JP4736439B2 - Nucleic acid immobilization carrier - Google Patents

Description

The present invention relates to a carrier on which a nucleic acid that selectively binds to a test substance is immobilized.

  Research on genetic information analysis of various organisms has been started, and information on many genes and their base sequences including human genes, proteins encoded by the gene sequences, and sugar chains that are secondarily produced from these proteins Is being revealed rapidly. The functions of macromolecules such as genes, proteins, and sugar chains whose sequences have been clarified can be examined by various methods. Mainly, with respect to nucleic acids, the relationship between various genes and their expression of biological functions can be examined by utilizing complementarity between various nucleic acids / nucleic acids such as Northern hybridization or Southern hybridization. With respect to proteins, the function and expression of proteins can be examined using protein / protein reactions, as typified by Western blotting.

  In recent years, a new analysis method or methodology called a DNA microarray method (DNA chip method) has been developed and attracted attention as a method for analyzing many gene expressions at once. In principle, these methods are the same as conventional methods in that they are nucleic acid detection and quantification methods based on nucleic acid / nucleic acid hybridization reactions, and include protein / protein or sugar chains / sugar chains and sugars. It can also be applied to protein and sugar chain detection and quantification based on chain / protein interactions. In addition, for antigen-antibody interaction, an ELISA method (Enzyme Linked Immuno Sorbent Assay) is a method for qualitative and quantitative analysis of an antigen by measuring the activity of the labeled enzyme by binding the antibody to the antigen and the enzyme-labeled antigen. Is mainly used. Also in this method, a fluorescent substance is used as a label, and qualitative / quantitative analysis is performed by detecting fluorescence from the label. Other examples include analytical methods that utilize the phenomenon of excitation energy transfer from fluorescent molecules to other molecules, such as the fluorescent energy transfer method (FRET) and the molecular beacon method. It is essential to detect weak fluorescence with high sensitivity. These techniques have a great feature in that a large number of DNA fragments, proteins, and sugar chains are arranged and fixed at high density on a flat glass substrate called a microarray or chip. As a specific method of using the microarray method, for example, a sample obtained by labeling a gene expressed in a cell to be studied with a fluorescent dye or the like is hybridized on a flat substrate piece, and complementary nucleic acids (DNA or RNA) are bound to each other. There are a method of reading the part at high speed with a high resolution analyzer and a method of detecting a response such as a current value based on an electrochemical reaction. Thus, the amount of each gene in the sample can be quickly estimated.

  As a technique for immobilizing nucleic acid on a substrate, poly-L-lysine, aminosilane, etc. are coated on a flat substrate such as a glass slide, and each nucleic acid is dispersed using a spotting device called a spotter. A method for immobilization has been developed (see, for example, Patent Document 1).

In addition, nucleic acid probes (nucleic acid immobilized on a substrate) used for DNA chips can reduce detection errors and synthesize from conventional cDNAs and fragments thereof of several hundred to several thousand bases. Because of the fact that it can be easily synthesized by a machine, oligo DNA (oligo DNA means that having up to 100 bases) is used as a nucleic acid probe. At this time, the oligo DNA and the glass substrate are bonded by a covalent bond (see, for example, Patent Document 2). As a method for immobilizing oligo DNA via a graft polymer on a solid substrate, there is a method of immobilizing terminal aminated DNA by covalent bonding with the graft polymer. (For example, Patent Documents 3 and 4)
JP 10-503841 A (Claims) JP 2001-108683 A (Claims and Examples) JP-A-2003-130878 (Claims and Examples) JP 2003-130879 A (Claims and Examples)

  However, when the concentration of the sample DNA is very low, there is a problem that the signal after hybridization becomes weak and detection becomes difficult. The problem to be solved by the present invention is to immobilize a polymer as described above on a solid surface, and covalently bond the acyl group bonded to the polymer and the terminal aminated DNA. It is to provide an immobilized substrate and a substrate for amplifying a hybridization signal.

(1) a nucleic acid-immobilized carrier obtained by immobilizing a nucleic acid on a solid surface, are covalently linked derived from a peptide or peptide derivative to the solid surface via one functional group of the solid surface, A nucleic acid- immobilized carrier, wherein a functional group bonded to a peptide or peptide derivative and a nucleic acid are immobilized by a covalent bond.
(2) The nucleic acid- immobilized carrier according to (1), wherein the solid surface is made of a resin .
(3) The nucleic acid- immobilized carrier according to (1), wherein the solid surface has the following general formula (1) as a structural unit.

(Wherein R ¹ , R ² and R ³ each represent an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 18 carbon atoms, or a hydrogen atom. X represents O, NR ³ or CH _2. Represents.)
(4) The nucleic acid- immobilized carrier according to any one of (1) to (3), wherein the functional group bonded to the peptide or peptide derivative is an acyl group .

According to the present invention, it is possible to provide a nucleic acid- immobilized carrier on which a nucleic acid is immobilized via a peptide or peptide derivative-derived substance bound to a solid surface, and a nucleic acid selective binding signal can be amplified.

The nucleic acid immobilization carrier of the present invention and the production method thereof are specifically described below.

As a result of repeated experiments, the present inventors formed a peptide or peptide derivative-derived material on a solid surface as shown in FIG. 1, and the acyl group and nucleic acid bound to the peptide or peptide derivative-derived material. Was found to be covalently immobilized to amplify the hybridization signal. Among these, an oligopeptide or a polypeptide formed from an amino acid can be more preferably used because of its ease of introduction onto a solid surface and cost. In addition, the peptide may be a peptide derivative that is protected with formylmethoxycarbonyl, t-butoxycarbonyl, or the like except for the site associated with the peptide bond. The degree of polymerization of the peptide or peptide derivative derived from the present invention is not particularly limited, but is preferably 1 to 1000 for ease of production. Those derived from this peptide or peptide derivative can be formed on the surface by the following reaction pathway. First, a functional group serving as a foothold is generated on the solid surface. Subsequently, a peptide or peptide derivative derived from a peptide having a functional group capable of binding to this functional group is bound. Then, the nucleic acid is immobilized on the solid substrate via the peptide or peptide derivative-derived substance by reacting with the nucleic acid having a functional group capable of binding to the functional group bonded to the peptide or peptide derivative-derived one. be able to. The functional group bonded to the peptide or peptide derivative-derived one may be the main chain end or the side chain end of the peptide or peptide derivative-derived one. In the case of a peptide or peptide derivative derived from a branched structure, it may be a branched end. As the type of the functional group, an acyl group (RCO-: R is a hydrocarbon) can be preferably used from the viewpoint of easy preparation and nucleic acid immobilization. Examples of acyl compounds having an acyl group include various carboxylates (COOM: M is a metal such as Na or K) or esters (RCOOR ') in which hydrogen atoms of aldehyde group (RCOH), carboxyl group (RCOOH), and carboxyl groups are substituted And acid amide (RCONH ₂ ), acid azide (RCON ₃ ), acid chloride (RCOCl) acid anhydride, and nitrile (RCN) substituted with a hydroxy group of the carboxyl group. Examples of acyl compounds that promote condensation reactions with amino groups and thiol groups include maleimide groups and succinimide groups. Specifically, the method for immobilizing peptides or peptide derivatives derived from the solid substrate is as follows. That is, by generating a carboxyl group on a solid surface, activating the carboxyl group with an ester activator, etc., and then reacting the amino group and the amino group of the amino acid having the carboxyl group with the activated carboxyl group on the surface A carboxyl group can be generated on the solid surface. Examples of the ester activator include succinimidyl 4- [maleimidophenyl] butyrate (SMPB), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate ( SMCC), N- (γ-maleimidobutyroxy) succinimide ester (GMBS), m-maleimidopropionic acid-N-hydroxysuccinimide ester (MPS) and N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), Examples thereof include N-hydroxysulfuccinimide (Sulfo-NHS) and N-hydroxysuccinimide (NHS). In general, various condensing agents such as dicyclohexylcarbodiimide, N-ethyl-5-phenylisoxazolium-3′-sulfonate are used to facilitate the reaction of these bonds. Among these, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium Chloride (DMT-MM) is one of the most effective condensing agents for the condensation reaction between a nucleic acid and a carboxyl group on the solid surface because of its low toxicity and relatively easy removal from the reaction system. . These condensing agents such as EDC may be used by mixing with a nucleic acid solution, or a solid surface on which a carboxyl group is formed on the surface is preliminarily immersed in an EDC solution to obtain a peptide or peptide derivative-derived one. The bonded carboxyl group may be activated.

The substrate of the nucleic acid immobilization carrier of the present invention is not particularly limited. For example, glass, silicon, gold as the metal, silver, platinum, copper, and the resin as polymethyl methacrylate, polyethyl methacrylate, polystyrene, polyacrylonitrile , Polyvinyl acetate, polycarbonate and the like. As a method of generating a functional group on glass or silicon, a hydroxyl group, a carboxyl group, or an amino group can be generated on the surface by dipping in a solution such as a silane coupling agent. Introducing a functional group onto a metal substrate can easily generate a hydroxyl group, a carboxyl group, or an amino group by immersing the substrate in a compound solution having a thiol group at the terminal.

  In addition, as a method of generating a functional group on the surface of the resin carrier, a carboxyl group, a hydroxyl group, or the like can be generated on the surface of the carrier by forcibly oxidizing by plasma treatment or the like. As other methods, methods such as hydrolysis of the side chain of the resin carrier can be used, and this method can be preferably used because it is a simple method.

Therefore, the resin of the nucleic acid immobilization carrier of the present invention is preferably a resin having a side chain structure, and specifically, the resin having a side chain structure is a vinyl such as polystyrene, polyacrylonitrile, polyvinyl acetate or the like. The resin having a structure represented by the following general formula (1) is particularly preferable.

(R ¹ , R ² and R ³ in the general formula (1) represent an alkyl group, an aryl group or a hydrogen atom.)
When the resin has the above structural unit, the side chain easily becomes a carboxyl group by hydrolysis of acid or alkali.

In the present invention, the preferred range of the average degree of polymerization of the resin represented by the general formula (1) is 100 to 10,000. Particularly preferably, it is 200 or more and 5000 or less. The number average degree of polymerization can be easily measured by measuring the molecular weight of the resin by a conventional method using GPC (gel permeation chromatograph). In the general formula (1), R ¹ and R ² represent an alkyl group, an aryl group or a hydrogen atom, and may be the same or different. The alkyl group may be linear or branched and preferably has 1 to 20 carbon atoms. The aryl group preferably has 6 to 18, more preferably 6 to 12 carbon atoms. The functional group X is arbitrarily selected from O, NR ₃ , or CH ₂ . R ₃ is a functional group defined in the same manner as R ¹ and R ² described above.

  Preferred examples of the resin containing various functional groups include polymethyl methacrylate (PMMA), polyethyl methacrylate (PEMA), and polypropyl methacrylate polyalkyl methacrylate (PMMA). Of these, polymethyl methacrylate is particularly preferred. Furthermore, polyvinyl acetate, polycyclohexyl methacrylate, polyphenyl methacrylate, or the like can also be used. Moreover, the copolymer of the structure which combined the said resin component or added the component of another 1 type or multiple types of resin to the component of the said resin can also be used. Examples of the other resin include polystyrene, polyacrylonitrile, and polyamide.

Means for generating carboxyl groups on the resin surface made of such materials include treatment with alkali, acid, etc., ultrasonic treatment in warm water, oxygen plasma, argon plasma, and a method of exposing the resin substrate to radiation. Although there are few damages on the surface of the resin, and it is easy to carry out, it is preferable to immerse the resin substrate in an alkali or acid to generate a carboxyl group on the surface. As a specific example, the resin substrate is immersed in an aqueous solution of sodium hydroxide or sulfuric acid (preferably the concentration is 1N to 20N), preferably at a temperature of 30 ° C. to 80 ° C. and held for 1 to 100 hours. That's fine .

Next, a preferred form of a specific method for using the nucleic acid-immobilized carrier of the present invention will be described. The nucleic acid-immobilized carrier of the present invention is preferably used for a solid called a microarray on which a large number of nucleic acids are immobilized. Here, the nucleic acid means a substance that can selectively bind to a test substance directly or indirectly, and a typical example is a single-stranded nucleic acid. This is because a single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a single-stranded nucleic acid having a base sequence complementary to the base sequence or a part thereof. The nucleic acid may be DNA, RNA or PNA. The nucleic acid used in the present invention may be a commercially available product or may be obtained from a living cell or the like. However, a nucleic acid called oligonucleic acid having a length of 10 to 100 bases can be easily synthesized with a synthesizer. Since it can be artificially synthesized and it is easy to modify the amino group at the end of the nucleic acid, it is preferable because it can be easily immobilized on a solid surface. Furthermore, if it is less than 20 bases, 20 to 100 bases are more preferable from the viewpoint that the stability of hybridization is low. In order to maintain the stability of hybridization, the range of 40 to 100 bases is particularly preferable. In addition to the amino group, an aldehyde group, an epoxy group, a carboxyl group, a hydroxyl group, or a thiol group can be preferably introduced into the functional group capable of terminal modification of a nucleic acid. In the terminal modification of the oligonucleic acid, an alkyl chain is introduced between these functional groups and the nucleic acid. Generally, the alkyl group has 3 to 18 carbon atoms. To immobilize the solid surface and the nucleic acid may be immobilized using a condensing agent such as EDC and DMT-MM as described above. When the acyl group of the polymer molecule on the solid surface is reacted with the amino group of the nucleic acid , the solid surface and the nucleic acid are immobilized by an amide bond, and the carboxyl group and the nucleic acid of the polymer on the solid surface are immobilized. When the hydroxyl group is reacted, the solid surface and the nucleic acid are immobilized by an ester bond. The temperature at which the sample containing the nucleic acid is allowed to act on the solid surface is preferably 5 ° C to 95 ° C, more preferably 15 ° C to 65 ° C. The treatment time is usually 5 minutes to 24 hours, preferably 1 hour or longer.

By immobilizing a nucleic acid on a solid surface via a peptide or peptide derivative-derived substance by the above-described method, the degree of spatial freedom of the immobilized nucleic acid is higher than when immobilized directly on the solid surface. For this reason, it is possible to obtain a nucleic acid- immobilized carrier that provides high hybridization efficiency with a specimen.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to these examples.

Comparative Example 1 and Example 1
(Preparation of DNA immobilization substrates 1 and 2)
A production scheme of the polymer molecule-immobilized PMMA substrate is shown in FIG. A polymethyl methacrylate (PMMA) plate (manufactured by Kuraray Co., Ltd .; Comograss extruded plate, thickness 1 mm, average molecular weight 30,000) was immersed in a 10N aqueous sodium hydroxide solution at 70 ° C. for 15 hours. Subsequently, it wash | cleaned in order of the pure water, 0.1N HCl aqueous solution, and a pure water. In this way, the side chain of PMMA on the substrate surface was hydrolyzed to generate a carboxyl group. Next, (A) 100 mg of N-hydroxysulfuximide (hereinafter abbreviated as Sulfo-NHS) and 350 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were added to 2-morpholinoethanesulfonic acid monohydrate. Dissolved in (MES) buffer (adjusted to pH 6.0 with 0.1 N sodium hydroxide). The PMMA substrate after hydrolysis was immersed in these mixed solutions and stirred with a micro stirrer. (B) After stirring for 1 hour, the substrate was immersed in an aqueous boric acid solution (adjusted to pH 8.3 with 1N sodium hydroxide) containing 80 mg of glycine, which is a kind of amino acid, and stirred with a micro stirrer. After stirring for 1 hour, a DNA-immobilized substrate 1 was prepared (Comparative Example 1) . Further, by repeating the operations (A) and (B) three more times, a DNA-immobilized substrate 2 in which 4 molecules of glycine were bonded to the PMMA surface was obtained (Example 1) .
Example 2
(Preparation of DNA-immobilized substrate 3)
A production scheme of the polymer molecule-immobilized PMMA substrate is shown in FIG.
In the same manner as in Example 1, carboxyl groups were generated on the surface of the PMMA substrate. Next, (a) Sulfo-NHS 100 mg and EDC 350 mg were dissolved in MES buffer (pH 6.0). The PMMA substrate after hydrolysis was immersed in these mixed solutions, and stirred and stirred for 1 hour with a micro stirrer. (B) 80 mg of glycine was dissolved in MES (pH 8.3), 350 mg of EDC was added and stirred for 1 hour to prepare a glycine polymer as a polypeptide. (C) The substrate was immersed in the glycine polymer solution prepared in (b) and stirred for 2 hours to obtain a DNA-immobilized substrate 3.

Example 3
(Preparation of DNA-immobilized substrate 4)
The operation (a) was again performed on the polymer molecule-immobilized PMMA substrate prepared in Example 2, and the succinimide group was modified at the polymer end to obtain the DNA-immobilized substrate 4.

Comparative Example 2
(Preparation of DNA-immobilized substrate 5)
Carboxyl groups were generated on the PMMA substrate by the same operation as in Example 1. Next, after the operation of (A), after stirring for 1 hour, the substrate was immersed in an aqueous boric acid solution (adjusted to pH 8.3 with 1N sodium hydroxide) containing 80 mg of alanine, which is an amino acid, and stirred with a micro stirrer. . After stirring for 1 hour, a DNA-immobilized substrate 5 was produced.

Example 4
(Preparation of DNA-immobilized substrate 6)
A glass substrate was used as the DNA immobilization substrate. A production scheme of a polymer molecule-immobilized glass substrate is shown in FIG.

  The slide glass was immersed in a 10N NaOH aqueous solution for 1 hour and then thoroughly washed with pure water. Next, after APS (3-aminopropyltriethoxysilane; manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in pure water at a ratio of 2% by weight, the slide glass was immersed for 1 hour and taken out from this solution. Dried at 110 ° C. for 10 minutes. In this way, amino groups were introduced on the surface of the glass.

  Subsequently, 5.5 g of succinic anhydride was dissolved in 335 ml of 1-methyl-2-pyrrolidone. 1M 50 ml sodium borate (3.09 g boric acid and sodium hydroxide for pH adjustment was added to make up to 50 ml with pure water. PH 8.0) was added to the succinic acid solution. The glass substrate was immersed in this mixed solution for 20 minutes. After soaking, it was washed with pure water and dried. In this way, the amino group on the surface of the glass substrate was reacted with succinic anhydride to introduce carboxylic acid on the glass surface. The DNA-immobilized substrate 6 was prepared by reacting with the glycine polymer in the same manner as in Example 2. This reaction scheme is shown in FIG.

Comparative Example 3
(Preparation of DNA-immobilized substrate 7)
For comparison, a carboxyl group was generated on the PMMA substrate by hydrolysis in the same manner as in Example 1 to obtain a DNA-immobilized substrate 7.

Comparative Example 4
(Preparation of DNA-immobilized substrate 8)
Carboxyl groups were produced by reacting succinic anhydride on the glass surface into which amino groups had been introduced in the same manner as in Example 5 to obtain a DNA-immobilized substrate 8.
(Immobilization of probe DNA)
The DNA of SEQ ID NO: 1 (60 bases, 5 ′ terminal amination) was synthesized.
5'-ACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCATCTGGA-3 '. The DNA of SEQ ID NO: 1 is aminated at the 5 ′ end.

These DNAs were dissolved in pure water at a concentration of 0.27 nmol / μl to obtain a stock solution. When spotting on the substrate, PBS (NaCl 8g, Na2HPO4 · 12H2O 2.9g, KCl 0.2g, KH2PO4 0.2g dissolved in pure water and made up to 1 liter was adjusted to pH 1 hydrochloric acid. In addition, the final concentration of the probe was adjusted to 0.027 nmol / μl at pH 5.5), and 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide (EDC) was added to bring the final concentration to 50 mg / ml. And about 200 nl of these mixed solutions were taken out and this was spotted on the board | substrate. Next, the substrate was sealed in a plastic container, incubated at 37 ° C. and 100% humidity for about 20 hours, and washed with pure water.

(Adjustment of sample DNA)
As the sample DNA, DNA of SEQ ID NO: 5 (968 bases) having a base sequence capable of hybridizing with the DNA-immobilized substrate was used. The adjustment method is shown below.

As the sample DNA, DNA (968 bp) having a base sequence capable of hybridizing with the DNA-immobilized substrate was used. The adjustment method is shown below.
Nucleic acids having the following sequences were synthesized.
5'-GGGCGAAGAAGTTGTCCATA-3 '(SEQ ID NO: 20 bases)
5'-GCAGAGCGAGGTATGTAGGC-3 '(SEQ ID NO: 3: 20 bases)
This was dissolved in pure water to a concentration of 100 μM. Next, pKF3 plasmid DNA (Takara Bio Inc. product number; 3100) (SEQ ID NO: 2264 bases) was prepared, which was used as a template, and the DNA reaction of SEQ ID NO: 2 and SEQ ID NO: 3 was used as a primer for PCR reaction ( Amplification was performed by Polymerase Chain Reaction.

  The conditions for PCR are as follows. That is, ExTaq 2 μl, 10 × ExBuffer 40 μl, dNTP Mix 32 μl (the above is manufactured by Takara Bio Inc .; attached to product number RR001A), SEQ ID NO: 2 solution 2 μl, SEQ ID NO 3 solution 2 μl, Template (SEQ ID NO: 4) 0.2 μl was added, and the volume was increased to 400 μl with pure water. These mixed solutions were divided into four microtubes, and PCR reaction was performed using a thermal cycler. This was purified by ethanol precipitation and dissolved in 40 μl of pure water.

  Subsequently, a 9-base random primer (manufactured by Takara Bio Inc .; product number 3802) was dissolved at a concentration of 6 mg / ml, and 2 μl was added to the DNA solution purified after the PCR reaction. This solution was heated to 100 ° C. and then rapidly cooled on ice. To this, 5 μl of the buffer attached to Klenow Fragment (Takara Bio Inc .; product number 2140AK) and 2.5 μl of dNTP mixture (dATP, dTTP, dGTP concentrations of 2.5 mM and dCTP concentration of 400 μM, respectively) were added. . Furthermore, 2 μl of Cy3-dCTP (manufactured by Amersham Pharmacia Biotech; product number PA53021) was added. To this solution, 10 U of Klenow Fragment was added and incubated at 37 ° C. for 20 hours to obtain a sample DNA (SEQ ID NO: 5: 968 bases) labeled with Cy3. This was further purified by ethanol precipitation and dried.

This labeled sample DNA is dissolved in 1% by weight BSA (bovine serum albumin), 5 × SSC (5 × SSC is 43.8 g of NaCl and trisodium citrate hydrate is dissolved in 22.1 g of pure water. 200 ml of NaCl, 43.8 g of NaCl and 22.1 g of trisodium citrate hydrate dissolved in pure water, and 1 ml of the mess up to 1 l. The solution is expressed as 10 × SSC, and the 5-fold diluted solution is expressed as 0.2 × SSC.), 0.1 wt% SDS (sodium dodecyl sulfate), 0.01 wt% salmon sperm DNA solution (each concentration is (Final concentration), dissolved in 400 μl to obtain a solution for hybridization.
(Hybridization)
The sample DNA was hybridized to each substrate on which the probe DNA obtained above was immobilized. Specifically, 10 μl of a hybridization solution was dropped on a carrier on which the probe nucleic acid prepared previously was immobilized, and a cover glass was placed thereon. In addition, the periphery of the cover glass was sealed with a paper bond so that the hybridization solution was not dried. This was placed in a plastic container and incubated at 65 ° C. and 100% humidity for 10 hours. After incubation, the cover glass was peeled off, washed and dried.
(Measurement)
The substrate after the above treatment was set on a DNA chip scanner (Axon Instruments GenePix 4000A), and measurement was performed with the laser output set to 33% and the photomultiplier voltage set to 500V. The results are shown in Table 1.

  Table 1 shows fluorescence signals after nucleic acid hybridization using DNA-immobilized substrates prepared by various methods.

DNA-immobilized substrate 1 of Comparative Example 1 DNA immobilized via glycine, as compared to the DNA-immobilized substrate 7 of Comparative Example 3 was DNA immobilized via the carboxyl group of the substrate surface, via a glycine-quarter element Highly efficient hybridization was observed for the DNA-immobilized substrate 2 of Example 1 immobilized with DNA and the DNA-immobilized substrate 3 of Example 2 immobilized with DNA via polyglycine . Further, from the result of the DNA-immobilized substrate 4 of Example 3, almost the same effect was confirmed even when the terminal of the peptide or peptide derivative-derived one was modified with a succinimide group. From the results of DNA-immobilized substrate 5 of Comparative Example 2, the amino acid is the same as glycine DNA-immobilized substrate 1 also Comparative Example 1 in the case of alanine was confirmed. Furthermore, the DNA-immobilized substrate 6 of Example 4 and the DNA-immobilized substrate 8 of Comparative Example 4 were compared, and even when a glass substrate was used, hybridization was achieved by immobilizing DNA via a glycine polymer. High efficiency was confirmed. Therefore, in any substrate, the peptide or peptide derivative-derived material is immobilized on a solid substrate, and the acyl group bound to the peptide or peptide derivative-derived material is condensed and immobilized on the DNA amino group. Thus, it was shown that the hybridization efficiency is dramatically improved.

Immobilization of nucleic acids via peptides or peptide derivatives from solid substrates Scheme 1 for immobilizing nucleic acid (DNA) on the surface of PMMA Scheme 2 for immobilizing nucleic acid (DNA) on the surface of PMMA Scheme 4 for immobilizing nucleic acid (DNA) on glass surface

Explanation of symbols

1 solid substrate 2 peptide or peptide derivative-derived 3 nucleic acid 4 PMMA substrate 5 succinimide group 6 glycine or alanine 7 terminal aminated DNA
8 Glycine polymer 9 Glass substrate 10 Succinic anhydride

Claims (4)

The nucleic acid to a solid surface an immobilized nucleic acid-immobilized carrier, those derived from peptides or peptide derivatives on the solid surface are covalently linked via one functional group of the solid surface, the peptide or A nucleic acid- immobilized carrier, wherein a functional group bonded to a peptide derivative-derived one and a nucleic acid are immobilized by a covalent bond. The nucleic acid- immobilized carrier according to claim 1, wherein the solid surface is made of a resin. The nucleic acid- immobilized carrier according to claim 1, wherein the solid surface has the following general formula (1) as a structural unit.

(Wherein R ¹ , R ² and R ³ each represent an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 18 carbon atoms, or a hydrogen atom. X represents O, NR ₃ or CH _2. Represents.) The nucleic acid- immobilized carrier according to any one of claims 1 to 3, wherein the functional group bonded to the peptide or peptide derivative is an acyl group.

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