JP2006262797A - Method for detection of genetic expression using capillary array - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing genetic expression, sensitively carried out by using a small amount of a nucleic acid probe, safety because the method is free from use of a radio isotope. <P>SOLUTION: The invention relates to the method for detection of genetic expression comprising a step immobilizing a probe which contains a first labeling substance and complimentary to the target gene, on a capillary array, a step producing a labeled nucleic acid by reopening an extension reaction by an extension reaction solution to take in a second labeling substance to a nuclei isolated from a cell, a step extracting the labeled nucleic acid, a step hybridizing the probe with the labeled nucleic acid by introducing the labeled nucleic acid into the capillary array, a step decomposing the probe which does not hybridizes by a single chain specific nuclease, a step removing the decomposed product from the capillary array and a step detecting remaining first and/or second labeling substance. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nuclear run on assay method for analyzing a gene that is being expressed in a target cell whose gene expression is to be measured.

A nuclear run on assay is known as a method for measuring the amount of gene transcription. This method is based on the phenomenon that new transcription initiation does not occur when transcription is continued in vitro using an isolated nucleus. In other words, this means that only RNA strands that have already started transcription before isolation of the nucleus are elongated. Therefore, if the transcript in the isolated nucleus is labeled with ³² P and the labeled RNA is detected with a specific probe, information such as how much the gene was transcribed at the moment the nucleus was isolated from the cell Can know. As a more specific application example, it is possible to know the influence on the gene expression of a cell to stimuli such as drugs, hormones, cytokines, and environmental pollutants.

Conventional nuclear run on assays are generally analyzed through the following steps. That is, plasmid DNA (about 5 to 10 μg) containing the gene sequence to be measured is alkali-treated to give single-stranded DNA, neutralized, and adsorbed on a filter that adsorbs nucleic acid such as a nitrocellulose filter. This filter is fixed by heat treatment at 80 ° C. for 3 hours. The DNA adsorbed on the filter serves as a probe for detecting an object to be tested. Next, nuclei are isolated from the cells to be tested, and radioisotopes such as ³² P-UTP are added to the isolated nuclei to synthesize labeled RNA. Next, labeled RNA is prepared and reacted with a filter adsorbed with the plasmid DNA to cause specific hybridization. After washing, hybridization is detected by autoradiography.

As described above, the conventional nuclear run on assay requires a large amount of nucleic acid. In addition, the labeling, radioisotopes such as ³² P are generally used, impact on the health and environmental exposure, and the like are concerned. In addition, handling of radioisotopes requires special facilities.

  An object of the present invention is to provide a nuclear run on assay for analyzing gene expression that can be carried out with high sensitivity with a small amount of nucleic acid probe and that can be carried out safely and simply because no radioisotope is used.

The present invention solves the problems of the present invention by the following means. That is,
A method for detecting a gene being expressed in a target cell whose gene expression is to be measured using a capillary array,
A solid phase immobilization step of immobilizing a probe complementary to a specific target gene and bound with a first labeling substance in a reaction section in a plurality of capillary structures included in the capillary array;
The nucleus isolated from the cells is allowed to react with an extension reaction solution prepared so that nucleotides bound to the second labeling substance are incorporated into the extension chain. A nucleic acid labeling step of producing a labeled nucleic acid labeled with a second labeling substance by performing an extension reaction of the RNA;
An extraction step of extracting the labeled nucleic acid into an appropriate solution after the above-mentioned step treatment;
After the above process, the labeled nucleic acid is introduced into the reaction section in the capillary array, so that the probe and the labeled nucleic acid corresponding to the target gene are hybridized to form a complementary double strand. A hybridization step to form nucleic acids;
After the above process, the probe that did not form a complementary double-stranded nucleic acid is an enzyme-treated solution containing a nucleolytic enzyme that specifically acts on a portion of a single-stranded nucleic acid that does not form a base pair. A decomposing step that specifically decomposes under appropriate reaction conditions;
A post-decomposition washing step for removing the degradation product of the probe containing the first labeling substance from the reaction part after the process step;
A detection step of detecting the first labeling substance and / or the second labeling substance remaining in the reaction part by a detecting means capable of detecting the first labeling substance and / or the second labeling substance after the above-mentioned process treatment; Comprising
Provided is a method characterized in that the above steps are performed in parallel for a plurality of samples under different condition control for each capillary.

  The nuclear run on assay for analyzing gene expression can be carried out with high sensitivity using a small amount of nucleic acid probe, and can be carried out safely and simply because no radioisotope is used. In addition, the assay can be processed simultaneously on a plurality of samples to be tested, while controlling various reaction conditions, and a large amount of results can be detected simultaneously. These processes and detections can be performed in a single structure. Can be done.

  Furthermore, regarding the pre-reaction probe used in the nuclear run on assay for analyzing gene expression, it is possible to easily test the immobilized state on the carrier that affects the quality.

  The present invention solves the problems of the present invention by performing a technique similar to a nuclease protection assay using a capillary array.

  The present invention will be briefly described with reference to FIG. Probe A and probe B in FIG. 1 are probes having sequences complementary to sequences contained in different target genes. The target in the figure means a nucleic acid having a sequence complementary to probe A. (A) in the figure shows a state in which the probe A and the probe B are solid-phased in the capillary array. (B) shows a situation in which a target nucleic acid complementary to probe A is hybridized with probe A. (C) shows a situation in which the probe B that does not hybridize with the target nucleic acid is decomposed, and the labeled nucleotide contained in the probe B is released. The present invention is a method for detecting whether or not a target nucleic acid is present in a sample by utilizing the fact that only the probe hybridized with the target nucleic acid remains in the capillary array.

Details of the present invention will be described below.
[Capillary array]
DNA microarrays are technologies that are expected to achieve mass analysis, improved detection sensitivity, sample saving by microfabrication, automation of data acquisition, and simplification by data processing. Currently, gene expression analysis methods currently performed using a DNA microarray are mainly analysis methods that compare differences in the expression ratios of specific genes present in two samples. This analysis method labels each of the two mRNA samples with a different fluorescent material by reverse transcription in the presence of a nucleotide (such as Cy3-dUTP or Cy5-dUTP) labeled with a fluorescent material that fluoresces at different wavelengths, They are hybridized competitively with DNA probes immobilized on the array, and the fluorescence of both is measured.

  A capillary array (also referred to as a DNA capillary, a DNA capillary array, etc.) is an improvement over the DNA microarray, and is disclosed in, for example, Japanese Patent Application Laid-Open Nos. 11-75812 and 2001-136963. Yes.

<Features of capillary array>
The capillary array can measure the same number of samples as the number of capillaries on a single substrate (for example, nucleic acids extracted from cells of each individual), and can simultaneously compare multiple samples. Moreover, it is easy to automate liquid feeding operations such as dispensing operations and cleaning operations. Furthermore, it is possible to control the temperature in the capillary for each capillary.

  The capillary array has features as exemplified in FIG. In the capillary array of FIG. 2, a capillary-shaped space 4 (hereinafter referred to as a capillary space) having a solution injection port 6 at one end, a solution discharge port 7 at the other end, and a probe 5 therein is provided on the substrate body 3. A plurality of (seven) are provided, the substrate upper part 2 is disposed so as to cover them, and the capillary array 1 is configured by connecting the inlet connecting part 8 and the outlet connecting part 9 in an integrated structure. Further, a heater 10 for controlling the temperature of the substrate is disposed below the capillary array 1. FIG. 2A is a view of the capillary array 1 as viewed from above. FIG. 2B is a view of the capillary array 1 as viewed from the side. FIG. 2 shows an example in which the heater is separate from the capillary array, but the heater, the sensor, and necessary wiring may be incorporated in the capillary array. Further, when temperature control is not required, a capillary array that does not include these may be used.

  The sample used for the experiment is injected into the capillary space 4 from the solution injection port 6 and discharged from the solution discharge port 7, but the reverse may be possible. Further, the plurality of solution discharge ports 7 may be connected to each other to form one common port. In this case, different samples can be introduced from the solution injection port 6 and incubated under the temperature control of the heater 10 for a required time to cause a biological reaction, and then the sample can be collected and removed from the common inlet / outlet in a lump. . Similarly, a solution having the same composition, such as a cleaning solution, can be injected into all the capillary spaces 4 from one channel through a common entrance / exit. In this way, all solutions such as a cleaning solution and a measurement reagent can be processed, so that it is easy to automate and an apparatus with high measurement processing capability can be configured. In addition, since all the reaction steps are completed by only one-way solution replacement, it is possible to fully automate, leading to a significant reduction in reaction time.

  As described above, the capillary array is a microarray having a plurality of capillary spaces, in which probes are solid-phased in one or more reaction portions existing in each capillary space. Usually, a plurality of the reaction parts are provided in the capillary space, and a probe is immobilized on each of them. The reaction unit may be formed by directly immobilizing the probe in a certain region in the capillary space. However, the probe is immobilized on a particle such as a bead, and the reaction unit is placed in the certain region in the capillary space. It may be formed by indirect solid phase immobilization. The granule may be any material as long as it can contact the probe solid-phased with the sample solution flowing through the capillary structure and can be fixed to a certain region in the capillary space. For example, magnetic beads or the like are preferably used, which can hold the beads themselves in a certain region in the capillary space by applying a magnetic force such as a magnet from the outside of the capillary array. In this case, various magnetic beads in which various probes are solid-phased can be filled in the capillary space in an appropriate order, for example, and fixed to the predetermined region by a magnetic force of a magnet or the like to function as a reaction unit. it can.

  The capillary space may be formed by appropriately combining appropriate members having openings at both ends thereof and allowing the solution to flow into the space. Therefore, the capillary space may be an internal space of an integral tubular structure, for example, but may be formed by joining a plurality of members. A capillary space formed by joining a plurality of members can be formed, for example, by covering a normal slide glass with a lid described below. That is, when the lid and the slide glass are joined, a capillary space is formed from the plurality of grooves existing on the bottom surface of the lid and the top surface of the slide glass. The probe is solid-phased on the slide glass surface in the capillary space. The lid includes a plurality of solution inlets for introducing the solution into the capillary space at one end of the upper surface, and a solution outlet that is paired at a position corresponding to the solution inlet at the other end. The outlet passes through the lid and opens at the bottom. When the slide glass and the lid are joined to the bottom surface of the lid, a plurality of grooves that connect the solution inlet and the solution outlet to the capillary space and form a plurality of capillary spaces, and the slide glass It is possible to use a structure provided with a joint where the lid and the lid are joined. The size of the capillary space is set to various sizes according to the application, but practically, the width is 10 μm to several mm, the depth is 1 μm to 500 μm, the length is several mm to 100 mm, and the space between the capillary spaces is 10 μm to Several millimeters are used. The flow path shape of the capillary space as described above may be various shapes such as a straight line and a curved line, and may be bent, folded, branched, merged, or widened in a tapered shape (or as necessary). (Narrow), via a protrusion (or microhole), or uphill (or downhill). If a plurality of capillaries are arranged in parallel, parallel liquid handling is easy. In particular, it is preferable to arrange them in a straight line in parallel (which may be two-dimensional or three-dimensional) in that the parallel processing can be easily monitored by any imaging means.

  The slide glass as the main body can be prepared by a technique usually used for immobilizing probes in a microarray. For example, the pretreatment method is performed by washing the slide glass with alkali or acid. Next, the necessary treatment is performed on the solid phase of the nucleic acid used as a probe. As the surface treatment, the array substrate is made positively charged with poly-L-lysine, the negatively charged DNA is adsorbed, an amino group is introduced into the substrate using an aminosilane agent, etc., and a crosslinking agent is added if necessary. By using it, the covalent bond between the probe and the slide glass can be realized. Alternatively, it can be produced by reacting a streptavidin-coated slide (Greiner Japan) with a biotin-labeled probe that specifically binds. After the surface treatment of the slide glass, a nucleic acid to be used as a probe is arranged on the substrate using a commercially available pin type arrayer or an inkjet arrayer. Next, a lid is attached to form a capillary space. A capillary array made from such a slide glass and a lid has the advantage that it can be disassembled and only the slide glass portion can be measured using a normal microarray measuring instrument.

  The capillary array has the features as described above, and the feature of the present invention that the process is performed in parallel on a plurality of samples under the condition control different for each capillary is the effect of the capillary array. It is based on.

<< Probe immobilized on capillary array >>
In the present invention, it is preferable to use a capillary array in which the probe to which the first labeling substance of the present invention is bound is present in a solid phase in the reaction part in the capillary array described above.

The probe only needs to have at least the following conditions. That is, the probe is
Has a sequence complementary to the specific target gene to be detected and can specifically hybridize with the sequence to form a complementary duplex;
Including a nucleotide appropriately labeled with a first labeling substance at an appropriate position in the probe;
Can be degraded by the nucleolytic enzymes used in the present invention;
And so on.

  As used herein, the term “appropriately labeled nucleotide” means that the formation of a complementary strand by hybridization and the digestion of a single-stranded nucleic acid by a nucleolytic enzyme are not affected by the presence of this label. For example, for hybridization, it is necessary not to interfere with normal base pairing. The “appropriate position” is a position on the solid phase surface side that is about 1/3 of the total length of the probe from the end opposite to the side on which the probe is immobilized. That is, for example, when the 5 'end of a 30 nucleotide probe is bound to the solid phase, it is preferable that the nucleotide near the 10th site from the 3' end is labeled with the first labeling substance.

  The chain length of the probe is appropriately selected according to the purpose within the range satisfying the above conditions, but is preferably in the range of 10-50 nucleotides, more preferably in the range of 20-40 nucleotides, most preferably 30 nucleotides. Front and back chain lengths are used. The probe can be synthesized and produced so as to contain a nucleotide appropriately labeled with a first labeling substance. As the nucleotide appropriately labeled with the first labeling substance, a nucleotide obtained by binding a fluorescent reagent to uridine (U), thymidine (T), cytidine (C), adenine (A) or the like can be used. As the fluorescent reagent, for example, Cy3, Cy5, FITC, rhodamine and the like can be used. Thus, since the probe of the present invention is labeled with the first labeling substance, the state of the solid phase can be confirmed by detecting the first labeling substance after the solid phase immobilization step. Therefore, the accuracy control of the capillary array can be performed by examining the amount of the solid phase and the homogeneity of the solid phase portion regarding the solid phase of the probe. In addition, since the amount of the immobilized probe and the spotted spot state can be known before a predetermined reaction, it is possible to overcome the accuracy control problems of conventional DNA microarrays in general. In the present invention, various measurable labeling substances other than fluorescent reagents (for example, for chemiluminescence, bioluminescence, electrochemiluminescence, electromagnetic energy, resonance energy, and nanosemiconductor emission) are used. You may change suitably.

[Nuclear run on assay]
In the present invention, the production of a labeled nucleic acid labeled with a second labeling substance can be performed with reference to a nuclear run on assay which is a conventional technique. The method for producing a labeled nucleic acid will be described below.

  First, the nuclei are isolated by lysing the plasma membrane in a lysis buffer containing a nonionic surfactant (NP-40). Next, an extension reaction solution containing a nucleotide to which the second labeling substance is bound and a nucleotide constituting other RNA in the form of a nucleotide triphosphate having three phosphate groups at the 5 ′ site of these nucleotides. Add. For example, in the case of 5-bromouridine, it may be contained in the extension reaction solution as 5-Bromouridin 5'-triphosphate. By adding the extension reaction solution and incubating, the RNA extension reaction that was in the process of extension during the isolation of the nucleus is started again. Next, DNase I is added to degrade the DNA. Next, the enzyme reaction is stopped by adding a stop solution containing SDS or the like. Next, RNA is extracted by a conventional RNA extraction method such as the AGPC method, but a kit such as an RNeasy kit (Qiagen) may be used. Further, it may be purified using affinity purification with a substance that specifically binds the second labeling substance. By such an operation, a labeled nucleic acid labeled with the second labeling substance is produced. The nucleotide to which the second labeling substance is bound may be any substance that can be detected separately from the first labeling substance, but 5-bromouridine or the like is preferably used. Alternatively, the nucleotide to which the second labeling substance is bound may be a nucleotide to which a fluorescent dye that can be detected at a wavelength different from that of the first labeling substance is bound. For example, if the first labeling substance is Cy3, it is possible to incorporate Cy5 into the RNA strand being transcribed using nucleotides to which Cy5 is bound. When the bound nucleotide is 5-bromouridine, it can be specifically detected by, for example, an anti-BrdU antibody. Moreover, the labeled nucleic acid incorporating 5-bromouridine can be purified by immunoprecipitation using this antibody.

[Use of nuclease]
The present invention also utilizes the principle of a nuclease protection assay. This assay is a method for examining the presence of a specific nucleic acid by utilizing a single-strand specific nucleolytic enzyme. That is, in this assay, the probe hybridized with the nucleic acid derived from the target gene forms a double-stranded nucleic acid, and as a result, this double-stranded nucleic acid escapes nuclease degradation and the probe is protected from degradation. In contrast, the unreacted probe (single-stranded DNA) that has not been hybridized is decomposed, and the nucleotide to which the first labeling substance is bound is released into the solution. And it is the method of measuring the presence frequency of a target gene by detecting the 1st labeling substance of the probe protected from decomposition | disassembly of a nuclease. Therefore, preparation of cDNA by reverse transcriptase and labeling for each specimen are not required, and assay in the state of mRNA can be performed, so that quantitativeness is improved and reagent costs can be reduced.

  The nuclease (nuclease) that can be used in the present invention will be described below. S1 nuclease is a single-strand-specific endonuclease, and both DNA and RNA are decomposed into 5'-P nucleotides (nucleotide monophosphate holding a phosphate at the 5 'position of the sugar) and finally Is an enzyme that degrades to 90% or more of 5′-P nucleotides. The enzyme also acts on a single-stranded portion in the double strand and decomposes this portion. Furthermore, the enzyme is also used for removing a single-stranded portion in DNA-DNA and DNA-RNA hybrids. Exonuclease I is a single-strand-specific exonuclease, which is an enzyme that hydrolyzes sequentially from the 3 'end of single-stranded DNA and decomposes into 5'-P nucleotides. This enzyme is used for removing primers after PCR. Other nucleases that specifically react with single strands include nuclease P1. A person skilled in the art can set appropriate conditions in consideration of the specificity and reaction conditions of each of these enzymes.

[Description of each process]
The method of the present invention is a method including a plurality of steps appropriately selected according to the purpose from each step described and explained below. The numbers given before each step are given for convenience during reference. The selected steps and the order of the steps can be appropriately changed within the scope of the present invention.

  1: A solid phase immobilization step in which a probe complementary to a specific target gene and bound with a first labeling substance is immobilized on reaction parts in a plurality of capillary structures provided in the capillary array. This step is a step of immobilizing a probe for detecting a target gene to be investigated in a reaction part of a capillary array. This step is carried out with reference to the description relating to the “capillary array” described above.

  2: An assay step in which the immobilized state of the probe immobilized on the reaction part in the plurality of capillary structures included in the capillary array in the above step is assayed by a detection means capable of detecting the first labeling substance. This step is a step of verifying whether or not the processing of the above step has been properly performed. Specifically, the reaction part is measured by a detection means capable of detecting the first labeling substance, and the solid phase state is assayed. The state of the solid phase includes, for example, the presence or absence of probe solid phase, the state of the reaction part such as homogeneity, or the state of the background signal emitted from a region other than the reaction part. In general, it is preferable to proceed to the next step after confirming the immobilized state of the probe in this step.

  3: The nucleus isolated from the cell is reacted with an extension reaction solution prepared so that the nucleotide to which the second labeling substance is bound is incorporated into the extension chain, and the extension is performed during the isolation of the nucleus. A nucleic acid labeling step of producing a labeled nucleic acid labeled with a second labeling substance by performing an RNA elongation reaction that was This step is a step of labeling RNA encoding a target gene to be detected, and is carried out with reference to the above-mentioned description relating to “nuclear run on assay”.

  4: Extraction step of extracting the labeled nucleic acid into an appropriate solution. This step is a step of extracting the RNA labeled in the above step, and is carried out with reference to the above description regarding the “nuclear run on assay”. The appropriate solution is appropriately selected depending on the next step. Next, for example, when shifting to the hybridization step, the solution should be a hybridization solution [for example, 5 × SSC solution (1 × SSC includes 0.15M NaCl, 0.015M sodium citrate), 50% formamide, 0.1% SDS]. preferable.

  5: A purification step for purifying the labeled nucleic acid by an appropriate purification means. This step is a step of purifying RNA, and is performed with reference to the above-mentioned description relating to “nuclear run on assay”. By this step, the labeled nucleic acid is highly purified. The purification means only needs to highly purify the labeled nucleic acid as a result, and may be a means based on a single purification method or a means based on a combination of a plurality of purification methods. Preferably, affinity purification using an antibody that specifically binds to the second labeling substance is used. As the next step, when moving to the following reverse transcription step, it is preferable to highly purify by this step.

  6: A reverse transcription step of reverse transcription of the labeled nucleic acid. In this step, the labeled nucleic acid is reverse transcribed into cDNA. Although this step is not essential, RNA may be converted to DNA by this step and then transferred to the next step. During this reverse transcription, it is preferable to label with the second labeling substance by the same operation as in the nucleic acid labeling step. When reverse transcription is performed in this step, the complementary double-stranded nucleic acid formed in the following hybridization step becomes double-stranded DNA. Conversely, when this step is not performed and reverse transcription is not performed, a hetero double-stranded nucleic acid in which RNA and DNA form a base pair is formed. In consideration of the type of such double-stranded nucleic acid, it is preferable to consider the conditions of the following steps. For example, it should be considered that the type of such double-stranded nucleic acid may affect the selection of the nucleolytic enzyme used in the degradation step.

  7: By introducing the labeled nucleic acid into the reaction section in the capillary array, the probe and the labeled nucleic acid corresponding to the target gene are hybridized to form a complementary double-stranded nucleic acid. Hybridization step. This step is a step of hybridizing the probe and the nucleic acid derived from the target gene. In order to achieve specific hybridization, this step is preferably performed by controlling the temperature of the capillary array according to the probe used. Various conditions including the temperature at the time of hybridization can be appropriately set by those skilled in the art.

  8: A non-binding nucleic acid washing step for washing the nucleic acid that has not been bound in the above step. This step is a step of washing and removing nucleic acids that did not form complementary double-stranded nucleic acids in the hybridization step. This step is also preferably performed at an appropriate temperature for the purpose of suppressing nonspecific binding. Various conditions including the temperature at the time of washing can be appropriately set by those skilled in the art.

  9: Appropriate reaction of the probe that did not form a complementary double-stranded nucleic acid with an enzyme treatment solution containing a nucleolytic enzyme that specifically acts on the portion of the single-stranded nucleic acid that does not form base pairs Decomposition process that specifically decomposes under conditions. This step is a step of decomposing a single-stranded nucleic acid present in the capillary array or a single-stranded portion that is not base-paired in the single-stranded nucleic acid and the double-stranded nucleic acid. This step is performed with reference to the description relating to “use of nucleolytic enzyme”.

  10: A reaction stopping step for stopping the decomposition reaction by subjecting the nucleolytic enzyme to an appropriate reaction stopping process. This step is performed in order to stop the decomposition step appropriately to stop further decomposition reaction. For this reaction termination treatment, an appropriate method is preferably selected according to the enzyme used. For example, the enzyme can be inactivated by a method such as addition of an inhibitor of the enzyme. Further, when it is not necessary to maintain the double strand formed in the hybridization step, a method such as addition of a denaturant or heat denaturation may be used. The object of this step can also be achieved by removing the enzyme by the following post-decomposition washing step without carrying out this step.

  11: A post-decomposition washing step for removing degradation products of the probe containing the first labeling substance from the reaction section. This step is a step of removing the solution remaining in the capillary array after the above process and washing the reaction part. In this step, at least the first labeling substance contained in the probe decomposed by the processing in the above step is excluded from the capillary array and does not affect the following detection step.

  12: A detection step of detecting the first labeling substance and / or the second labeling substance remaining in the reaction part by a detection means capable of detecting the first labeling substance and / or the second labeling substance. This step forms a complementary double-stranded nucleic acid, and as a result, detects the probe and labeled nucleic acid protected from degradation by the nucleolytic enzyme by a detection method based on the first labeling substance and / or the second labeling substance. This is a detecting step. Since the detection means is a measurement means capable of detecting the first labeling substance and the second labeling substance, it is preferably selected as appropriate according to the type of the two labeling substances. When a fluorescent reagent is used as both the first labeling substance and the second labeling substance, the first labeling substance and the second labeling substance are preferably different fluorescent reagents that emit fluorescence having wavelengths that can be distinguished from each other. For example, if Cy3 and Cy5 are used as the first labeling substance and the second labeling substance, respectively, fluorescence signals derived from both can be detected independently. Alternatively, if the affinity substance that specifically binds to the first labeling substance and the second labeling substance is labeled with Cy3 and Cy5, respectively, fluorescence signals derived from both can be detected independently. As the affinity substance, an antibody that specifically binds to the first labeling substance or the second labeling substance is preferably used. An appropriate combination of the first labeling substance and the second labeling substance can be selected by those skilled in the art in consideration of various requirements such as the purpose of implementation.

  From the results detected by the method of the present invention including the above steps, any of the following results A to C may occur. Each of A to C will be described below.

A: Only one of the first labeling substance and the second labeling substance is detected:
Normally this situation does not occur.
B: Both the first labeling substance and the second labeling substance are detected:
This state is a state indicating that the target gene was transcribed when the nucleus was isolated. That is, it shows a situation in which the probe and the labeled nucleic acid corresponding to the target gene were appropriately hybridized, so that both were not decomposed.
C: Neither is detected:
This state is a state indicating that the target gene was not transcribed when the nucleus was isolated. That is, it indicates that the probe was decomposed because appropriate hybridization did not occur.

The above information can be acquired from the measurement result.
However, this information is described on the assumption that all processes have been appropriately processed. Therefore, when it is implemented without including some steps, it may be different from the following description. For example, when the purification step is not performed, it is conceivable that only the first labeling substance is detected. This is because it is expected to occur when the probe and the unlabeled nucleic acid specifically hybridize. Even in this case, the probe is not decomposed. In this case, the unlabeled nucleic acid is a nucleic acid complementary to the target gene, but may have existed before the isolation of the nucleus. One skilled in the art can estimate the expected results when performing the method of the present invention including selected steps.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

[Example 1]
In this example, Cy3 was used as the first labeling substance, 5-bromouridine was used as the second labeling substance, and S1 nuclease was used as the nuclease.
In this example, it is tested whether only the hybridized probe is adequately protected by measuring the fluorescence at the wavelength of Cy3.

<Materials and methods>
<< Preparation of RNA >>
HeLa cells as a cultured cell line were detached with 0.05% trypsin-0.53 mM EDTA, washed twice with cold PBS, and 0.5 mL of lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl ₂ , 0.5% NP-40) and left in ice for 10 minutes. The cells are then centrifuged to separate the nuclear fraction (pellet), which is suspended in 100 μL suspension buffer (50 mM Tris-HCl pH 8.3, 40% glycerol, 5 mM MgCl ₂ , 0.1 mM EDTA). It became cloudy. Next, 100 μL of the extension reaction solution [10 mM Tris-HCl pH 8.0, 5 mM MgCl ₂ , 300 mM KCl, 0.5 mM ATP, CTP, GTP and 0.1 mM 5-bromouridine 5′-triphosphate (Sigma)] In addition, the mixture was reacted at 30 ° C. for 30 minutes. DNase I was added to 20 μg / mL, and the mixture was further reacted at 30 ° C. for 30 minutes. Then, 200 μL of a stop solution (20 mM Tris-HCl, 2% SDS, 10 mM EDTA, 200 μg / mL proteinase K) was added and reacted at 42 ° C. for 30 minutes. The solution after the stop solution treatment was treated with an RNeasy kit (Qiagen) according to the method of use, and RNA was recovered. Thereafter, labeled RNA labeled with 5-bromouridine was purified by immunoprecipitation using an anti-BrdU antibody (Boehringer Mannheim) and protein G beads (Amersham). Next, RNA bound to the antibody was recovered in a hybridization solution [5 × SSC solution (0.15 M NaCl, 0.015 M sodium citrate), 50% formamide, 0.1% SDS] to obtain an RNA solution.

<< Preparation of capillary array >>
On the other hand, a DNA probe PRH-HPRT1M.BD {biotin-GGGGGCTATAAATTCTTTGC (T-Cy3), which is a streptavidin-coated slide (Gliner Japan), labeled with biotin at the 5 'end and Cy3 at the 10th T from the 3' end. ) GACCTGCTG} was spotted at 10 nM using a spotting apparatus, and solidified by biotin-avidin reaction. The sequence of this DNA probe is a sequence complementary to a part of the HPRT gene that has been confirmed to be expressed in the HeLa cells. Cover the slide with the above-mentioned lid (having a plurality of capillary-shaped grooves having a length of 3 to 4 cm, a width of 1 mm, and a height of 0.1 to 0.2 mm with holes through which solutions can be taken in and out), and a capillary array It was.

<< Hybridization and nuclease treatment >>
Next, the RNA solution was put into the capillaries of the capillary array and hybridized at 42 ° C. for 1 hour. After hybridization, the unreacted RNA was removed from the capillary by washing thoroughly with 1 × SSC solution. Thereafter, an enzyme-treated solution [30 mM sodium acetate (pH 4.6), 280 mM NaCl, 1 mM ZnSO ₄ , 1200 units / ml S1 nuclease (where 1 unit is heat-denatured calf thymus-derived DNA as a substrate, 37 ° C., pH 4.6) The enzyme activity is such that 1 μg of acid-soluble substance is produced in 1 minute)] was placed in a capillary and reacted at 37 ° C. for 1 hour. After the reaction, the plate was sufficiently washed with 0.1 × SSC to remove unreacted substances, and fluorescence was measured.

<Results and discussion>
The results of measuring the fluorescence of the capillary array are shown in Table 1 (Table 1 shows relative values with the strongest fluorescence intensity being 1).
As is clear from Table 1, the fluorescence intensity in the capillary array was very low under conditions where no RNA sample was present. This result shows that the DNA probe remained single-stranded and was decomposed and removed by a subsequent washing operation. In contrast, Cy3 wavelength fluorescence was observed when treated with RNA samples. This result shows that as a result of proper double strand formation, the Cy3-labeled base was retained in the capillary array without nuclease degradation.

[Example 2]
In this example, Cy3 was used as the first labeling substance, 5-bromouridine was used as the second labeling substance, and S1 nuclease was used as the nuclease.
In this example, it is tested whether only the hybridized probe is adequately protected by measuring the fluorescence at the wavelength of Cy3. Further, the presence of the labeled nucleic acid is assayed by reacting 5-bromouridine with a Cy5-labeled anti-BrdU antibody and measuring the fluorescence at the wavelength of Cy5. Further, from the measurement results of both (detection results of Cy3 and Cy5), the relationship between the probe in the reaction part in the capillary array and the labeled nucleic acid is estimated.

<Materials and methods>
<< Preparation of RNA >>
HeLa cells as a cultured cell line were detached with 0.05% trypsin-0.53 mM EDTA, the cells were washed twice with cold PBS, and 0.5 mL of a buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl ₂ , 0.5% NP-40) and left in ice for 10 minutes. The cells are then centrifuged to separate the nuclear fraction (pellet), which is suspended in 100 μL suspension buffer (50 mM Tris-HCl pH 8.3, 40% glycerol, 5 mM MgCl ₂ , 0.1 mM EDTA). did. Next, 100 μL of the reaction solution [10 mM Tris-HCl pH 8.0, 5 mM MgCl ₂ , 300 mM KCl, 0.5 mM ATP, CTP, GTP and 0.1 mM 5-bromouridine 5′-triphosphate (Sigma)] was added. And reacted at 30 ° C. for 30 minutes. DNase I was added to 20 μg / mL, and the mixture was further reacted at 30 ° C. for 30 minutes. Then, 200 μL of a stop solution (20 mM Tris-HCl, 2% SDS, 10 mM EDTA, 200 μg / mL proteinase K) was added and reacted at 42 ° C. for 30 minutes. It was treated with RNeasy kit (Qiagen) according to the usage method, and RNA was recovered. Thereafter, labeled RNA labeled with 5-bromouridine was purified by immunoprecipitation using an anti-BrdU antibody (Boehringer Mannheim) and protein G beads (Amersham). Next, RNA bound to the antibody was recovered in a hybridization solution [5 × SSC solution (0.15 M NaCl, 0.015 M sodium citrate), 50% formamide, 0.1% SDS] to obtain an RNA solution.

<< Preparation of capillary array >>
On the other hand, a DNA probe PRH-HPRT1M.BD {biotin-GGGGGCTATAAATTCTTTGC (T-Cy3), which is a streptavidin-coated slide (Gliner Japan), labeled with biotin at the 5 'end and Cy3 at the 10th T from the 3' end. GACCTGCTG} was spotted at 10 nM using a spotting apparatus, and immobilized on a biotin-avidin reaction. The sequence of this DNA probe is a sequence complementary to a part of the HPRT gene that has been confirmed to be expressed in the HeLa cells. Cover the slide with the above-mentioned lid (having a plurality of capillary-shaped grooves having a length of 3 to 4 cm, a width of 1 mm, and a height of 0.1 to 0.2 mm with holes through which solutions can be taken in and out), and a capillary array It was.

<< Hybridization and nuclease treatment >>
Next, the RNA solution was placed in the capillaries of the capillary array and hybridized at 42 ° C. for 1 hour. After hybridization, the unreacted RNA was removed from the capillary by washing thoroughly with 1 × SSC solution. Thereafter, an enzyme-treated solution [30 mM sodium acetate (pH 4.6), 280 mM NaCl, 1 mM ZnSO ₄ , 1200 units / ml S1 nuclease (where 1 unit is heat-denatured calf thymus-derived DNA as a substrate, 37 ° C., pH 4.6) The enzyme activity is such that 1 μg of acid-soluble substance is produced in 1 minute)] was placed in a capillary and reacted at 37 ° C. for 1 hour. After the reaction, the unreacted material was removed by washing thoroughly with 0.1 × SSC. Thereafter, RNA containing 5-bromouridine was reacted with an anti-BrdU antibody (Boehringer Mannheim), washed, reacted with a Cy5-labeled antibody against the anti-BrdU antibody, washed again, and fluorescence at wavelengths of Cy3 and Cy5 was measured (Table). 2).

<Results and discussion>
The results of measuring the fluorescence of the capillary array are shown in Table 2 (Table 2 shows relative values with the strongest fluorescence intensity being 1).
As can be seen from Table 2, the fluorescence intensity in the capillary array was very low under conditions where no RNA sample was present. This result shows that the DNA probe remained single-stranded and was decomposed and removed by a subsequent washing operation. In contrast, Cy3 wavelength fluorescence was observed when treated with RNA samples. This result shows that as a result of proper double strand formation, the Cy3-labeled base escapes nuclease degradation and is retained in the capillary array.

  Furthermore, the fluorescence of Cy5 was also measured in the reaction part where the fluorescence of the wavelength of Cy3 was observed. This result indicates that RNA incorporating 5-bromouridine 5′-triphosphate of the RNA sample formed an appropriate double strand with the DNA probe, so that both nucleic acids remained undegraded. . Thus, the feasibility of the present invention was confirmed to be sufficiently quantitative, sensitive and reproducible in mRNA level experiments. Therefore, the method of the present invention can be applied not only to expression frequency analysis but also to universal arrays (JP 2002-318992, JP 2002) effective for SNP (single molecule polymorphism) analysis, molecular computers (DNA computers), and the like. (See -335999), and can be expected to be versatile.

It is a figure explaining the outline | summary of this invention. It is a figure explaining the typical example of a capillary array.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Capillary array board | substrate 2 ... Board | substrate upper part 3 ... Board | substrate body 4 ... Capillary structure 5 ... Probe 6 ... Solution injection port 7 ... Solution discharge port 8 ... Injection port connection part 9 ... Discharge port connection part 10 ... Heater

Claims (9)

A method of detecting a gene that is being expressed in a target cell for measuring gene expression using a capillary array,
A solid phase immobilization step of immobilizing a probe complementary to a specific target gene and bound with a first labeling substance in a reaction section in a plurality of capillary structures included in the capillary array;
The nucleus isolated from the cells is allowed to react with an extension reaction solution prepared so that nucleotides bound to the second labeling substance are incorporated into the extension chain. A nucleic acid labeling step of producing a labeled nucleic acid labeled with a second labeling substance by performing an extension reaction of the RNA;
An extraction step of extracting the labeled nucleic acid into an appropriate solution after the above-mentioned step treatment;
After the above process, the labeled nucleic acid is introduced into the reaction section in the capillary array, so that the probe and the labeled nucleic acid corresponding to the target gene are hybridized to form a complementary double strand. A hybridization step to form nucleic acids;
After the above process, the probe that did not form a complementary double-stranded nucleic acid is an enzyme-treated solution containing a nucleolytic enzyme that specifically acts on a portion of a single-stranded nucleic acid that does not form a base pair. A decomposing step that specifically decomposes under appropriate reaction conditions;
A post-decomposition washing step for removing the degradation product of the probe containing the first labeling substance from the reaction part after the process step;
A detection step of detecting the first labeling substance and / or the second labeling substance remaining in the reaction part by a detecting means capable of detecting the first labeling substance and / or the second labeling substance after the above-mentioned process treatment; Comprising
A method characterized in that the above steps are advanced in parallel for a plurality of samples under different condition control for each capillary.   The method according to claim 1, further comprising the step of immobilizing a probe immobilized on a reaction portion in a plurality of capillary structures provided in a capillary array in the step, following the step of immobilization. An assay step of assaying by a detection means capable of detecting the first labeling substance.   The method according to claim 1 or 2, further comprising a purification step of purifying the labeled nucleic acid by an appropriate purification means following the extraction step.   4. The method according to claim 3, further comprising a reverse transcription step of reverse transcription of the labeled nucleic acid following the purification step.   The method according to any one of claims 1 to 4, further comprising a non-binding nucleic acid washing step for washing the nucleic acid that has not been bound in the step after the hybridization step.   The method according to any one of claims 1 to 5, further comprising a reaction stopping step for stopping the decomposition reaction by performing an appropriate reaction stopping process on the nucleolytic enzyme following the decomposition step. A method comprising:   The method according to claim 1, wherein the first labeling substance is Cy3 or Cy5.   The method according to any one of claims 1 to 7, wherein the nucleotide to which the second labeling substance is bound is 5-bromouridine.   The method according to any one of claims 1 to 8, wherein the nucleolytic enzyme is selected from the group consisting of S1 nuclease, exonuclease I, and nuclease P1.

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