WO2005078082A1 - Méthode de fabrication d'une sonde dna pour protéine membranaire de codage acide nucléique, méthode de détection de l'expression de protéine membranaire de codage de gêne et support d'immobilisation de sonde dna - Google Patents

Méthode de fabrication d'une sonde dna pour protéine membranaire de codage acide nucléique, méthode de détection de l'expression de protéine membranaire de codage de gêne et support d'immobilisation de sonde dna Download PDF

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WO2005078082A1
WO2005078082A1 PCT/JP2005/001914 JP2005001914W WO2005078082A1 WO 2005078082 A1 WO2005078082 A1 WO 2005078082A1 JP 2005001914 W JP2005001914 W JP 2005001914W WO 2005078082 A1 WO2005078082 A1 WO 2005078082A1
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Prior art keywords
dna
membrane protein
probe dna
base sequence
probe
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PCT/JP2005/001914
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English (en)
Japanese (ja)
Inventor
Masaharu Seno
Ya To
Hiroshi Okamura
Kouichi Hirayama
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Toyo Kohan Co., Ltd.
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Publication of WO2005078082A1 publication Critical patent/WO2005078082A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • the present invention provides a method for preparing a probe DNA for detecting the expression of a gene encoding a membrane protein, a method for detecting the expression of the gene using the probe DNA, and a method for detecting the expression of the gene using the probe DNA. And a method for detecting the expression of the gene using the carrier.
  • Membrane proteins comprise about 30% of all proteins in eukaryotic cells, and play an important role in receptor and ion channels, transporters, etc., in signal transduction and substance transport through cell membranes. I have. Membrane proteins are broadly divided into two types based on their structure. One is a superficial membrane protein bound to the cell membrane surface and the other is an integral membrane protein embedded in the lipid bilayer of the cell membrane. The structure of integral membrane proteins is composed of extracellular, intracellular and transmembrane domains. The transmembrane region is rich in hydrophobic amino acids and is thought to form an ⁇ -helix within the membrane.
  • this domain is used as a signal sequence that initiates membrane permeation of the nascent peptide chain following the C-terminus during biosynthesis of the membrane protein, or a stop transfer that stops permeation Serves as an array.
  • domains having respective functions are alternately arranged, and extracellular regions and intracellular regions are alternately arranged via these domains.
  • membrane proteins are specifically expressed in cancer cells, and these are considered to be useful in developing various diagnostic agents and anticancer agents as tumor antigens. It is said that 70% of drugs used in the treatment of various diseases and disorders are membrane proteins, and by analyzing the expression pattern of membrane proteins, tissue-specific therapeutic drug targets can be analyzed. It is expected to clarify.
  • Methods for detecting an interaction between specific proteins include an immunoprecipitation method and a Western blotting method, but these methods are not suitable for comprehensive analysis. Although yeast two-hybrid analysis can perform exhaustive analysis, it often detects many false positives and false negatives, making it difficult to immediately determine true positives and true negatives (Non-Patent Document 1) .
  • a method of analyzing the expression of a membrane protein As a method of analyzing the expression of a membrane protein, a method of analyzing a membrane protein itself can be considered.
  • membrane proteins are difficult to purify because of their high hydrophobicity, and because of the special environment of being present in the lipid bilayer of the cell membrane, their tertiary structure is complex, and they are less than water-soluble proteins that exist in cells. Its expression analysis is very difficult. Methods of analysis by RT-PCR or Northern plotting are not suitable for comprehensive analysis and are insufficient for expression analysis of extremely diverse membrane proteins as described above.
  • the method using a cDNA microarray has problems in that it takes time and effort to isolate cDNA, and the amount of DNA to be immobilized becomes unstable.
  • Non-patent document 1 edited by Tadaomi Takebashi et al., Bio Manual UP series, Experimental method for intermolecular interaction of proteins, Yodosha, 1996
  • An object of the present invention is to provide a method for rapidly and comprehensively detecting the expression of a gene encoding a membrane protein.
  • nucleic acid encoding a membrane protein can be specifically detected by using, as a probe, a DNA comprising a partial region containing at least a part of the exon sequence and containing a certain amount of cytosine and guanine, or a DNA complementary to the partial region. They found what they could do and completed the present invention.
  • the present invention includes the following inventions.
  • the partial region comprising the sequence and containing cytosine and guanine at a ratio of 40 to 60% in total is selected to prepare DNA comprising the partial region or DNA comprising a base sequence complementary to the partial region.
  • a partial region of a gene encoding a membrane protein wherein at least 5% of its nucleotide sequence is composed of a sequence in exon including a portion encoding a region essential for membrane binding in the membrane protein, and A carrier having immobilized thereon a probe DNA selected from a DNA comprising the partial region containing cytosine and guanine at a total ratio of 40 to 60% and a DNA comprising a nucleotide sequence complementary to the partial region.
  • (e) DNA consisting of a base sequence having homology to the DNA of (a)-(d) and specifically hybridizing to a nucleic acid encoding a corresponding membrane protein.
  • the carrier is a solid support having a functional group capable of covalently bonding to a probe DNA on a substrate and at least one surface layer selected from diamond, soft diamond, a carbon-based material, and a carbide.
  • the carrier for immobilizing the probe DNA according to (3) or (4).
  • a probe DNA selected from the group consisting of the following DNAs (a) to (e): (a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 11-385
  • (e) DNA consisting of a base sequence having homology to the DNA of (a)-(d) and specifically hybridizing to a nucleic acid encoding a corresponding membrane protein.
  • FIG. 1 shows spot positions of the 385 types of probe DNAs shown in Table 1 on a carrier.
  • the symbols shown in the left two columns of Table 1 correspond to the spot positions in FIG.
  • FIG. 2 shows the results of Example 1, in which probe DNA immobilized on a carrier was hybridized with cDNA labeled with Cy3 fluorescence and a fluorescence image was taken with FLA-8000.
  • Figure 3 shows Example 2. After probe cDNA on the carrier was hybridized with fluorescently labeled cDNA, the plate was washed with ultrapure water at 95 ° C for 30 minutes, and the fluorescent image was obtained with FLA-8000. The result of shooting is shown.
  • Figure 4 shows Example 2, in which probe-DNA on the carrier was hybridized with fluorescently labeled cDNA, washed with ultrapure water at 95 ° C for 30 minutes, and then rehybridized. The results of taking a fluorescence image with the FLA-8000 after performing the show are shown.
  • the probe DNA is a partial region of a gene encoding a membrane protein, and usually has a base sequence of 5% or more, preferably 60% or more, more preferably 80% or more, and still more preferably Is 90% or more, most preferably 95% or more is the exon sequence that encodes a region essential for membrane binding in a membrane protein, and cytosine and guanine are usually combined in a total of 40 to 60%, preferably 45 to 55%.
  • DNA comprising the partial region at a ratio of a DNA complementary to the DNA is also used as a probe DNA.
  • the membrane protein has a meaning usually used in the art, that is, a protein constituting a biological membrane.
  • Membrane proteins include surface-expressed proteins attached to the surface of biological membranes and integral membrane proteins buried inside, for example, receptors, transporters, membrane-bound enzymes, and cytoforce receptors.
  • Family ion channel, extracellular matrix protein, cell adhesion factor, membrane-bound cell growth factor, more specifically tyrosine kinase-type growth factor receptor, squamous cell carcinoma antigen (SCCA1), CD34, EGF family , EGF receptor family, type I and type II TGF beta receptor family, interleukin receptor family, TNF receptor family, Note h family, EGF-CFC family and the like.
  • SCCA1 squamous cell carcinoma antigen
  • EGF family EGF receptor family
  • type I and type II TGF beta receptor family interleukin receptor family
  • TNF receptor family Note h family, EGF-CFC family and the like.
  • the region essential for membrane binding means a region in a membrane protein that is responsible for binding between the membrane protein and a cell membrane.
  • Such regions include, but are not limited to, transmembrane regions that are usually composed of hydrophobic amino acids, regions that contain amino acids to which GPI anchors that bind to membranes through lipids, and the like. is not.
  • transmembrane region and the region containing the amino acid to which the GPI anchor binds in the membrane protein information that has already been published can be specified, or if known information cannot be obtained, the known information can be used.
  • the search can be performed using a prediction program or the like.
  • the base length of the probe DNA of the present invention is a length capable of hybridizing with the nucleic acid encoding the membrane protein, and is usually 10 to 5000 bases, preferably 15 to 500 bases, more preferably 20 to 100 bases, More preferably, it is 40 to 70 bases.
  • the probe DNA of the present invention is preferably selected from the group consisting of the following (a) to (e):
  • (e) DNA consisting of a base sequence having homology to the DNA of (a)-(d) and specifically hybridizing to a nucleic acid encoding a corresponding membrane protein.
  • a part of the DNA means at least 15 bases, more preferably at least 20 bases, and most preferably 30 to 60 bases in the base sequence represented by SEQ ID NO: 11-385. It means a portion consisting of a base sequence having a continuous base length.
  • a nucleotide sequence having homology to DNA is usually about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology.
  • Nucleic acids encoding membrane proteins include both DNA and RNA, and are preferably cDNA.
  • the term "specifically hybridizes” means that the hybridized state with the nucleic acid encoding the membrane protein can be maintained under stringent hybridization conditions.
  • the stringent hybridization conditions are, for example, conditions in which the sodium concentration is 15 to 40 mM, preferably 15 to 20 mM, and the temperature is 50 to 70 ° C, preferably 60 to 65 ° C. . In particular, the case where the sodium concentration is 16-17 mM and the temperature is 65 ° C. is most preferable.
  • the present invention also relates to a carrier having the probe DNA immobilized thereon.
  • the probe DNA-immobilized carrier is useful for detecting the expression of a membrane protein.
  • Immobilization of the probe DNA to the carrier can be performed by a method known in the art.
  • the spotting solution is prepared by dissolving or dispersing the probe DNA to be immobilized and its PCR product (DNA fragment obtained by amplifying DNA by PCR) in a solvent. By incubating the spotting solution after spotting on the carrier, the probe DNA can be immobilized on the carrier.
  • the solvent for dissolving or dispersing the probe DNA include polar organic solvents such as distilled water, SSC (saline-sodium citrate), PBS (phosphate buffered saline), and sodium bicarbonate (NaHCO), for example, dimethylsulfoxyl.
  • One or more solvents selected from the group consisting of chill-2-pyrrolidone, dioxane, ethyl acetate and the like can be used. In the present invention, it is preferable to use PBS.
  • spotting is performed on the surface of the carrier surface-treated with polycations (polylysine, polyethyleneimine, etc.), and the DNA is charged.
  • polycations polylysine, polyethyleneimine, etc.
  • a method of electrostatically bonding a carrier on a carrier is used.
  • a method for treating the surface of the carrier a method using a silane coupling agent having an amino group, an aldehyde group, an epoxy group, or the like is also used. In this case, an amino group, an aldehyde group, and the like are introduced on the carrier surface by a covalent bond, and thus are more stably present on the carrier surface than in the case of using a polycation.
  • a probe DNA into which a reactive group has been introduced may be synthesized, and the DNA may be spotted on the surface-treated carrier surface and covalently bonded.
  • PDC p-phenylene diisothiocyanate
  • Spotting is performed by dispensing a spotting solution containing probe DNA into a 96- or 384-well plastic plate, and dropping the dispensed solution onto a carrier using a spot device or the like.
  • a spot device a pin-type device is usually used in which a pin holds a sample solution, the pin is brought into contact with the carrier surface, and the solution is transferred to the carrier surface to form a spot.
  • pin tip shapes including solid pin types (especially those with no grooves) and tile pin types (things with grooves like a fountain pen). Can also be used. Preferably, it is of the pin type.
  • spot devices that use the ink jet method using the principle of an ink jet printer or the capillary method using a capillary tube can be used.
  • the spotting amount of the spotting solution per spot can be appropriately determined by those skilled in the art, but is usually in the range of lpL—1 ⁇ L, and preferably in the range of lOOpL—IOOnL. Spot sizes are usually in the range of 50-300 / zm in diameter. The distance between the spots is usually in the range of 0-1.5 mm, preferably 100-700 ⁇ m In the range. After the spotting, it is preferable that the incubation is usually performed at room temperature to 100 ° C, preferably 70 to 80 ° C, for usually 3 hours or less, preferably 0.5 to 1.5 hours.
  • the carrier is washed to remove unimmobilized DNA and the like.
  • the washing solution those commonly used in the art can be used.
  • an aqueous solution containing 2 ⁇ SSC, 0.2% SDS (sodium dodecyl sulfate) can be used. In this way, a carrier having hundreds of thousands of spots can be obtained.
  • the probe DNA-immobilized carrier of the present invention usually has at least 10 spots or more, preferably 50-1000 spots, more preferably 1000-2000 spots of probe DNA immobilized thereon.
  • For one type of probe DNA usually 1 to 10 spots, preferably 3 to 5 spots are spotted.
  • the carrier for immobilizing the probe DNA those usually used in the art can be used.
  • noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductive materials such as graphite and carbon typified by carbon fiber; single-crystal silicon, amorphous silicon, Semiconductor materials typified by silicon carbide, silicon oxide, silicon nitride, etc .; composite materials of these semiconductor materials typified by SOI (silicon on insulator); glass, quartz glass, alumina, sapphire , Ceramics, forsterite, photosensitive glass and other inorganic materials; polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinyl chloride, polyvinyl acetate, and polyvinyl acetate.
  • examples include organic materials such as polymers, polyphenylene oxide, and polysulfone.
  • a carrier for immobilizing probe DNA a functional group capable of covalently bonding to a nucleic acid on a substrate, and diamond, soft diamond, a carbon-based material, and a carbide It is preferable to use a solid support having at least one surface layer selected.
  • Examples of the material of the substrate used for the solid support include silicone, glass, fiber, wood, paper, ceramics, and plastics (eg, 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, Shiraidani vinyl resin ), Synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon; metals such as gold, silver, copper, aluminum, tungsten, and molybdenum; And the like, in which the above-mentioned resin is mixed and formed as a binder; That a material obtained by powder raw materials such as metal powder or ceramic powder with a press molding machine and sintered at high temperature and the like.
  • plastics eg, polyester resin, polyethylene resin, polypropylene resin, ABS resin.
  • nylon acrylic resin, flu
  • the solid support of the present invention has a surface layer on a substrate. With this surface layer, a compound for introducing a functional group capable of covalently binding to a nucleic acid can be firmly fixed on a substrate.
  • the surface layer is formed of at least one kind selected from diamond, soft diamond, carbon-based material and carbide.
  • the diamond, soft diamond, carbon-based material and carbide include synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (eg, diamond-like carbon), amorphous carbon, and carbon-based material (eg, graphite, fullerene, Carbon nanotubes), a mixture thereof, or a laminate thereof, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, And carbides such as chromium carbide and vanadium carbide.
  • the soft diamond is a general term for an incomplete diamond structure which is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. In the present invention, it is preferable to use a soft diamond.
  • the substrate is formed of at least one material selected from diamond, soft diamond, a carbon-based material, and carbide
  • the surface is formed by applying a surface treatment to at least one material selected from diamond, soft diamond, carbonaceous materials and carbides. Form a layer.
  • An example of a substrate that has been subjected to a surface treatment is a substrate in which soft diamond is formed on a slide glass.
  • diamond-like carbon is used for hydrogen gas 0-9
  • It is preferably prepared by ionization deposition in a mixed gas containing 9% by volume and 100-1% by volume of methane gas remaining.
  • the thickness of the surface layer formed by the surface treatment is preferably from lnm to 100m.
  • the surface treatment of the substrate is performed by a known method, for example, a microwave plasma CVD (Chemical Vapor Deposit) method, an ECRCVD (Electric Cyclotron Resonance Chemical Vapor Deposit) method, an ICP (Inductive Coupled Plasma) method, a DC sputtering method, It can be performed by ECR (Electric Cyclotron Resonance) sputtering, ion plating, arc plating, EB (Electron Beam) evaporation, resistance heating evaporation, ionization evaporation, arc evaporation, laser evaporation, or the like.
  • a microwave plasma CVD Chemical Vapor Deposit
  • ECRCVD Electrical Cyclotron Resonance Chemical Vapor Deposit
  • ICP Inductive Coupled Plasma
  • DC sputtering method It can be performed by ECR (Electric Cyclotron Resonance) sputtering, ion plating, arc plating, EB (Electron Beam) evapor
  • a surface layer may be formed by forming a laminate or a composite of the above-mentioned substrate material (for example, a composite of diamond and another substance (for example, a two-phase body)).
  • the shape and size of the substrate are not particularly limited.
  • Examples of the shape include a plate shape, a thread shape, a sphere shape, a polygonal shape, and a powder shape.
  • a single layer of Ti, Au, Pt, Nb, Cr, TiC, TiN or the like or a composite film thereof may be formed as a reflective layer on the front surface or the back surface of the substrate.
  • the thickness of the reflective layer is preferably 10 nm or more, more preferably 100 nm or more, because it is necessary that the thickness of the reflective layer be uniform throughout.
  • the surface is intentionally roughened with Ra (jIS B 0601) in the range of lnm-100Onm.
  • Ra jIS B 0601
  • Such a roughened surface is advantageous in that the surface area of the substrate increases and a large amount of probe DNA can be immobilized at a high density.
  • the solid support of the present invention is provided with an electrostatic layer for electrostatically attracting nucleic acids. May be.
  • the electrostatic layer is not particularly limited as long as it is capable of electrostatically attracting the nucleic acid and improving the amount of immobilized nucleic acid.
  • a compound having a positive charge such as an amino group-containing compound can be used. Can be formed.
  • the amino group-containing compound may be an unsubstituted amino group (1-NH) or a compound having 1 carbon atom.
  • the amino group is contained by introducing the amino group-containing compound into a film forming apparatus at the time of surface treatment. A carbon-based film is formed.
  • the electrostatic layer is formed without being covalently bonded to the surface layer, the non-substituted or mono-substituted substrate is formed on the substrate in order to increase the affinity between the electrostatic layer and the surface layer, that is, the adhesion.
  • the compound having an amino group and the carbon compound it is preferable to introduce a functional group capable of covalently binding to the nucleic acid.
  • the carbon compound used here is not particularly limited as long as it can be supplied as a gas.
  • the vapor deposition method the ionization vapor deposition method is preferable.
  • the working pressure is preferably 0.1 to 50 Pa, and the acceleration voltage is preferably 200 to 1000 V. .
  • the electrostatic layer is formed by covalent bonding with the surface layer
  • the surface of the substrate having the surface layer is chlorinated by irradiating ultraviolet rays in chlorine gas to chlorinate the surface, and then the amino group-containing layer is formed.
  • the compounds for example, polyallylamine, polylysine, 4,4,4 "-triaminotriphenylmethane Then, a polyvalent amine such as triamterene is reacted to introduce an amino group into the terminal not bonded to the substrate, whereby an electrostatic layer can be formed.
  • a reaction for introducing a functional group capable of covalently binding to a nucleic acid for example, introduction of a carboxyl group using a dicarboxylic acid or a polycarboxylic acid
  • a substrate is immersed in a solution containing the above-mentioned compound having an unsubstituted or monosubstituted amino group, and then a functional group capable of covalently binding to a nucleic acid is introduced.
  • the solvent for the solution include water, N-methylpyrrolidone, and ethanol.
  • the substrate When a carboxyl group is introduced into the substrate provided with the electrostatic layer using a dicarboxylic acid or a polycarboxylic acid, the substrate may be activated in advance with N-hydroxysuccinimide and Z or carbodiimides, Alternatively, it is preferable to carry out the reaction in the presence of N-hydroxysuccinimide and / or carposimides.
  • the thickness of the electrostatic layer is preferably lnm-500m.
  • the solid support of the present invention has a functional group capable of covalently binding to a nucleic acid.
  • the functional group can be formed by chemically modifying the substrate surface.
  • Examples of the functional group include a carboxyl group, an active ester group, a haloformyl group, a hydroxyl group, a sulfate group, a cyano group, a nitro group, a thiol group, and an amino group.
  • Examples of the compound used to introduce a carboxyl group as a functional group include compounds represented by the formula: X—R 1 —COOH (wherein, X is a halogen atom, and R 1 is a divalent C 11 -C 12) A hydrocarbon group.), For example, chloroacetic acid, fluoroacetic acid, bromoacetic acid, odoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chloroacrylic acid Dibenzoic acid represented by the formula: HOOC—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 and malonic acid , Succinic acid, maleic acid, fumaric acid, phthalic acid; polycarboxylic acids such as polyacrylic acid, polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; formula: R 3
  • X—OC—R 5 —COOH (where X is a halogen atom, R 5 is a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms)
  • monohalides of dicarboxylic acids for example, succinic monochloride, malonic monochloride; and acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.
  • the carboxyl group introduced as described above can be converted to a dehydration condensing agent such as cyanamide percarboimide (for example, 1- [3- (dimethylamino) propyl] 3ethylcarposimide) and a compound such as N-hydroxysuccinimide.
  • a dehydration condensing agent such as cyanamide percarboimide (for example, 1- [3- (dimethylamino) propyl] 3ethylcarposimide) and a compound such as N-hydroxysuccinimide.
  • Examples of the compound used to introduce a haloformyl group as a functional group include, for example, a compound represented by the formula: X—OC—R 6 —CO—X (where X is a halogen atom, R 6 is a single bond or carbon number) Dino and dilide of dicarboxylic acid such as succinic chloride and malonic chloride.
  • the compound used for introducing a hydroxyl group as a functional group includes, for example, a compound represented by the formula: HO
  • R 7 represents a divalent hydrocarbon group having 11 to 12 carbon atoms), such as hydroxy acid or phenolic acid.
  • Examples of the compound used to introduce an amino group as a functional group include amino acids.
  • polycarboxylic acids such as polyacrylic acid, polymethacrylic acid, trimellitic acid, and butanetetracarboxylic acid can also be used to improve hydrophilicity.
  • the probe DNA can be firmly fixed, so that highly reliable diagnosis and detection sensitivity can be achieved.
  • the method for detecting the expression of the gene encoding the membrane protein of the present invention is not particularly limited as long as it is based on the hybridization of the probe DNA of the present invention and a nucleic acid encoding the membrane protein.
  • Methods known in the art can be used. For example, Northern blotting, RNase protection assay, Southern blotting in cDNA cloning, colony Z plaque hybridization, microarray, etc. Is mentioned.
  • the probe DNA immobilized on a carrier is brought into contact with a sample cDNA obtained by reverse transcription of sample-derived mRNA, and the probe DNA is contacted with the probe DNA.
  • the probe DNA is contacted with the probe DNA.
  • the expression of a gene encoding a membrane protein is detected by detecting the presence or absence of expression of a gene encoding a membrane protein, and by detecting a change in the expression level of a gene encoding a membrane protein. Detecting and quantifying the expression level of a gene encoding a membrane protein is also encompassed.
  • the sample cDNA can be prepared by a method known in the art. For example, total RNA isolated from a sample is added to a reaction mixture containing an RT primer and an appropriate buffer. After incubation for primer annealing, RT buffer, dNTPs, DTT, aminoallyl dUTP, RNase inhibitor and reverse transcriptase are further added. After incubation to complete the reverse transcription of the RNA, the RT product is reacted with a reactive labeling reagent. Alternatively, labeled unlabeled dNTPs may be incubated in the PCR reaction mixture without labeling the primers. PCR amplification can be performed in a DNA thermal cycler according to conventional techniques.
  • the label is not particularly limited as long as it can be incorporated into a nucleic acid.
  • the label include fluorescent labels (CyDye such as Cy3 and Cy5, FITC, RITC, rhodamine, texathread, TET, TAMRA, FAM , HEX, ROX, etc.) and radioactive labels ( ⁇ -32P, y-32P, 35S, etc.).
  • the sample solution is prepared by dissolving in a buffer such that the nucleic acid concentration in the solution is usually 0.01 to 100 g, preferably 1 to 50 g.
  • This solution is dropped on the carrier on which the probe DNA prepared above is immobilized, and hybridized by incubation.
  • Hybridization can be performed according to a known method or a method analogous thereto, such as the method described in Molecular Cloning 2 (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989).
  • the incubation is carried out usually at 50 to 70 ° C, preferably 55 to 65 ° C, and usually for 120 hours, preferably 5 to 16 hours.
  • the carrier can be washed and dried, and then the hybridization can be detected by reading the label derived from the hybridized nucleic acid.
  • the probe DNA-immobilized carrier of the present invention can dissociate the nucleic acid once hybridized by hybridizing with the nucleic acid derived from the sample and then washing the carrier, and further re-hybridize the nucleic acid. Can be done.
  • a solid support having a functional group capable of covalently binding to DNA on a substrate and at least one surface layer selected from diamond, soft diamond, carbonaceous materials and carbides as a carrier, nucleic acid dissociation and noise The reproducibility of the reduction reaction can be improved.
  • the dissociation of the hybridized nucleic acid is usually performed at room temperature to 100 ° C, preferably 90 to 100 ° C, more preferably 95 to 100 ° C, with a sodium concentration of 1 to 15 mM, usually 10 to 60 mM. Minutes, preferably 10 to 40 minutes, more preferably 30 to 35 minutes.
  • aqueous solution for washing ultrapure water, sodium hydrogen carbonate solution, SSC solution, PBS solution and the like can be used. In the present invention, it is preferable to carry out washing for 10 to 30 minutes using ultrapure water at 95 to 100 ° C.
  • a diamond-like carbon layer is coated on a 3 mm square silicon substrate, an amino group is introduced by ammonia plasma treatment, modified with a carboxyl group, activated with N-hydroxysuccinimide, and immobilized for immobilization of probe DNA.
  • a carrier was made.
  • 385 types of 60mer DNAs whose 5 'ends were aminated at 5 pmol / ⁇ L were spotted on this carrier as probe DNAs.
  • the DNA fragment was dissolved at a concentration of 5 M in a final concentration of 20% PEG (polyethylene glycol) solution to obtain a spotting solution.
  • Table 1 shows the 385 types of probe DNAs immobilized on the carrier.
  • the column with the gene name is the general name of the protein encoded by the gene, and the name registered with the gene sequence in the gene database, or the protein sequence data It is a name registered on one base together with its amino acid sequence.
  • the symbols A to D in the rightmost column labeled “Creation method” indicate the method for preparing the probe DNA, and the meanings of the symbols are as follows.
  • A The region coding for the transmembrane region in the gene coding for each membrane protein was also created by selecting a region with a cytosine and guanine content of 0-60%.
  • TMP transmembrane region prediction program
  • FIG. 1 shows the spot positions of these probe DNAs on the carrier.
  • cDNA was synthesized by performing a reverse transcription reaction from 4 ⁇ g of PolyA + RNA extracted from the liver of BALBZc mouse, and Cy3 fluorescent labeling and column purification were performed. Thereafter, a sample solution was prepared using 5 ⁇ SSCZ0.5% SDS solution as a buffer, using 16 / z L of cDNA in 20 / z L of column eluate. This was dropped onto a probe DNA-immobilized carrier, and hybridization was performed at 55 ° C. for 16 hours in a champer.
  • Example 1 After observing the fluorescence image in Example 1, the carrier was washed with ultrapure water at 95 ° C (5 minutes, 30 minutes), the cDNA was dissociated, and the fluorescence image was observed again with a fluorescence scanner.
  • Figure 3 shows the results after washing for 30 minutes. Since the dissociation of the once hybridized cDNA was confirmed, the cDNA was hybridized again in the same procedure as above, and the fluorescence image was observed.
  • Figure 4 shows the results. Further, the row of R in Table 1 (the second row from the right) shows the ratio of the fluorescence intensity in the second hybridization to the fluorescence intensity in the first hybridization.
  • Probe DNA having a value of R of 0.5 or more, preferably 0.5 to 2.0, more preferably 0.6 to 1.5 is particularly suitable, and the immobilization of such probe DNA is particularly preferable. Is preferred Industrial availability
  • the probe DNA-immobilized carrier of the present invention can be used repeatedly, and the surface cost of the probe is also very advantageous. Further, according to the present invention, a nucleic acid encoding a membrane protein can be specifically detected, and therefore, expression of a gene encoding a membrane protein can be rapidly and comprehensively detected.

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Abstract

: L'intention est de fournir une méthode de détection rapide et détaillée de l'expression d'un gêne codant une protéine membranaire. Notamment, une méthode de fabrication d'une sonde DNA pour un acide nucléique codant une protéine membranaire qui comprend la sélection d'une zone partielle du gêne codant la protéine membranaire, dans laquelle 5% ou plus de la séquence de base comprend une séquence exon codant une zone essentiellement nécessaire pour la liaison membrane dans la protéine membranaire et le contenu total des quantités de cytosine et de guanine de 40 à 60%, et la fabrication d'une DNA comprenant la séquence de base de la zone partielle telle décrite ci-dessus ou une séquence de base complémentaire de cette zone partielle.
PCT/JP2005/001914 2004-02-13 2005-02-09 Méthode de fabrication d'une sonde dna pour protéine membranaire de codage acide nucléique, méthode de détection de l'expression de protéine membranaire de codage de gêne et support d'immobilisation de sonde dna WO2005078082A1 (fr)

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JP2004037041A JP2005224189A (ja) 2004-02-13 2004-02-13 膜タンパク質をコードする核酸に対するプローブｄｎａを作成する方法、膜タンパク質をコードする遺伝子の発現を検出する方法、及びプローブｄｎａ固定化担体
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JP2006217821A (ja) * 2005-02-08 2006-08-24 Toyo Kohan Co Ltd 膜タンパク質をコードする核酸に対するプローブｄｎａを作成する方法、膜タンパク質をコードする遺伝子の発現を検出する方法、及びプローブｄｎａ固定化担体
JP2009050182A (ja) * 2007-08-24 2009-03-12 Okayama Univ ヒトおよびラットの細胞表面マーカー遺伝子を結合したｄｎａアレイとその用途

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WO2002095065A2 (fr) * 2001-05-18 2002-11-28 Azign Bioscience A/S Matrices de recepteurs couples a la proteine g

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WO2002095065A2 (fr) * 2001-05-18 2002-11-28 Azign Bioscience A/S Matrices de recepteurs couples a la proteine g

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MONTROSE-RAFIZADEH C. ET AL: "Cellular differentiation regulates expression of Cl- transport and cystic fibrosis transmembrane conductance regulator mRNA in human intestinal cells", J BIOL CHEM, vol. 266, no. 7, 5 March 1991 (1991-03-05), pages 4495 - 4499, XP002987990 *
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