JP2006217821A - Method for making probe dna for nucleic acid encoding membrane protein, method for detecting expression of gene encoding membrane protein, and probe dna-immobilized carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly and comprehensively detecting the expression of a gene encoding a membrane protein. <P>SOLUTION: This method for making a probe DNA for a nucleic acid encoding a membrane protein comprises selecting a partial region which is a partial region of the gene encoding the membrane protein, in which ≥5% of the base sequence of the partial region comprises an exon sequence encoding a region essential to a membrane bond in the membrane protein and which contains cytosine and guanine in a total amount of 40 to 60%, and then making the DNA comprising the base sequence of the partial region or a base sequence complementary to the partial sequence. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  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, a carrier on which the probe DNA is immobilized, and the method The present invention relates to a method for detecting expression of the gene using a carrier.

  There are about 30% of all proteins in eukaryotic cells. Membrane proteins play important functions in information transmission and mass transport through cell membranes as receptors, ion channels, transporters, and the like. Membrane proteins are roughly divided into two types based on their structures. 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 an integral membrane protein is composed of an extracellular region, an intracellular region, and a transmembrane region. The transmembrane region is rich in hydrophobic amino acids and is thought to form an α-helix in the membrane. In the case of a protein having a plurality of transmembrane regions, when the membrane protein is biosynthesized, this domain is a signal sequence for starting transmembrane passage of the nascent peptide chain following the C-terminal side, or stop transfer for stopping permeation thereof. Functions as an array. In many cases, domains having respective functions are alternately arranged, and extracellular regions and intracellular regions are alternately arranged through the domains.

  Some membrane proteins are specifically expressed in cancer cells, and these are considered useful for developing various diagnostic agents and anticancer agents as tumor antigens. And 70% of the target of drugs used for treatment of various diseases and diseases is said to be membrane protein. By analyzing the expression pattern of membrane protein, target of tissue-specific therapeutic drug It is expected to clarify.

  As a method of analyzing the expression of the membrane protein, a method of analyzing the membrane protein itself can be considered. However, membrane proteins are difficult to purify because of their high hydrophobicity, and because they are in a special environment that exists in the lipid bilayer of cell membranes, the three-dimensional structure is complex, compared to water-soluble proteins present in cells. Its expression analysis is very difficult. Methods of analysis by RT-PCR method or Northern blot method are unsuitable for exhaustive analysis, and are insufficient for expression analysis of extremely diverse membrane proteins as described above. In addition, the method using a cDNA microarray has problems that it takes time and labor to isolate cDNA, and the amount of DNA to be immobilized becomes unstable.

  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.

  As a result of intensive studies to solve the above problems, the present inventors have determined that at least a part of an exon sequence that encodes a region that is a partial region of a gene that encodes a membrane protein and is essential for membrane binding in the membrane protein. And a nucleic acid encoding a membrane protein can be specifically detected by using DNA consisting of a partial region containing a certain amount of cytosine and guanine or a DNA complementary to the partial region as a probe. It came to complete.

That is, the present invention includes the following inventions.
(1) A partial region of a gene encoding a membrane protein, wherein 5% or more of the base sequence consists of a sequence in an exon containing a region encoding a region essential for membrane binding in the membrane protein, and cytosine and guanine A membrane protein comprising: selecting the partial region containing a total of 40 to 60% and preparing a DNA comprising the partial region or a DNA comprising a base sequence complementary to the partial region A method for preparing a probe DNA for a nucleic acid.
(2) A method for detecting the expression of a gene encoding a membrane protein by hybridizing a probe DNA prepared by the method according to (1) and a nucleic acid derived from a sample.
(3) A partial region of a gene encoding a membrane protein, wherein 5% or more of the base sequence is composed of a sequence in an exon containing a region encoding a region essential for membrane binding in the membrane protein, and cytosine and guanine A carrier on which a probe DNA selected from DNA consisting of the partial region and DNA consisting of a base sequence complementary to the partial region is fixed.
(4) Probe DNA immobilization carrier on which a probe DNA selected from the group consisting of the following DNAs (a) to (e) is immobilized:
(a) DNA comprising the base sequence represented by any of SEQ ID NOs: 1 to 1015
(b) DNA comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(c) DNA having 10 to 5000 bases including a part of the DNA consisting of the base sequence represented by any of SEQ ID NOs: 1 to 1015
(d) DNA of 10 to 5000 bases including a part of DNA consisting of a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(e) DNA consisting of a base sequence having homology with the DNAs of (a) to (d) and specifically hybridizing with a nucleic acid encoding a corresponding membrane protein.
(5) The support is a solid support having a functional group capable of covalently bonding to the probe DNA on the substrate and at least one surface layer selected from diamond, soft diamond, a carbon-based material, and carbide. (3) Or the probe DNA fixed carrier as described in (4).
(6) A method for detecting the expression of a gene encoding a membrane protein using the probe DNA-immobilized carrier according to any one of (3) to (5).
(7) Probe DNA selected from the group consisting of the following DNAs (a) to (e):
(a) DNA comprising the base sequence represented by any of SEQ ID NOs: 1 to 1015
(b) DNA comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(c) DNA having 10 to 5000 bases including a part of the DNA consisting of the base sequence represented by any of SEQ ID NOs: 1 to 1015
(d) DNA of 10 to 5000 bases including a part of DNA consisting of a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(e) DNA consisting of a base sequence having homology with the DNAs of (a) to (d) and specifically hybridizing with a nucleic acid encoding a corresponding membrane protein.

  According to the present invention, a nucleic acid encoding a membrane protein can be specifically detected, and thus the expression of a gene encoding the membrane protein can be detected quickly and exhaustively.

Preparation of probe DNA In one embodiment of the present invention, the probe DNA is a partial region of a gene encoding a membrane protein, and is usually 5% or more, preferably 60% or more, more preferably 80% or more of the base sequence, More preferably 90% or more, most preferably 95% or more consists of an exon sequence encoding a region essential for membrane binding in a membrane protein, and cytosine and guanine are usually 40-60% in total, preferably 45-55% It consists of the partial region contained at a ratio of In the present invention, DNA complementary to the DNA is also used as probe DNA.

  In the present invention, the membrane protein has a meaning usually used in this technical field, that is, a protein constituting a biological membrane. As membrane proteins, there are membrane surface proteins attached to the surface of biological membranes, membrane integral proteins buried inside, for example, receptors, transporters, membrane-bound enzymes, cytokine receptor 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 TGFβ receptor family, interleukin receptor family, TNF receptor family, Notch family, EGF-CFC family and the like. In the present invention, a probe for a nucleic acid encoding a membrane protein that characterizes the surface properties of the cell among the above proteins, that is, a membrane protein that is highly expressed in the cell membrane of the target cell, is preferably prepared.

  In the present invention, the membrane protein is preferably selected from the membrane proteins described in Table 1 below. The base sequence of DNA encoding each membrane protein is registered in GenBank with the accession numbers shown in Table 1 (http://www.ncbi.nlm.nih.gov).

  The region essential for membrane binding means a region responsible for binding between the membrane protein and the cell membrane in the membrane protein. Examples of such a region include a transmembrane region usually composed of a hydrophobic amino acid, a region containing an amino acid to which a GPI anchor that binds to a membrane via a lipid, and the like are not limited thereto. Absent.

  A transmembrane region in a membrane protein and a region containing an amino acid to which a GPI anchor binds can be identified from information that has already been published, or when known information cannot be obtained, a known prediction program or the like is used. Can be searched.

  The base length of the probe DNA of the present invention is a length capable of hybridizing with a nucleic acid encoding a membrane protein, and is usually 10 to 5000 bases, preferably 15 to 500 bases, more preferably 20 to 100 bases, still more preferably. 40-70 bases.

The probe DNA of the present invention is preferably selected from the group consisting of the following DNAs (a) to (e):
(a) DNA comprising the base sequence represented by any of SEQ ID NOs: 1 to 1015
(b) DNA comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(c) DNA having 10 to 5000 bases, more preferably DNA having 20 to 100 bases, including a part of the DNA consisting of the base sequence represented by any of SEQ ID NOs: 1 to 1015
(d) DNA having 10 to 5000 bases, more preferably DNA having 20 to 100 bases, including a part of DNA consisting of a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(e) DNA consisting of a base sequence having homology with the DNAs of (a) to (d) and specifically hybridizing with a nucleic acid encoding a corresponding membrane protein.

  Here, the part of the DNA is at least 15 bases long, more preferably at least 20 bases long, and most preferably 30-60 bases long in the DNA consisting of the base sequence represented by any of SEQ ID NOs: 1 to 1015. It means a part consisting of a continuous base sequence.

  A base sequence having homology with DNA is usually a base sequence having a homology or identity of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more. It means a base sequence that specifically hybridizes with a nucleic acid encoding the corresponding membrane protein. Nucleic acids encoding membrane proteins include both DNA and RNA, preferably cDNA.

  To specifically hybridize means to be able to maintain a hybridized state with a nucleic acid encoding a membrane protein 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, it is most preferable that the sodium concentration is 16 to 17 mM and the temperature is 65 ° C.

Probe DNA Immobilization Carrier The present invention also relates to a carrier on which the probe DNA is immobilized. The probe DNA-immobilized carrier is useful for detecting the expression of a membrane protein.

The probe DNA can be immobilized on a carrier by a method known in the art. A spotting solution is prepared by dissolving or dispersing a probe DNA to be immobilized and a PCR product thereof (DNA fragment obtained by amplifying DNA by a PCR method) in a solvent. By incubating the spotting solution after spotting on the carrier, the probe DNA can be immobilized on the carrier. Examples of 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), sodium bicarbonate (NaHCO ₃ ), such as dimethyl sulfoxide, dimethylformamide, One or more solvents selected from tetrahydrofuran, trifluoroacetic acid, triethylamine, 1-methyl-2-pyrrolidone, dioxane, ethyl acetate and the like can be used. In the present invention, it is preferable to use PBS.

  For immobilization of the probe DNA, a spotter device provided in the DNA chip preparation device is used to spot the carrier surface that has been surface-treated with a polycation (polylysine, polyethyleneimine, etc.) and use the charge of the DNA. In general, a method of electrostatic coupling on a carrier is used. Further, as 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, since an amino group, an aldehyde group, and the like are introduced to the support surface by a covalent bond, they are stably present on the support surface as compared with the case of using a polycation.

  It is also possible to synthesize probe DNA into which a reactive group has been introduced, spot the DNA on the surface of the surface-treated carrier, and covalently bond it. For example, there are a method of reacting an amino group-introduced DNA in the presence of PDC (p-phenylene diisothiocyanate) on an amino group-introduced slide glass, and a method of reacting an aldehyde group-introduced DNA on the slide glass.

  Spotting is performed by dispensing a spotting solution containing the probe DNA into a 96-hole or 384-hole plastic plate and dropping the dispensed solution onto a carrier using a spot device or the like. As the spot device, a pin type device is generally used in which a sample solution is held on a pin, the pin is brought into contact with the carrier surface, and the solution is transferred to the carrier surface to form a spot. There are various types of pin tips such as solid pin type (especially those without grooves) and quill pin types (those with grooves like a fountain pen). Can also be used. Preferably, it is a quill pin type. In addition to the pin method, a spot device using an ink jet method using the principle of an ink jet printer or a capillary method using a capillary tube can also 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 1 pL to 1 μL, and preferably in the range of 100 pL to 100 nL. The spot size is usually in the range of 50 to 300 μm in diameter. The distance between the spots is usually in the range of 0 to 1.5 mm, preferably in the range of 100 to 700 μm. After spotting, it is usually preferable to incubate at room temperature to 100 ° C, preferably 70 to 80 ° C, usually for 3 hours or less, preferably 0.5 to 1.5 hours.

  After the probe DNA is immobilized on the carrier, the carrier is washed in order to remove non-immobilized DNA and the like. As the cleaning solution, those commonly used in the art can be used, and for example, an aqueous solution containing 2 × SSC, 0.2% SDS (sodium dodecyl sulfate) can be used. In this way, a carrier having hundreds to tens of thousands of spots can be obtained.

  In the probe DNA immobilization carrier of the present invention, usually at least 10, preferably at least 50, more preferably at least 100, further preferably at least 500, most preferably at least 1000 types of probe DNA are immobilized. . The probe DNA-immobilized carrier of the present invention usually has at least 10 spots, preferably 50 to 1000 spots, more preferably 1000 to 2000 spots of probe DNA immobilized thereon. For one type of probe DNA, spotting is usually performed at 1 to 10 spots, preferably 3 to 5 spots.

Carriers for immobilizing probe DNA As carriers for immobilizing probe DNA, those commonly used in the art can be used. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductor materials such as graphite and carbon typified by carbon fiber; single crystal silicon, amorphous Semiconductor materials typified by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these semiconductor materials typified by SOI (silicon-on-insulator), etc .; glass, quartz glass, alumina, sapphire, ceramics, Inorganic materials such as stellite and photosensitive glass; polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl alcohol Tar, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide and polysulfone, etc. An organic material etc. are mentioned.

  In the present invention, as a carrier for immobilizing probe DNA, a solid support having a functional group capable of covalently binding to a nucleic acid on a substrate and at least one surface layer selected from diamond, soft diamond, carbonaceous material and carbide. It is preferable to use the body.

  Examples of the material of the substrate used for the solid support include, for example, silicone, glass, fiber, wood, paper, ceramics, plastic (for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), nylon. , Acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methyl pentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin), synthetic diamond, high pressure synthetic diamond, natural diamond, soft diamond (eg diamond-like carbon) Amorphous carbon; Gold, silver, copper, aluminum, tungsten, molybdenum and other metals; Binder the resin to the metal powder, ceramic powder, etc. Include those those raw materials such as the metal powder or ceramic powder and powder with a press molding machine and sintered at a high temperature; that as mixing, those bound form.

  The solid support of the present invention has a surface layer on a substrate. By this surface layer, a compound for introducing a functional group capable of covalently bonding with a nucleic acid can be firmly immobilized on the substrate.

  The surface layer is formed of at least one selected from diamond, soft diamond, carbon-based material, and carbide. Examples of diamond, soft diamond, carbon-based material, and carbide include 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 a laminate thereof, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, Carbides such as vanadium carbide can be mentioned. 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. In the present invention, it is preferable to use soft diamond.

  When the substrate is made of at least one material selected from diamond, soft diamond, carbon-based material and carbide, it is not necessary to form a new surface layer on the substrate, but the substrate is made of other materials. In the case of forming the surface layer, a surface treatment is performed to form a surface layer formed of at least one material selected from diamond, soft diamond, a carbon-based material, and carbide.

As an example of the substrate subjected to the surface treatment, 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 thickness of the surface layer formed by the surface treatment is preferably 1 nm to 100 μm.

  The surface layer is formed on the substrate by a known method such as 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.

  Alternatively, a surface layer may be formed by forming a laminate or a composite of the above substrate materials (for example, a composite of diamond and another substance (for example, a two-phase body)).

  Although the shape and size of the substrate are not particularly limited, examples of the shape include a flat plate shape, a thread shape, a spherical shape, a polygonal shape, a powder shape, and the like. ˜100 mm, length 0.1˜100 mm, thickness 0.01˜10 mm.

  Alternatively, a single layer of Ti, Au, Pt, Nb, Cr, TiC, TiN, or a composite film thereof may be formed on the front or back surface of the substrate as a reflective layer. Since the thickness of the reflective layer needs to be uniform throughout, it is preferably 10 nm or more, more preferably 100 nm or more.

  When glass is used as the substrate, the surface thereof is preferably intentionally roughened in the range of 1 nm to 1000 nm with Ra (JIS B 0601). Such a roughened surface is advantageous in that the surface area of the substrate is increased and a large amount of probe DNA can be immobilized at a high density.

  The solid support of the present invention may be provided with an electrostatic layer in order to attract nucleic acid electrostatically.

  The electrostatic layer is not particularly limited as long as it attracts nucleic acid electrostatically and improves the amount of nucleic acid immobilized. For example, the electrostatic layer is formed using a positively charged compound such as an amino group-containing compound. be able to.

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 ).

  In the case where the electrostatic layer is formed without being covalently bonded to the surface layer, for example, when the surface treatment is performed, the amino group-containing compound is introduced into the film forming apparatus to form a carbon-based film containing an amino group. Form a film. In addition, when the electrostatic layer is formed without being covalently bonded to the surface layer, the above-mentioned non-substituted or mono-substituted is formed on the substrate in order to increase the affinity between the electrostatic layer and the surface layer, that is, the adhesion. It is preferable to introduce a functional group capable of being covalently bonded to a nucleic acid after vapor deposition of the compound having an 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.

  In the case where the electrostatic layer is formed by covalent bonding with the surface layer, for example, the substrate having the surface layer is irradiated with ultraviolet rays in chlorine gas to chlorinate the surface, and among the amino group-containing compounds, for example, By reacting a polyamine such as polyallylamine, polylysine, 4,4 ′, 4 ″ -triaminotriphenylmethane, triamterene, etc., and introducing an amino group at the terminal not bonded to the substrate, A layer can be formed.

  In addition, when a reaction for introducing a functional group capable of covalently bonding to a nucleic acid (for example, introduction of a carboxyl group using dicarboxylic acid or polyvalent carboxylic acid) in a solution is performed in a solution, the substrate Is immersed in a solution containing the compound having an unsubstituted or monosubstituted amino group, and then a functional group capable of covalently bonding to a nucleic acid is preferably introduced. 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. This improves the amount of nucleic acid immobilized.
The thickness of the electrostatic layer is preferably 1 nm to 500 μm.

  The solid support in the present invention has a functional group that can be covalently bonded 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.

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 carboxyl group introduced as described above is active with a dehydrating condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and a compound such as N-hydroxysuccinimide. Can be esterified.

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 a carbon number of 1 to 12). 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.

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

  By using the solid support as described above as a carrier, the probe DNA can be firmly immobilized, so that diagnosis with high detection sensitivity and reliability is possible.

Detection of Expression of Gene Encoding Membrane Protein The method for detecting the expression of the gene encoding the membrane protein of the present invention is particularly limited as long as it is based on hybridization of the probe DNA of the present invention and a nucleic acid encoding the membrane protein. Rather, methods known in the art can be used. Examples include Northern blotting, RNase protection assay, Southern blotting in colony cloning, colony / plaque hybridization, microarray, and the like.

  In one embodiment of the detection method of the present invention, the above-described probe DNA immobilized on a carrier is brought into contact with a sample cDNA obtained by reverse transcription of mRNA derived from the sample, and the sample cDNA hybridizes with the probe DNA. Is detected to measure the level of mRNA encoding the corresponding membrane protein in the sample, thereby detecting the expression level of the gene encoding the membrane protein.

  In the present invention, the expression of a gene encoding a membrane protein is detected by detecting the presence or absence of expression of the gene encoding the membrane protein, detecting a change in the expression level of the gene encoding the membrane protein, and the membrane protein Quantifying the expression level of the gene encoding

  Sample cDNA can be prepared by methods known in the art. For example, total RNA isolated from a sample is added to a reaction mixture containing RT primers and an appropriate buffer. After incubation for primer annealing, RT buffer, dNTP, DTT, aminoallyl dUTP, RNase inhibitor and reverse transcriptase are further added. After incubation to complete reverse transcription of the RNA, the RT product is reacted with a reactive labeling reagent. Alternatively, labeled 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. For example, fluorescent labels (CyDye such as Cy3 and Cy5, FITC, RITC, rhodamine, Texas red, TET, TAMRA, FAM, HEX, ROX Etc.), radiolabels (α-32P, γ-32P, 35S, etc.) and the like.

The sample solution is prepared by dissolving in a buffer so that the nucleic acid concentration in the solution is usually 0.01-100 μg, preferably 1-50 μg. This solution is dropped on the carrier on which the probe DNA prepared above is immobilized, and hybridized by incubation. Hybridization methods analogous to known methods or to, for ^{example, Molecular Cloning 2 nd (J. Sambrook} et al., Cold Spring Harbor Lab. Press, 1989) can be carried out according to the method described in.

In the detection method of the present invention, the incubation is usually performed at 50 to 70 ° C., preferably 55 to 65 ° C., usually 1 to 20 hours, preferably 5 to 16 hours. Finally, the carrier can be washed and dried, and then hybridization can be detected by reading the label derived from the hybridized nucleic acid.

  The probe DNA immobilization carrier of the present invention can be dissociated once hybridized with a sample-derived nucleic acid and then washed with the carrier, and further hybridized with the nucleic acid again. it can. Nucleic acid dissociation and hybridization by using as a carrier a solid support having a functional group capable of covalently bonding to DNA on a substrate and at least one surface layer selected from diamond, soft diamond, carbonaceous material and carbide. The reproducibility of the reaction can be improved.

  The dissociation of the hybridized nucleic acid is usually a solution having a sodium concentration of 1 to 15 mM at room temperature to 100 ° C, preferably 90 to 100 ° C, more preferably 95 to 100 ° C, and usually 10 to 60 minutes, preferably 10 to 10 minutes. It can be carried out by washing for 40 minutes, more preferably 30 to 35 minutes. As an aqueous solution for washing, ultrapure water, sodium hydrogen carbonate solution, SSC solution, PBS solution, or the like can be used. In the present invention, it is preferable to perform washing for 10 to 30 minutes using ultrapure water at 95 to 100 ° C.

(Example 1) Preparation of probe DNA immobilization carrier A 3 mm square silicon substrate was coated with a diamond-like carbon layer, amino groups were introduced by ammonia plasma treatment, modified with carboxyl groups, and then esterified with N-hydroxysuccinimide. A carrier for immobilizing probe DNA was prepared. A microarray spotter SPBIO2000 (Hitachi Soft) was used as a probe, and spotted as a probe DNA were 6015 DNAs aminated at 1015 kinds of 5 ′ ends and adjusted to 5 pmol / μL. The DNA fragment was dissolved in a final concentration of 20% PEG (polyethylene glycol) solution at a concentration of 5 μM to obtain a spotting solution.

  Table 1 below shows 1015 types of probe DNA immobilized on a carrier. The number of the column described as SEQ ID NO. In Table 1 corresponds to the sequence number of each probe DNA shown in the sequence table. The column described as the gene name is a general name of the protein encoded by the gene, and is a name registered with the gene sequence in the gene database, or a name registered with the amino acid sequence in the protein sequence database. The symbols A to D shown in the rightmost column “creation method” indicate the preparation method of the probe DNA, and the meaning of each symbol is as follows.

A: A region having a cytosine and guanine content of 40-60% selected from a region encoding a transmembrane region in a gene encoding each membrane protein B: Transmembrane in a gene encoding each membrane protein A region coding for a region of cytosine and guanine selected from a region coding region and its surrounding region C: a region encoding a transmembrane region in a gene encoding each membrane protein, Searched with a transmembrane region prediction program (TMpred, (K. Hofmann & W. Stoffel (1993) Biol. Chem. Hoppe-Seyler 374, 166)), cytosine and guanine content from 40 to 60 were obtained from the obtained region. %: D: selected from the region coding for the region containing the amino acid to which the GPI anchor binds in each membrane protein. Those content of Nin created by selecting 40 to 60% of the area p: positive control also shows CG content in these probe DNA at%.

SEQ ID NOs: 1-1015: synthetic oligonucleotides

Claims (7)

  A partial region of a gene encoding a membrane protein, wherein 5% or more of the base sequence is composed of a sequence in an exon including a portion encoding a region essential for membrane binding in the membrane protein, and cytosine and guanine are combined in total. A probe for a nucleic acid encoding a membrane protein, comprising selecting the partial region contained at a ratio of 40 to 60% and preparing DNA consisting of the partial region or DNA consisting of a base sequence complementary to the partial region A method for producing DNA.   A method for detecting the expression of a gene encoding a membrane protein by hybridizing a probe DNA prepared by the method according to claim 1 and a nucleic acid derived from a sample.   A partial region of a gene encoding a membrane protein, wherein 5% or more of the base sequence is composed of a sequence in an exon including a portion encoding a region essential for membrane binding in the membrane protein, and cytosine and guanine are combined in total. A carrier on which a probe DNA selected from DNA consisting of the partial region and DNA consisting of a base sequence complementary to the partial region contained at a ratio of 40 to 60% is immobilized. Probe DNA immobilization carrier on which a probe DNA selected from the group consisting of the following DNAs (a) to (e) is immobilized:
(a) DNA comprising the base sequence represented by any of SEQ ID NOs: 1 to 1015
(b) DNA comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(c) DNA having 10 to 5000 bases including a part of the DNA consisting of the base sequence represented by any of SEQ ID NOs: 1 to 1015
(d) DNA of 10 to 5000 bases including a part of DNA consisting of a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(e) DNA consisting of a base sequence having homology with the DNAs of (a) to (d) and specifically hybridizing with a nucleic acid encoding a corresponding membrane protein.   The support is a solid support having a functional group capable of covalently bonding to the probe DNA on a substrate and at least one surface layer selected from diamond, soft diamond, carbon-based material, and carbide. The probe DNA immobilization carrier.   A method for detecting the expression of a gene encoding a membrane protein using the probe DNA-immobilized carrier according to any one of claims 3 to 5. Probe DNA selected from the group consisting of the following DNAs (a) to (e):
(a) DNA comprising the base sequence represented by any of SEQ ID NOs: 1 to 1015
(b) DNA comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(c) DNA having 10 to 5000 bases including a part of the DNA consisting of the base sequence represented by any of SEQ ID NOs: 1 to 1015
(d) DNA of 10 to 5000 bases including a part of DNA consisting of a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 1015
(e) DNA consisting of a base sequence having homology with the DNAs of (a) to (d) and specifically hybridizing with a nucleic acid encoding a corresponding membrane protein.