converted into a mouse kidney-derived cDNA library using a commercially available kit, SuperScript plasmid system for cDNA synthesis and plasmid cloning (Invitrogen), and introduced into E. coli XL2-Blue strain (STRATAGENE). A library clone was obtained.

  Next, 1,000 E. coli colonies were transplanted from the mouse kidney-derived cDNA library clone described above into 150 μL of CIRCLEGROW medium (BIO 101) together with 1/500 amount of M13KO7 helper phage (GIBCO BRL). And cultured for 16 hours in a 37 ° C. incubator. The cultured cells were centrifuged using a centrifuge to precipitate the cells and collect the culture supernatant. This culture supernatant contains M13KO7 helper phage, which retains mouse cDNA-derived single-stranded DNA generated from the aforementioned mouse kidney-derived cDNA library clone.

  Next, the aforementioned mouse kidney-derived single-stranded DNA was amplified as mouse kidney-derived cDNA by a PCR reaction using a primer DNA complementary to a vector sequence linked to mouse kidney-derived cDNA. That is, 1 μL of the above culture supernatant, 3 μL of 10 × Z-Taq buffer (Takara Bio), and 2.5 μL of 2.5 mM dNTP, and 0.5 μL of 10 pmol / μL M13 forward primer (sequence; 5′-GTTTTCCCAGTCACGACGGT ) (SEQ ID NO: 10), and 0.5 [mu] L of 10 pmol / [mu] L M13 reverse primer (SEQ ID NO: 5'-AGCGGAATAACAATTTCACAC) (SEQ ID NO: 11), and 0.25 [mu] L TaKaRaZ-Taq DNA polymerase (Takara Bio), and 22 25 μL of sterile distilled water was mixed in a 0.2 mL microtube and the thermal cycler GeneAmp PCR System 9700 (Applied Biosystem). The PCR reaction was performed in a PCR reaction using a total of 35 cycles, with one cycle consisting of 3 steps of 96 ° C for 30 seconds, 48 ° C for 30 seconds, and 72 ° C for 4 minutes. Mouse cDNA was obtained.

  Next, the amplified mouse kidney-derived cDNA was purified by the glass bead method. That is, the amplified mouse cDNA was adsorbed onto glass beads by suspending the entire PCR reaction solution after amplification and 150 μL of 6M sodium iodide solution in SILICA beads (SIGMA) activated by hydrochloric acid treatment. Next, this suspension was filtered on MultiScreen-GV (MILLIPORE), and 100 μL of a solution containing 20 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, and 50% ethanol was added to perform filtration. Further, 25 μL of TE Buffer Solution was added to carry out the filtrate, and the final filtrate was collected to obtain a purified amplified mouse kidney-derived cDNA.

  The sequence and significance of the purified amplified mouse kidney cDNA was then determined by cycle sequencing reactions and the BLAST algorithm. That is, using Big Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems), 4 μL of purified amplified mouse kidney-derived cDNA was reacted according to the manual attached to the kit, and this reaction was performed on the website of MILLIPOREt: Purified according to the “Die Terminator and Sequencing Reaction Cleanup Using Multiscreen 96 Well Plate” method in www.millipore.com/nihon\analytical\jppubdbase.nsf/docs/tn053JA.html). The base sequence of the purified reaction solution was determined using ABI PRISM 3700 Genetic Analyzer (Applied Biosystems). Each determined nucleotide sequence was compared with the mouse gene sequence registered in the GenBank gene sequence database using the BLAST algorithm, and the gene name and the registration number in GenBank were assigned to the aforementioned mouse kidney-derived cDNA.

  Next, the duplicated amplified mouse kidney-derived cDNA was removed from the amplified mouse kidney-derived cDNA purified based on the meaning information, and 25 μL of ethanol was added to the purified amplified mouse kidney-derived cDNA without duplication. The precipitate was collected by centrifugation and dissolved in 20 × SSC diluted 6-fold in 30 μL with ultrapure water, and finally 190 kinds of selectively binding substances derived from mouse kidney were obtained.

  As the confirmation probe, 1 μL of Cy3-dUTP (GE Healthcare) and Cy5-dUTP (company) were dissolved in 20 × SSC diluted 6 times with 30 μL of ultrapure water.

  Next, the above-mentioned selective binding substance mouse kidney-derived cDNA and confirmation probes Cy3-dUTP and Cy5-dUTP were aligned and stored in a 96-well microtiter plate (Corning) as shown in FIG. That is, 8 columns with symbols A, B, C, D, E, F, G, H and (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 A 96-well microtiter plate of 8 columns and 12 rows, in which each section is uniquely encoded by a combination of 12 character strings with a symbol and a symbol, is shown in FIG. In all sections except B2 of FIG. 1 and C3 section of FIG. 1 (b), 190 kinds of selectively binding substances mouse kidney-derived cDNA are stored in each section so that the contents are not mixed independently, Cy3-dUTP, which is one of the confirmation probes, is stored in section B2 in FIG. 1 (a), and Cy5-dUTP, which is one of the confirmation probes, is stored in section C3 in FIG. 1 (b). Mouse kidney-derived cDNA that is a binding substance, and for confirmation A lobe Cy3-dUTP, and was also to obtain a container constituting the DNA chip clone set for consisting Cy5-dUTP is confirmation probe.

  Next, using a container constituting a DNA chip clone set and GMS417 Arrayer (Affymetrix), the DNA chip clone set was coated with poly L-lysine in the positional relationship shown in FIG. 3) in distilled water at 95 ° C. for 5 minutes in a 400 mL solution containing 10 mg / mL succinic anhydride and 85% N-methyl-2-pyrrolidone and 0.2 M sodium borate. The substrate was immersed in 95% ethanol for 1 minute in that order and then air-dried to obtain the substrate shown in FIG.

  Next, the excitation light of Cy3 and Cy5, which are fluorescent substances, was measured using GenePix 4000B (Axon Instruments), a laser scanner for DNA chips, with a laser output of 100% and a photomultiplier voltage setting of 500. The measurement data was stored as a 16-bit Tiff format image and confirmed using GenePixPro 5.0 (Axon Instruments), which is DNA chip image analysis software.

  At this time, as shown in FIGS. 4 (a), 4 (b), and 4 (c), the container shown in FIGS. 1 (a) and 1 (b) is shown in FIG. 4 (a). Assuming that the DNA chip clone set is aligned and placed on the substrate in the correct order and orientation, the orientations of FIGS. 1 (a) and 1 (b) are correct but the order is reversed. 1 (a) and 1 (b) are correct, but the direction is reversed, as shown in FIG. 2 (c), and FIG. 1 (a) or 1 (b) is not used. In the above-mentioned image, the confirmation probes Cy3-dUTP and Cy3-dUTP, which are aligned and stored in the B2 section of FIG. 1A and the C3 section of FIG. It was possible to detect from the alignment state.

  Next, mouse kidney-derived polyadenine-added RNA was extracted and purified from the mouse kidney by the method described above, placed in two 0.2 mL microtubes of 5 μg each, and CyScribe First-Strand cDNA Labeling Kit (GE Healthcare) was used. And labeled according to the manual attached to the kit to obtain two kinds of labeled mouse kidney-derived cDNAs. At this time, one mouse kidney-derived polyadenine-added RNA was labeled with Cy3-dUTP, and the other was labeled with Cy3-dUTP. Two kinds of labeled mouse kidney-derived cDNAs were added with 480 μL of TE Buffer Solution, purified using Microcon-YM30 (Amicon) according to the attached manual, and concentrated to 6 μL. To this concentrate is added 2.5 μL of 10 mg / mL polyadenine (Roche), 0.5 μL of 10% SDS solution, 3 μL of 0.4% BSA, and 15 μL of formamide, and 3 μL of 20 × SSC, After heating at ° C for 2 minutes, the mixture was allowed to stand at room temperature in the dark for 30 minutes to obtain a target liquid. After that, the region where the DNA chip clone set on the substrate where the above-mentioned confirmation probe is detected is arranged and covered with a Gap cover glass (Matsunami Glass Industrial Co., Ltd.), and the target solution is added between the substrate and the Gap cover glass. The reaction was performed at 42 ° C. for 16 hours using a hybridization chamber GAPS coated slide (Corning) according to the attached manual. After the reaction, the substrate was diluted 10-fold with ultrapure water containing 0.1% SDS for 10 minutes in 20 × SSC, diluted 200-fold with ultrapure water containing 0.1% SDS for 10 minutes, and ultrapure water was added for 200 minutes with ultrapure water. The substrate was immersed in 20 × SSC diluted twice for 5 minutes in order, and the substrate was dried by centrifugation at 200 × g for 5 minutes. When the dried substrate was measured using the above-described GenePix4000B and GenePixPro5.0, the probe for alignment arranged on the substrate was detected, but the noise of the substrate itself was an equivalent value.

  Therefore, the confirmation probe arranged on the substrate can detect the difference in the order, direction and type of the containers constituting the DNA chip clone set, and the detected substrate can be selectively bonded to the target solution. The detection value of the confirmation probe that can be used for the reaction with the substance was a value that does not affect the reaction between the target liquid and the selective binding substance on the substrate.

Schematic which shows an example of the container which stored and stored this selective binding substance and confirmation probe by this invention. Fig.1 (a) and FIG.1 (b) show an example of two different types of containers. A selective binding substance 11 and a confirmation probe 12 are stored in the container 10, and the aligned arrangement state of the selective binding substance 11 is confirmed after storage. The confirmation probe 12 is stored in alignment so that it is different for each container so as to uniquely determine the type and orientation of the container, and is asymmetric in all containers. Schematic which shows an example of the positional relationship of a board | substrate and a container when manufacturing the board | substrate of this invention. The positional relationship between the substrate 13 and the container 10 is shown. The broken line arrows in the figure indicate the movement of the selective binding substance 11 or the confirmation probe 12 from the container to the substrate. Schematic which shows an example of the board | substrate with which the selective binding substance 11 of this invention and the probe 12 for a confirmation are arrange | positioned. The substrate 13 has an arrangement region 14 in which the selective binding substance 11 and the confirmation probe 12 are arranged. Schematic which shows an example of the arrangement | positioning area | region on the board | substrate which arranged the selective binding substance and the confirmation probe on the board | substrate using the container of Fig.1 (a) and FIG.1 (b). 4A is an example in which FIG. 1B is arranged next to FIG. 1A, FIG. 4B is an example in which FIG. 1A is arranged next to FIG. 1B, FIG. FIG. 1C shows an example in which FIG. 1B is arranged after FIG. 1A, but unlike FIG. 4A, the direction of FIG. 1A is rotated by 180 degrees.

Explanation of symbols

10... 11 selective binding substance and container in which 12 confirmation probes are aligned and stored, 11... Selective binding substance, 12... Confirmation probe, 13 .. substrate, 14. region

Claims (6)

A substrate having a selective binding substance immobilized thereon, wherein a confirmation probe for confirming the arrangement of the selective binding substance is disposed on the substrate. 2. The substrate according to claim 1, wherein the confirmation probe is disposed at a specific location on the substrate that is adjacent to the selective binding substance but does not overlap the arrangement of the selective binding substance. The confirmation probe is adjacent to the selective binding substance, but is arranged at a specific location on the substrate that does not overlap with the arrangement of the selective binding substance, and the positional information of the confirmation probe is the selective binding property. The substrate according to claim 1, which shows an arrangement state of substances. The confirmation probe is adjacent to the selective binding substance, but is arranged at a specific location on the substrate that does not overlap with the arrangement of the selective binding substance, and the positional information of the confirmation probe is the selective binding property. The substrate according to claim 1, wherein the substrate shows a structure of a substance. 2. The method for manufacturing a substrate according to claim 1, wherein the selective binding substance and the confirmation probe are aligned and stored in a container, the alignment storage state in the container is confirmed, and the container is preliminarily stored. A method of manufacturing a substrate, comprising the step of aligning and arranging the selective binding substance and the confirmation probe at a predetermined position on the substrate by using them in the order and orientation. 6. The method according to claim 5, wherein the step of detecting the positional relationship of the confirmation probe and the step of detecting that the containers have been used in the correct order and orientation from the positional relationship of the verification probe on the substrate. A method for manufacturing and managing the substrate.