JP4524392B2 - Method for regenerating carrier for immobilizing probe polynucleotide - Google Patents

Description

  The present invention relates to a method for regenerating a carrier on which a probe polynucleotide is immobilized, and a reagent and kit therefor.

  In addition to clarifying the genetic structures of a wide variety of organisms, attempts are being made to elucidate their gene functions on a genome scale, and technological development for efficient analysis of gene functions is rapidly progressing. Yes. A microarray is a high-density array in which a large number of polynucleotides such as DNA are aligned and fixed on a substrate such as a slide glass for each predetermined region. Determination of nucleic acid base sequences, gene expression, mutation, polymorphism, etc. It is very useful for simultaneous analysis. Analysis of genetic information using this microarray is extremely useful for drug discovery research, disease diagnosis and development of preventive methods, etc., so further development is desired by the microarray production technology and the analysis system of the obtained data. ing.

  In detection using a microarray, first, a polynucleotide sample labeled with a radioisotope or a fluorescent dye is hybridized to a probe polynucleotide arranged on the carrier surface at a high density. At this time, molecules having a base sequence complementary to the probe polynucleotide in the target polynucleotide sample hybridize complementarily to the probe polynucleotide. The signal derived from the label on the hybridized target polynucleotide molecule is then measured to identify the hybridized probe polynucleotide. Specifically, the radiation intensity or fluorescence intensity on the microarray is measured by RI or a fluorescence image scanner, and the obtained image data is analyzed. Therefore, according to the microarray, thousands to tens of thousands of molecules can be simultaneously hybridized and detected, and a large amount of gene information can be obtained in a short time with a small amount of sample.

  In order to immobilize probe polynucleotides in a microarray at a high density and firmly, for example, reactive active groups such as active ester groups that form covalent bonds with polynucleotides are introduced on the surface of the carrier, so that the probe polynucleotides are covalently bonded. (See Patent Documents 1 to 3) and the like.

  Since microarrays are expensive, it is desirable that the target polynucleotides are hybridized and analyzed, and then the hybridized polynucleotides are removed and regenerated for reuse. However, it has been difficult to completely remove the polynucleotide once hybridized to the probe polynucleotide, and therefore, it is substantially impossible to reuse the microarray.

WO 00/22108 WO 01/75447 WO 02/12891

  An object of the present invention is to provide means for removing and regenerating a target polynucleotide hybridized to a probe polynucleotide after using a carrier on which the probe polynucleotide is immobilized for analysis.

  As a result of investigations to solve the above problems, the present inventors contacted a polynucleotide sample with a carrier on which a probe polynucleotide is immobilized, hybridized the target polynucleotide, and removed unreacted polynucleotide. Then, by applying an aqueous dispersion containing at least one selected from the group consisting of a humectant, preferably a gelling agent and a sugar, to the carrier, the target polynucleotide can be efficiently used in the subsequent carrier washing step. It was found that it was removed, and the present invention was completed.

That is, the present invention includes the following inventions.
(1) A method for regenerating a carrier in a method for analyzing a polynucleotide sample using a carrier on which a probe polynucleotide is immobilized,
The method comprising: applying an aqueous dispersion containing a humectant to a carrier in which a target polynucleotide is hybridized to a probe polynucleotide; and washing the carrier.
(2) The method according to (1), wherein the humectant is at least one selected from the group consisting of a gelling agent and a saccharide.
(3) A method of removing a target polynucleotide hybridized with a probe polynucleotide immobilized on a carrier,
The method comprising: applying an aqueous dispersion containing a humectant to a carrier in which a target polynucleotide is hybridized to a probe polynucleotide; and washing the carrier.
(4) The method according to (3), wherein the humectant is at least one selected from the group consisting of a gelling agent and a saccharide.

(5) A method for analyzing a polynucleotide sample using a carrier on which a probe polynucleotide is immobilized,
a) contacting a polynucleotide sample with a probe polynucleotide immobilized on a carrier to hybridize the target polynucleotide contained in the polynucleotide sample to the probe polynucleotide;
b) removing unreacted polynucleotide;
c) applying an aqueous dispersion containing a humectant to a carrier; and d) measuring a signal based on hybridization.
(6) The method according to (5), wherein the humectant is at least one selected from the group consisting of a gelling agent and a saccharide.
(7) A method for regenerating a carrier by washing the carrier after the step d) according to (5) or (6).
(8) A reagent for promoting regeneration of a carrier in a method for analyzing a polynucleotide sample using a carrier on which a probe polynucleotide is immobilized, the reagent comprising an aqueous dispersion containing a humectant.
(9) The reagent according to (8), wherein the humectant is at least one selected from the group consisting of a gelling agent and a saccharide.
(10) A polynucleotide by contacting a polynucleotide sample with a probe polynucleotide immobilized on a carrier, hybridizing a target polynucleotide contained in the polynucleotide sample to the probe polynucleotide, and measuring a signal based on hybridization A kit for analyzing a sample, comprising the reagent according to (8) or (9).

  According to the present invention, after carrying out an analysis using a carrier on which a probe polynucleotide is immobilized, the carrier can be regenerated and reused.

In one embodiment, the present invention relates to a method for regenerating a carrier in a method for analyzing a polynucleotide sample using a carrier on which a probe polynucleotide is immobilized,
Applying an aqueous dispersion containing at least one selected from the group consisting of a humectant, preferably a gelling agent and a sugar, to the carrier in which the target polynucleotide is hybridized to the probe polynucleotide, and washing the carrier The method comprising the steps of:

  In the present invention, to regenerate a carrier, that is, a carrier on which a probe polynucleotide is immobilized, means to remove the target polynucleotide hybridized to the probe polynucleotide immobilized on the carrier. This allows the carrier to be used again for analysis.

Accordingly, in one embodiment, the present invention provides a method for removing a target polynucleotide hybridized to a probe polynucleotide immobilized on a carrier, comprising:
Applying an aqueous dispersion containing at least one selected from the group consisting of a humectant, preferably a gelling agent and a sugar, to the carrier in which the target polynucleotide is hybridized to the probe polynucleotide, and washing the carrier The method comprising the steps of:

In other words, the present invention is a method for promoting the removal of a target polynucleotide hybridized to a probe polynucleotide immobilized on a carrier,
Applying the carrier in which the target polynucleotide is hybridized to the probe polynucleotide to an aqueous dispersion containing at least one selected from the group consisting of a humectant, preferably a gelling agent and a saccharide. .

  In the present specification, the polynucleotide includes oligonucleotides, and also includes nucleic acids including DNA and RNA, and nucleic acid derivatives. DNA includes single-stranded DNA and double-stranded DNA. Examples of nucleic acid derivatives include artificial nucleic acids modified with phosphodiester sites; artificial nucleic acids modified with glycosyl bonds and hydroxyl groups at furanose sites; artificial nucleic acids modified with nucleobase sites; and structures other than sugar / phosphate skeletons Artificial nucleic acid used, etc. are mentioned. More specifically, phosphorothioate type, phosphorodithioate type, phosphorodiamidate type, methylphosphonate type or methylphosphonothioate in which the oxygen atom of the phosphate site is substituted with a sulfur atom -Type artificial nucleic acid; substituent-modified type on furanose ring, pyranose type with 1-carbon increase in sugar ring skeleton, or polycyclic sugar skeleton type artificial nucleic acid; pyrimidine C-5 modified base type, purine C-7 Examples thereof include artificial nucleic acids having a position-modified base type or a ring-expanded modified base type. In addition, those having a label added thereto, and those having a reactive functional group added to be immobilized on a carrier are also included.

  In the present specification, the probe polynucleotide has a meaning usually used in the art, and is a polynucleotide used for detecting a target gene, specifically a polynucleotide corresponding to the target gene or a fragment thereof. A polynucleotide that hybridizes. As the probe polynucleotide, synthetic oligonucleotides, cDNA and genomic DNA, fragments thereof, and those obtained by denaturing them (for example, those obtained by modifying a single strand into a double strand) are used. The probe polynucleotide usually has a length of 20 to 5000 bases, preferably 20 to 1500 bases.

  In this specification, a target polynucleotide has the meaning normally used in this technical field, and refers to the polynucleotide which is a detection target. A target may be referred to as a target. Usually, a polynucleotide derived from a test sample to be hybridized to a probe and a polynucleotide enzymatically synthesized based on the polynucleotide, specifically, mRNA, cDNA, aRNA, fragments thereof, and their denatured A thing etc. are used.

  Hybridization or hybridization has the meaning normally used in the art, and polynucleotides having complementary sequences, for example, single-stranded DNAs, single-stranded RNAs, or single-stranded It means that DNA and single-stranded RNA form a double strand under appropriate conditions.

The method of analyzing a polynucleotide sample using a carrier on which a probe polynucleotide is immobilized refers to an analysis method commonly used in the art, and is usually performed by the following steps:
For contacting a polynucleotide sample with a probe polynucleotide immobilized on a carrier to hybridize the target polynucleotide contained in the polynucleotide sample to the probe polynucleotide, removing unreacted polynucleotide, and hybridization. Measuring a signal based thereon.

  The polynucleotide sample is not particularly limited as long as it can contain a target polynucleotide that hybridizes to a probe polynucleotide immobilized on a carrier. For example, blood, serum, urine, tears, cells, organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue, chondrocytes, synovial macrophages, endothelial cells, skin, extracts and debris thereof, and Examples include biological samples such as cultured cells, embryonic cells, stem cells, animal cell extracts, plant extracts, prokaryotic cell extracts, and eukaryotic single cell extracts. The polynucleotide sample includes a sample derived from the above biological sample, for example, a sample in which a specific nucleic acid is amplified. Usually, hybridization is detected by using a polynucleotide sample labeled with the contained polynucleotide. A polynucleotide sample (hereinafter also referred to as a hybridization solution) is prepared by dissolving or dispersing the biological sample and a sample derived therefrom in an aqueous medium such as SSC (saline-sodium citrate). be able to.

  Measuring a signal based on hybridization means, in other words, measuring a signal indicating hybridization between a probe polynucleotide and a target polynucleotide, usually a signal based on a label. Depending on the detection method, labeling method and type of label.

The labeling method and the type of label are not particularly limited as long as hybridization between the probe polynucleotide and the target polynucleotide can be detected, and is known in the art. For example, when a target polynucleotide is synthesized or amplified, a substrate (mainly UTP) to which a label such as a radioactive label or a fluorescent label is covalently bound is incorporated, and hybridization can be detected by the label. As the label, those usually used in the art, for example, a radioactive label, a fluorescent label, a chemiluminescence, a luminescence and a photosensitizer can also be used. Examples of fluorescent labels include CyDye such as Cy3 and Cy5, FITC, RITC, rhodamine, Texas Red, TET, TAMRA, FAM, HEX, ROX, and the like. As radioactive labels, α- ³² P, γ- ³² P , ³⁵ S and the like. Furthermore, an enzyme, an enzyme fragment, an enzyme inhibitor, an antibody, a catalyst or the like may be bound.

  For example, when a fluorescent label is used as the label, the fluorescent signal is detected by a suitable detector. As the detector, for example, a fluorescence scanning apparatus in which a fluorescence laser microscope, a cooled CCD camera, and a computer are connected is used, and the fluorescence intensity on the carrier can be automatically measured. A confocal laser or a non-focus laser may be used instead of the CCD camera. Thereby, image data is obtained. From the obtained data, target polynucleotides having complementarity to the probe polynucleotide immobilized on the carrier can be identified, and gene expression profiles can be created based on the target polynucleotides. The sequence can also be determined.

  Hybridization of the target polynucleotide to the probe polynucleotide can be carried out by spotting the polynucleotide sample on a carrier on which the probe polynucleotide is immobilized and incubating the sample. For example, spotting can be performed by dispensing a sample into a plastic plate having 96 holes or 384 holes and dropping the sample using a spot device. Incubation is preferably carried out at a temperature ranging from room temperature to 100 ° C. for a time ranging from 1 to 24 hours.

  It is preferable to remove unreacted polynucleotide after completion of hybridization. For example, washing can be performed using a mixed solution of a surfactant solution and a buffer to remove unreacted polynucleotide. As the surfactant, sodium dodecyl sulfate (SDS) is preferably used. As the buffer solution, a citrate buffer solution, a phosphate buffer solution, a borate buffer solution, a Tris buffer solution, a Good buffer solution, or the like can be used, and a citrate buffer solution is preferably used.

  The carrier regeneration method of the present invention is the above analysis method, wherein the target polynucleotide is at least selected from the group consisting of a humectant, preferably a gelling agent and a carbohydrate, on the carrier hybridized to the probe polynucleotide. It includes a step of applying an aqueous dispersion containing one kind. The above step is performed after the hybridization reaction. Preferably, after completion of hybridization, it is carried out after removing unreacted polynucleotide and before measuring the signal.

  The present inventors apply an aqueous dispersion containing at least one humectant, preferably at least one selected from the group consisting of a gelling agent and a saccharide, to the carrier after analysis. It has been found that the removal of the target polynucleotide hybridized with the probe polynucleotide is promoted in the regeneration step. Therefore, the aqueous dispersion can be used as a reagent for promoting the regeneration of the carrier in the method of analyzing a polynucleotide sample using the carrier on which the probe polynucleotide is immobilized.

  The aqueous dispersion can be prepared by dispersing or dissolving at least one humectant, preferably at least one selected from the group consisting of a gelling agent and a saccharide, in a solvent containing water as a main component. In the present invention, the aqueous dispersion includes an aqueous solution. As the solvent, a solvent containing 70% or more of water, preferably 80% or more, more preferably 90% or more is used. As the solvent, preferably pure water, more preferably ultrapure water is used. The aqueous dispersion of the present invention uses a humectant at a saturation concentration or less.

  In this specification, the humectant is a substance having an effect of providing moisture to a substance or tissue by contacting, penetrating or binding to a certain substance or tissue, or a substance having an effect of retaining moisture. Defined and includes gelling agents and carbohydrates. Specifically, glycerin, xylitol (xylit), diglycerin, dipropylene glycol (DPG), sorbitol (sorbit), sodium DL-pyrrolidonecarboxylate, 3-methyl-1,3-butanediol, 1,3-butylene Polyols such as glycol (1, 3BG), propylene glycol, polyethylene glycol (carbowax), polyglycerol, mixed isomerized sugar, amino acid, L-aspartic acid, sodium L-aspartate, DL-alanine, L- Arginine, L-isoleucine, L-lysine, L-lysine hydrochloride, L-glycine (aminoacetic acid), L-glutamine, L-glutamic acid, sodium L-glutamate, γ-aminobutyric acid (piperidine acid), L- Threonine, L-serine, L-tyrosine, L- Liptophan, L-valine, L-histidine, L-histidine hydrochloride, L-hydroxyproline (L-oxyproline), L-phenylalanine, L-proline, L-leucine, L-cysteine, L-cystine, L- Methionine, DL-pyrrolidonecarboxylic acid (PCA), DL-pyrrolidonecarboxylate sodium solution (PCA soda), lactic acid, sodium lactate (solution), urea, uric acid, acidic mucopolysaccharide, hyaluronic acid, sodium hyaluronate, chondroitin sulfate sodium , Glucuronic acid, collagen, soluble collagen (water-soluble collagen), collagen hydrolyzate (hydrolyzed collagen), hydrolyzed collagen liquid, hydrolyzed collagen powder, hydrolyzed collagen ethyl, hydrolyzed collagen hexadecyl, collagen amino acid, atelocolla Gen, gelatin, hydrolyzed gelatin powder, elastin, water-soluble elastin, hydrolyzed elastin (elastin hydrolyzate), intercellular lipid, sphingolipid (ceramide), keratin, hydrolyzed keratin, keratin amino acid, nucleic acid, deoxyribonucleic acid (DNA) ), Ribonucleic acid (RNA), guanosine, guanine, phosphate, ATP (adenosine triphosphate), phosphoflavin sodium phosphate, phospholipid, lecithin and other biological system moisturizing ingredients, albumin (dried deglycated egg white), serum albumin, Non-fat dry milk, whey (whey, lactic acid bacteria fermentation liquid), lactose, casein, milk sugar protein, lactoferrin, hydrolyzed silk, silk amino acid, silk extract, silk powder, sericin, hydrolyzed conchiolin liquid (pearl protein drawing liquid) , Conchiolin powder, chitin, chitosa , Glucosamine (chitosamine), royal jelly, honey, glucose (glucose), soy protein, soy protein hydrolyzate, soy phospholipid (soy lecithin), soy lysophospholipid (lysolecithin), egg yolk lecithin (yolk phospholipid), natto extract (Soybean fermentation metabolite), alpha hydroxy acid (AHA), glycolic acid, malic acid, seaweed extract, brown algae extract, sulfur-containing aluminum silicate (marine clay), magnesium chloride, marine collagen, yeast extract, dry yeast, pullulan, Moisturizing ingredients composed of animal and plant ingredients such as sulfakinin and trehalose can be mentioned.

  In the present specification, the gelling agent is not particularly limited as long as the phase change from solid to liquid at about 10 to 90 ° C. is observed in the substance itself or a mixture of the substance and a solvent. The gelling agent usually has a moisturizing effect on the substance itself or a mixture of the substance and a solvent. Specifically, gelatin, collagen, agar, carrageenan, pectin, alginic acid, sodium alginate, mannan, konjac, konjac mannan, glucomannan, chitosan, xanthan gum, tamarind seed polysaccharide, gellan gum, caraya gum, acacia gum, pullulan, curdlan Xyloglucan, guar gum, gum arabic, locust bean gum, carboxymethyl cellulose polyvalent metal salt, DPPC (Dipalmitoyl phosphatidylcholine), poly (N-isopropylacrylamide), poly (ε-caprolactone) and the like. A single gelling agent may be used, or a plurality of types may be used in combination. For example, if the gelling agent is gelatin, the aqueous dispersion of the present invention is 0.05% (W / V) or more and 3.0% (W / V) or less, preferably 0.5% (W / V). The gelling agent is contained in an amount of 2.0% (W / V) or less, more preferably 1.0% (W / V) or more and 1.5% (W / V) or less.

  In this specification, saccharides include monosaccharides, oligosaccharides (such as disaccharides, trisaccharides and tetrasaccharides) and polysaccharides, and reduced derivatives thereof (sugar alcohols, deoxysugars, glycals), alkylated derivatives (alkylated sugars). ), Sulfated derivatives (sulfated sugars), oxidized derivatives (aldonic acid, uronic acid, aldaric acid, etc.) and dehydrated derivatives (glycosene, anhydrosugar, etc.). Carbohydrates usually have a moisturizing effect in the substance itself or in a mixture of the substance and a solvent. As the saccharide, a water-soluble saccharide is preferably used. Moreover, what has the property to melt | dissolve in a solvent slowly at room temperature is preferable. Specific examples of carbohydrates include monosaccharides such as glucose, galactose, mannose, arabinose, ribose, fructose, xylose, sorbose, α, α-trehalose (trehalose), alkylated trehalose, sulfated trehalose, β, β-trehalose (Isotrehalose), α, β-trehalose (neotrehalose), trehalosamine, kojibiose, nigerose, maltose, maltitol, maltulose, isomaltose, isomaltulose (palatinose), sophorose, laminarabiose, cellobiose, gentiobiose, galactosucrose, lactose , Lactosamine, lactose diamine, lactobionic acid, lactitol, lactulose, melibiose, neolactose, primeverose, lutinose, sylabiose, sclaro , Sucrose (sucrose), turanose, vicyanose, cellotriose, cacotriose, gentianose, isomaltotriose, isopanose, maltotriose, manninotriose, melezitose, panose, planteose, raffinose, soratriose, umbelliferose, lycotetraose , Maltotetraose, stachyose, maltopentaose, verbascose, maltohexaose, α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), δ -Oligosaccharides such as cyclodextrin (cyclomaltononaose) and their derivatives, polysaccharides such as starch hydrolysates and dextran, and ethylene glycol, diethylene glycol, polyethylene glycol, dipro Glycol, polypropylene glycol, glycerin, diglycerin, polyglycerin, 1,3-butylene glycol, erythritol, pentitol, hexitol, xylitol, such as sugar alcohols such as sorbitol and mannitol. Carbohydrates may be used alone or in combination of two or more. For example, if the carbohydrate is trehalose, the aqueous dispersion of the present invention has a concentration of 0.1% (W / V) to 30% (W / V), preferably 1.0% (W / V) to 25%. (W / V) or less, More preferably, it contains 5% (W / V) or more and 20% (W / V) or less of carbohydrates.

  The aqueous dispersion of the present invention may contain one kind of humectant, preferably any one of a gelling agent and a saccharide, and may contain a plurality of humectants. In the present invention, the aqueous dispersion may appropriately contain components other than the humectant, such as a buffering agent and a surfactant.

  The method for applying the aqueous dispersion to the carrier is not particularly limited as long as the aqueous dispersion can be applied to the entire surface on which the probe polynucleotide is immobilized. For example, the support may be applied by immersing the carrier in an aqueous dispersion. In that case, after the immersion, the aqueous dispersion can be uniformly applied to the surface of the carrier by subjecting the carrier to a centrifugal treatment. The aqueous dispersion may be applied by dropping or spraying on the surface of the carrier.

  The method for applying the aqueous dispersion to the carrier is not particularly limited as long as the aqueous dispersion can be applied to the entire surface on which the probe polynucleotide is immobilized, and the water is retained on the surface of the carrier.

  The carrier coated with the aqueous dispersion is subjected to measurement of a signal based on hybridization to analyze a polynucleotide sample, and then the carrier is washed to regenerate the carrier, that is, the polynucleotide hybridized to the probe polynucleotide Can be removed. The conditions in this washing step can be set as appropriate based on the type of aqueous dispersion used. Usually, cleaning is performed using an aqueous cleaning solution. The aqueous cleaning liquid refers to a cleaning liquid mainly composed of water, and may contain a surfactant, a buffering agent, or the like. As the aqueous cleaning liquid, preferably ultrapure water is used. Specifically, the cleaning can be performed by using an aqueous cleaning liquid having a boiling point or lower, preferably 30 ° C. or higher and 100 ° C. or lower, more preferably 60 ° C. or higher and 100 ° C. or lower.

  The washing time can be appropriately set according to the step of measuring a signal based on hybridization. In addition, the signal based on hybridization usually refers to measuring a signal based on a label, and the step of measuring the signal is not particularly limited as long as it can detect hybridization between the probe polynucleotide and the target polynucleotide. .

In other words, the present invention also provides a method for analyzing a polynucleotide sample using a carrier on which a probe polynucleotide is immobilized,
a) contacting a polynucleotide sample with a probe polynucleotide immobilized on a carrier to hybridize the target polynucleotide contained in the polynucleotide sample to the probe polynucleotide;
b) removing unreacted polynucleotide;
c) applying an aqueous dispersion containing at least one humectant, preferably at least one selected from the group consisting of a gelling agent and a saccharide, to a carrier; and d) measuring a signal based on hybridization. The method comprising the steps of: By carrying out step c) in the method for analyzing a polynucleotide sample, the carrier can be easily regenerated by washing after the analysis.

  As the carrier for immobilizing the probe polynucleotide, those commonly used in the art can be used, and the shape and material are not particularly limited. For example, a flat plate such as a microarray, a particle such as a bead, and a thread may be used, and a flat plate such as a microarray is preferably used. Examples of the material include metals such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium, and nickel; alloys such as stainless steel, hastelloy, inconel, monel, and duralumin; and the above metals and ceramics. Laminate; Silicon; Glass material such as glass, quartz glass, fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite and photosensitive glass; plastic (for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile) Butadiene Styrene resin), nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin).

  In the present invention, a carrier having an electrostatic layer for electrostatically attracting a polynucleotide on a substrate and a functional group capable of covalently bonding to the polynucleotide is preferably used. Here, the material of the substrate is not particularly limited, and examples thereof include the above materials. The shape and size of the substrate are not particularly limited, and examples of the shape include a flat plate shape, a thread shape, a spherical shape, a polygonal shape, a powder shape, and the size is usually 0.1 mm in width when a flat plate shape is used. ˜100 mm, length 0.1˜100 mm, thickness 0.01˜10 mm.

  The substrate may be subjected to a surface treatment. For surface treatment, synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene, carbon nanotube), a mixture thereof, or It is preferable to use a laminate of them. Further, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may be used. Here, the soft diamond is a generic term for an imperfect diamond structure that is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. The thickness of the surface treatment layer is preferably 1 nm to 100 μm.

  As an example of the surface-treated substrate, a substrate obtained by forming a soft diamond film on a slide glass can be given. Such a substrate is preferably prepared by ionized vapor deposition of diamond-like carbon in a mixed gas containing 0 to 99% by volume of hydrogen gas and 100 to 1% by volume of the remaining methane gas.

  The surface treatment layer of the substrate is formed by a known method, for example, microwave plasma CVD (Chemical Vapor Deposit) method, ECRCVD (Electric Cyclotron Resonance Chemical Vapor Deposit) method, ICP (Inductive Coupled Plasma) method, DC sputtering method, ECR (Electric Cyclotron Resonance) A sputtering method, an ion plating method, an arc ion plating method, an EB (Electron Beam) vapor deposition method, a resistance heating vapor deposition method, an ionization vapor deposition method, an arc vapor deposition method, a laser vapor deposition method, or the like.

  The carrier of the present invention is preferably provided with an electrostatic layer in order to attract the polynucleotide electrostatically. The electrostatic layer is not particularly limited as long as it attracts polynucleotides electrostatically and improves the amount of immobilized polynucleotides. For example, a positively charged compound such as an amino group-containing compound is used. Can be formed.

Examples of the amino group-containing compound include an unsubstituted amino group (—NH ₂ ), or a compound having an amino group (—NHR; R is a substituent) monosubstituted by an alkyl group having 1 to 6 carbon atoms, for example, Ethylenediamine, hexamethylenediamine, n-propylamine, monomethylamine, dimethylamine, monoethylamine, diethylamine, allylamine, aminoazobenzene, aminoalcohol (eg, ethanolamine), acrinol, aminobenzoic acid, aminoanthraquinone, amino acid (glycine, alanine) , Valine, leucine, serine, threonine, cysteine, methionine, phenylalanine, tryptophan, tyrosine, proline, cystine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, histidine ), Aniline, or polymers thereof (for example, polyallylamine, polylysine) or copolymers; polyamines (polyvalent amines) such as 4,4 ′, 4 ″ -triaminotriphenylmethane, triamterene, spermidine, spermine, and putrescine ).

  The electrostatic layer may be formed without being covalently bonded to the substrate or the surface treatment layer, or may be formed to be covalently bonded to the substrate or the surface treatment layer.

  In the case where the electrostatic layer is formed without being covalently bonded to the substrate or the surface treatment layer, for example, when the surface treatment layer is formed, the amino group-containing compound is introduced into the film forming apparatus to thereby form an amino group. A carbon-based film containing is formed. Ammonia gas may be used as the compound introduced into the film forming apparatus. Further, the surface treatment layer may be a multi-layer structure in which a film containing an amino group is formed after the adhesion layer is formed, and in this case, it may be performed in an atmosphere containing ammonia gas.

  In the case where the electrostatic layer is formed without being covalently bonded to the substrate or the surface treatment layer, the affinity between the electrostatic layer and the substrate or the surface treatment layer, that is, the adhesion is improved on the substrate. It is preferable to introduce a functional group capable of covalently bonding to a polynucleotide after vapor-depositing the compound having an unsubstituted or monosubstituted amino group and a carbon compound. The carbon compound used here is not particularly limited as long as it can be supplied as a gas. For example, methane, ethane, and propane which are gases at normal temperature are preferable. As a method of vapor deposition, ionized vapor deposition is preferable, and as conditions for ionized vapor deposition, it is preferable that an operating pressure is 0.1 to 50 Pa and an acceleration voltage is in a range of 200 to 1000 V.

  When the electrostatic layer is formed by covalently bonding to the substrate or the surface treatment layer, for example, the surface of the substrate or the substrate to which the surface treatment layer is applied is chlorinated by irradiating ultraviolet rays in chlorine gas, and then the amino group Among the contained compounds, for example, a polyamine such as polyallylamine, polylysine, 4,4 ′, 4 ″ -triaminotriphenylmethane, and triamterene is reacted to form an amino group at the terminal not bonded to the substrate. By introducing, an electrostatic layer can be formed. After chlorination of the substrate surface, amino groups may be introduced by UV irradiation in ammonia gas.

  In addition, when a reaction for introducing a functional group capable of covalently bonding to a polynucleotide (for example, introduction of a carboxyl group using a dicarboxylic acid or a polycarboxylic acid) in a solution is performed in a solution, It is preferable to introduce a functional group that can be covalently bonded to the polynucleotide after the substrate is immersed in the solution containing the compound having an unsubstituted or monosubstituted amino group. Examples of the solvent for the solution include water, N-methylpyrrolidone, and ethanol.

  When a carboxyl group is introduced into a substrate on which an electrostatic layer has been applied using dicarboxylic acid or polyvalent carboxylic acid, the substrate is activated with N-hydroxysuccinimide and / or carbodiimide in advance, or the reaction is N -It is preferably carried out in the presence of hydroxysuccinimide and / or carbodiimides.

  In the case where an electrostatic layer is formed by immersing the substrate in a solution containing a compound having an unsubstituted or monosubstituted amino group, when polyallylamine is used as the amino group-containing compound, the substrate is in close contact with the substrate. And the amount of immobilized polynucleotide is further improved.

The thickness of the electrostatic layer is preferably 1 nm to 500 μm.
As described above, it is preferable to chemically modify the surface of the substrate on which the electrostatic layer has been applied in order to introduce a functional group that can be covalently bonded to the polynucleotide.

  Examples of the functional group include a carboxyl group, an active ester group, a haloformyl group, a hydroxyl group, a cyano group, a nitro group, a thiol group, and an amino group.

As a compound used for introducing a carboxyl group as a functional group, for example, the formula: X—R ¹ —COOH (wherein X is a halogen atom, R ¹ is a divalent hydrocarbon group having 1 to 12 carbon atoms) For example, chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC Dicarboxylic acid represented by —R ² —COOH (wherein R ² represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, Fumaric acid, phthalic acid; polyacrylic acid, polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid and other polyvalent carboxylic acids; formula: R ³ —CO—R ⁴ —COOH (formula In which R ³ represents a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms, and R ⁴ represents a divalent hydrocarbon group having 1 to 12 carbon atoms). A monohalide of a dicarboxylic acid represented by: X—OC—R ⁵ —COOH (wherein X represents a halogen atom, R ⁵ represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), for example, Examples include succinic acid monochloride, malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

  The active ester group means an ester group having a highly acidic electron-attracting group on the alcohol side of the ester group and activating a nucleophilic reaction, that is, an ester group having a high reaction activity. Those commonly used in the fields of synthesis, such as polymer chemistry and peptide synthesis.

  Actually, the ester group has an electron-attracting group on the alcohol side of the ester group and is activated more than the alkyl ester. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc. Known as. Specific examples of the active ester group include a p-nitrophenyl group, an N-hydroxysuccinimide group, a succinimide group, a phthalimide group, and a 5-norbornene-2,3-dicarboximide group.

  For example, the N-hydroxysuccinimide group is formed by adding a dehydration condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) to the carboxyl group introduced as described above. It can be produced by reacting a compound such as N-hydroxysuccinimide.

Examples of the compound used for introducing a haloformyl group as a functional group include, for example, the formula: X—OC—R ⁶ —CO—X (wherein X is a halogen atom, R ⁶ is a single bond or 1 to 12 carbon atoms). And dicarboxylic acid dihalides such as succinic acid chloride and malonic acid chloride.

As a compound used for introducing a hydroxyl group as a functional group, for example, represented by the formula: HO—R ⁷ —COOH (wherein R ⁷ represents a divalent hydrocarbon group having 1 to 12 carbon atoms). Hydroxy acids or phenolic acids.

  Examples of the compound used for introducing an amino group as a functional group include amino acids.

  In the above compound, the carboxyl group is condensed with the amino group of the electrostatic layer to form an amide bond.

  Among the above-mentioned compounds, polycarboxylic acids such as polyacrylic acid, polymethacrylic acid, trimellitic acid, and butanetetracarboxylic acid can be used for improving hydrophilicity.

  In the above step, the treatment with the amino group-containing compound, the functional group introduction treatment, and the active esterification treatment are preferably repeated several times.

  The polynucleotide can be immobilized on the carrier by dissolving or dispersing the probe polynucleotide in an appropriate solvent and spotting the probe on the carrier. For example, a solution containing the probe polynucleotide is dispensed onto a 96-hole or 384-hole plastic plate, and the dispensed solution is dropped on a carrier using a spot device or the like to perform spotting. As the spot device, a spot device using a pin method, an ink jet method, a capillary method using a capillary tube, or the like can be used.

  The present invention also includes contacting a polynucleotide sample with a probe polynucleotide immobilized on a carrier, hybridizing a target polynucleotide contained in the polynucleotide sample to the probe polynucleotide, and measuring a signal based on hybridization. A kit for analyzing a polynucleotide sample, comprising: a reagent for promoting regeneration of a carrier comprising an aqueous dispersion containing at least one selected from the group consisting of a gelling agent and a saccharide. . The kit is characterized by containing a reagent for promoting the regeneration of the carrier in addition to a kit for analyzing a known polynucleotide sample. The kit of this invention can be comprised by each element used for the well-known and official kit except including the reagent for promoting the reproduction | regeneration of the said support | carrier. For example, an enzyme for degrading or fragmenting a polynucleotide, a buffer solution, a matrix solvent, a washing buffer, a sample diluent, a reaction stop solution, a standard substance and the like can be included. Further, an instruction manual for explaining the assay method can be combined with the kit of the present invention.

Example 1 Preparation of Fluorescent Labeled cDNA (Target Polynucleotide) For preparation of fluorescently labeled cDNA, CyScribe First-Strand cDNA Labeling Kit manufactured by Amersham Biosciences was used.

  A heat block of 70 ° C., 42 ° C., and 37 ° C. or a thermostat was prepared in advance, and the mRNA sample and each reagent were dissolved and prepared on ice. Next, the following system including mRNA samples was assembled. This system was sufficient to carry out subsequent hybridizations three times.

  After assembling the above reaction system, the mixture was kept at 70 ° C. for 5 minutes, transferred to room temperature, and left for 10 minutes. Reagents were further added to the reaction solution as described below to form a reaction system. The subsequent operations were performed under light-shielding conditions as much as possible.

  After assembling the above reaction system, incubation was carried out at 42 ° C. for 90 minutes, and fluorescently labeled cDNA was synthesized by reverse transcription reaction. Next, 2 μl of 2.5 M NaOH was added and incubated at 37 ° C. for 15 minutes to decompose unreacted mRNA. Next, 10 μl of 2M HEPES was neutralized to obtain a fluorescently labeled cDNA solution.

  Next, the fluorescence-labeled cDNA was purified using a Cyscribe GFX Purification Kit manufactured by Amersham Biosciences. A GFX spin column was set in the collection tube, 500 μl of Capture buffer was added to the GFX spin column, a fluorescence labeled cDNA solution was further added, and mixed by pipetting. Centrifugation was performed at 13,000 rpm for 30 seconds, and the filtrate in the collection tube was discarded. The GFX spin column was set again in the same collection tube, and 600 μl of Wash buffer was added. Centrifugation was performed at 13,000 rpm for 30 seconds, and the filtrate in the collection tube was discarded. The washing process was repeated twice more. The GFX spin column was set again in the same collection tube, and centrifuged at 13,000 rpm for 10 seconds to completely remove the wash buffer attached to the column. A GFX spin column was set in a new 1.5 ml microtube, and 22 μl of Elution buffer that had been kept at 65 ° C. in advance was added to the center of the filter and left for 10 minutes. Centrifugation was performed at 13,000 rpm for 1 minute, and the filtrate was recovered in the microtube. The elution process was repeated twice more and collected in the same microtube. A total of 66 μl of fluorescence labeled cDNA solution was obtained by the above operation. The obtained fluorescently labeled cDNA solution was dispensed in 22 μl aliquots into new microtubes, dried to dryness with a speed bag and stored at −20 ° C. in the dark.

Example 2 Production of Microarray (Probe Polynucleotide Immobilization Carrier) By immersing a slide glass of 25 mm (width) × 75 mm (length) × 1 mm (thickness) in a polyallylamine aqueous solution (0.1 g / l), An electrostatic layer was formed. Thereafter, polyacrylic acid as a polyvalent carboxylic acid was condensed to the amino group of the electrostatic layer in the presence of 0.1 M 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide. Then, an activation solution in which 0.1 M 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride and 20 mM N-hydroxysuccinimide were dissolved in 300 ml of 0.1 M phosphate buffer (pH 6). A carrier having an electrostatic layer and an N-hydroxysuccinimide group on the surface of the glass substrate was produced by activating by dipping in the substrate for 30 minutes.

  A probe polynucleotide corresponding to the fluorescence-labeled cDNA obtained above was spotted on the obtained carrier to obtain a microarray in which the probe polynucleotide was immobilized on the carrier.

Example 3 Regeneration Method Using Gelatin (1) Pretreatment of Microarray The microarray produced in Example 2 was immersed in 2 × SSC / 0.2% SDS and allowed to stand at room temperature for 15 minutes. Thereafter, the microarray was immersed in 2 × SSC / 0.2% SDS that had been heated to 90 ° C. in advance, heated for 5 minutes, rinsed with ultrapure water, centrifuged at 300 rpm for 3 minutes, and the moisture on the surface of the macroarray. Was removed.

(2) Hybridization 18 μl of 5 × SSC / 0.5% SDS solution was added to the dried fluorescently labeled cDNA to prepare a hybridization solution. The pretreated microarray was set in a hybridization cassette (ArrayIt), a cover glass with a gap of 24 × 24 mm was placed so as to match the spot position, and the hybridization solution was poured by capillary action. 30 μl each of purified water and 5 μl of 3 × SSC were dropped at two points away from the cover glass on the glass in the recesses in the cassette to prevent drying during hybridization. Hybridization was performed at 60 ° C. for 16 hours.

(3) Washing (removal of unreacted polynucleotide)
The microarray was immersed in 2 × SSC / 0.2% SDS to wash the cover glass, and washed with 2 × SSC / 0.2% SDS for 15 minutes. This was washed with 2 × SSC and centrifuged at 300 rpm for 3 minutes to remove moisture on the surface of the macroarray. In the control experiment, the fluorescence intensity was measured at this stage in the same manner as (5) below (FIG. 1a).

(4) Gelatin coating This microarray was immersed in 0.2 × SSC / 1 mM EDTA / 1.5% (W / V) gelatin previously heated to 50 to 60 ° C. and immediately dissolved at 300 rpm. Centrifugation was carried out for 3 minutes, and the mixture was allowed to stand at room temperature in the dark to gelatinize the gelatin. As gelatin, “Gelatin 21” manufactured by Nitta Gelatin Co., Ltd. was used.

(5) Measurement of fluorescence and quantification of fluorescence intensity The fluorescence of the fluorescence-labeled cDNA hybridized to the spot on the microarray was measured with a microarray scanner GeneTAC UC-4 (manufactured by Genomic Solutions). Quantification of fluorescence intensity was performed using ScanAlyze ver. 2.5 was used (FIG. 1b).

(6) Regeneration of carrier A solution prepared by heating ultrapure water (about 50 mL) in a 50 mL Falcon tube on a boiling water bath was prepared in advance. The microarray whose fluorescence intensity was measured was immersed in ultrapure water heated on the boiling water bath and washed for 15 minutes. This operation was repeated a total of 4 times to remove the fluorescently labeled cDNA hybridized to the spot on the microarray. That is, the heat cleaning process with ultrapure water was performed for a total of 60 minutes. Thereafter, the microarray was centrifuged at 300 rpm for 3 minutes to remove moisture on the surface of the macroarray, and fluorescence was measured with a microarray scanner in the same manner as in (5) (FIG. 2).

  Eight spots: ROW4 COL6, ROW4 COL7, ROW5 COL4, ROW5 COL5 on grid 1 and ROW4 COL6, ROW4 COL7, ROW5 COL4, ROW5 COL5 (see Figure 7) on grid 2 and when applied The results of measuring the fluorescence intensity after the first hybridization and the first regeneration in the absence (control) are shown in Table 3 and FIG. 1c below.

(7) The steps (1) to (3) were repeated for the gelatin-coated microarray, the second hybridization after the regeneration was performed, and the fluorescence was measured with a microarray scanner (FIG. 3a). The carrier was again regenerated (6) and fluorescence was measured with a microarray scanner (FIG. 3b).

result:
After the regeneration step of (6), hybridized fluorescently labeled cDNA remains in the carrier that was not gelatin-coated (FIG. 2a), whereas the carrier that was gelatin-coated was hybridized. It can be seen that the fluorescently labeled cDNA was almost completely removed (FIG. 2b). Furthermore, it was shown that after the regeneration step, the fluorescently labeled cDNA rehybridizes to the probe polynucleotide on the carrier (FIG. 3a) and can be regenerated again (FIG. 3b). Therefore, according to the present invention, it was shown that the probe polynucleotide-immobilized carrier can be regenerated and reused.

Example 4 Regeneration Method Using Trehalose (1) Pretreatment of Microarray The microarray produced in Example 2 was immersed in 2 × SSC / 0.2% SDS and allowed to stand at room temperature for 15 minutes. Thereafter, the microarray was immersed in 2 × SSC / 0.2% SDS that had been heated to 90 ° C. in advance, heated for 5 minutes, rinsed with ultrapure water, centrifuged at 300 rpm for 3 minutes, and the moisture on the surface of the macroarray. Was removed.

(2) Hybridization 18 μl of 5 × SSC / 0.5% SDS solution was added to the dried fluorescently labeled cDNA to prepare a hybridization solution. The pretreated microarray was set in a hybridization cassette (ArrayIt), a cover glass with a gap of 24 × 24 mm was placed so as to match the spot position, and the hybridization solution was poured by capillary action. 30 μl each of purified water and 5 μl of 3 × SSC were dropped at two points away from the cover glass on the glass in the recesses in the cassette to prevent drying during hybridization. Hybridization was performed at 60 ° C. for 16 hours.

(3) Washing (removal of unreacted polynucleotide)
The microarray was immersed in 2 × SSC / 0.2% SDS to wash the cover glass, and washed with 2 × SSC / 0.2% SDS for 15 minutes. This was washed with 2 × SSC and centrifuged at 300 rpm for 3 minutes to remove moisture on the surface of the macroarray. In the control experiment, the fluorescence intensity was measured at this stage in the same manner as (5) below (FIG. 4a).

(4) Trehalose coating This microarray was immersed in a 2 × SSC / 20% (W / V) trehalose solution, immediately centrifuged at 300 rpm for 3 minutes, and allowed to stand at room temperature, protected from light. As trehalose, Sigma D-(+)-Trehalose dehydrate (T9531): α-D-glucopyranosyl-α-D-glucopyranoside was used.

(5) Measurement of fluorescence and quantification of fluorescence intensity The fluorescence of the fluorescence-labeled cDNA hybridized to the spot on the microarray was measured with a microarray scanner GeneTAC UC-4 (manufactured by Genomic Solutions). Quantification of fluorescence intensity was performed using ScanAlyze ver. 2.5 was used (Figure 4b).

(6) Regeneration of carrier A solution prepared by heating ultrapure water (about 50 mL) in a 50 mL Falcon tube on a boiling water bath was prepared in advance. The microarray whose fluorescence intensity was measured was immersed in ultrapure water heated on the boiling water bath and washed for 15 minutes. This operation was repeated a total of 4 times to remove the fluorescently labeled cDNA hybridized to the spot on the microarray. That is, the heat cleaning process with ultrapure water was performed for a total of 60 minutes. Thereafter, the microarray was centrifuged at 300 rpm for 3 minutes to remove moisture on the surface of the microarray, and fluorescence was measured with a microarray scanner in the same manner as in (5) (FIG. 5).

  Trehalose coating for 8 spots surrounded by red line, namely ROW4 COL6, ROW4 COL7, ROW5 COL4, ROW5 COL5 on grid 1 and ROW4 COL6, ROW4 COL7, ROW5 COL4, ROW5 COL5 on grid 2 (see Figure 7) Table 4 and FIG. 4c below show the results of measurement of the fluorescence intensity after the first hybridization and the first regeneration in the case where the measurement was performed and the case where the control was not performed (control).

(7) For the microarray coated with trehalose, the above steps (1) to (5) were repeated, the second hybridization after regeneration was performed, and the fluorescence was measured with a microarray scanner (FIG. 6a). The carrier was regenerated (6) again (in the second regeneration, washing was performed only once for 15 minutes), and fluorescence was measured with a microarray scanner (FIG. 6b).

result:
After the regeneration step of (6), the carrier that was not subjected to trehalose coating was left with hybridized fluorescently labeled cDNA (FIG. 5a), whereas the carrier that was coated with trehalose was hybridized. It can be seen that the fluorescently labeled cDNA was almost completely removed (FIG. 5b). Furthermore, it was shown that after the regeneration step, the fluorescently labeled cDNA re-hybridizes to the probe polynucleotide on the carrier (Fig. 6a) and can be regenerated again (Fig. 6b). Therefore, according to the present invention, it was shown that the probe polynucleotide-immobilized carrier can be regenerated and reused.

The fluorescence image measured after the 1st hybridization about the support | carrier (b) with which gelatin coating was performed, and the support | carrier (a) which was not performed is shown. Moreover, the measurement result of fluorescence intensity is shown (c). The fluorescence image measured after the 1st reproduction | regeneration processing about the support | carrier (b) with which gelatin coating was performed, and the support | carrier (a) which was not performed is shown. The fluorescence images measured for the gelatin-coated carrier after the second hybridization (a) and after the second regeneration treatment (b) are shown. The fluorescence image measured after the 1st hybridization about the support | carrier (b) with which the trehalose coating was performed, and the support | carrier (a) which was not performed is shown. Moreover, the measurement result of fluorescence intensity is shown (c). The fluorescence image measured after the 1st reproduction | regeneration processing is shown about the support | carrier (b) with which trehalose coating was performed, and the support | carrier (a) which was not performed. The fluorescence images measured after the second hybridization (a) and after the second regeneration treatment (b) for the carrier coated with trehalose are shown. The spot position on the carrier on which the probe polynucleotide is immobilized is shown. The spot positions of grid 1 and grid 2 are the same. In both grids 1 and 2, ROW1 COL6 and ROW1 COL7 are control spots on which Cy3 oligonucleotides are immobilized.

Claims (4)

A method for regenerating a carrier in a method for analyzing a polynucleotide sample using a carrier on which a probe polynucleotide is immobilized,
A step of applying an aqueous dispersion containing a humectant to a carrier in which the target polynucleotide is hybridized to the probe polynucleotide; and a step of washing the carrier, wherein the humectant is selected from the group consisting of a gelling agent and a saccharide. Said method comprising at least one selected. A method for removing a target polynucleotide hybridized to a probe polynucleotide immobilized on a carrier, comprising:
A step of applying an aqueous dispersion containing a humectant to a carrier in which the target polynucleotide is hybridized to the probe polynucleotide; and a step of washing the carrier, wherein the humectant is selected from the group consisting of a gelling agent and a saccharide. Said method comprising at least one selected. A method for analyzing a polynucleotide sample using a carrier on which a probe polynucleotide is immobilized, comprising:
a) contacting a polynucleotide sample with a probe polynucleotide immobilized on a carrier to hybridize the target polynucleotide contained in the polynucleotide sample to the probe polynucleotide;
b) removing unreacted polynucleotide;
c) applying an aqueous dispersion containing a humectant to a carrier; and d) measuring a signal based on hybridization, wherein the humectant is selected from the group consisting of a gelling agent and a carbohydrate. Said method comprising a species.   A method for regenerating a carrier by washing the carrier after the step d) according to claim 3.

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