JP4161053B2 - DNA chip and target DNA detection method. - Google Patents

Description

  The present invention relates to a DNA chip and a method for detecting a target DNA using the DNA chip.

  A DNA chip has been developed as a means for efficiently analyzing a gene function of an organism. A DNA chip is a device capable of obtaining expression information of various genes by immobilizing DNA on a substrate and utilizing hybridization. In the manufacture of a DNA chip, it is necessary to immobilize DNA serving as a probe on a substrate. However, immobilization of DNA on the substrate surface is very difficult, and various methods have been proposed so far. However, DNA that functions on the substrate surface cannot be efficiently introduced, and hybridization selectivity and efficiency are improved. Is a problem. Further, the immobilization technique is complicated and there is a problem of cost.

  For example, as a method for immobilizing DNA, an attempt has been made to bind thiol to single-stranded DNA and immobilize the thiolated single-stranded DNA on a metal substrate. However, this method has a problem that the immobilized DNA strand collapses due to its affinity with the substrate, and the selectivity is low and the efficiency of hybridization is not high.

On the other hand, after immobilizing thiolated single-stranded DNA, attempts to increase surface coverage and activity by removing non-single-stranded DNA present on the surface by using non-specific adsorption using alkanethiol (Non-patent Document 1). However, there has been a problem that introduction of an alkyl group takes a lot of labor.
There has also been an attempt to immobilize a DNA chain having a double-stranded part and a single-stranded part on the substrate surface on the double-stranded part side (Patent Document 1). This method prevents the DNA strand from falling down by making the double-stranded portion dense, and contains a DNA strand having only a double strand, and in a place away from the substrate, a space around the single-stranded portion. This makes it easy to use for hybridization. However, this method has a problem that the process is complicated, for example, it is necessary to prepare and fix double-stranded DNA and single-stranded and double-stranded DNA in advance. In addition, since the alkanethiol reagent used for the second stage of immobilization has a stronger immobilization force on the surface than thiolated DNA, it can easily undergo an exchange reaction with the thiolated DNA immobilized on the substrate surface.

  In order to solve such a problem, the present inventors have developed a DNA chip in which probe DNA is bonded to a polymer and the polymer is immobilized on a gold substrate (Non-patent Document 2). According to this method, since the polymer has hydrophobicity and the DNA strand is hydrophilic, the DNA strand can be exposed to the solution side without falling down on the substrate surface. Moreover, since the introduction of DNA into the polymer can be easily controlled, there is an advantage that the introduction density of DNA strands onto the substrate can be easily controlled.

  However, in the case of this method, it is necessary to prepare a number of polymers corresponding to the type of probe, and there is a problem that the type of probe sequence cannot be easily selected. In addition, there is a problem that the linker connecting the polymer and the DNA chain is short and the degree of freedom of the probe is limited, and further improvement in the selectivity of hybridization has been desired.

JP 2003-43037 A J. et al. Am. Chem. Soc. 1998, 120, 9787-9792 Analytical Sciences, January 2003, vol. 19, 177-179

  An object of the present invention is to solve the problems associated with the prior art as described above, and to provide a DNA chip having excellent hybridization selectivity and efficiency. It is another object of the present invention to provide a DNA chip in which the introduction of a DNA strand to be a probe is simple and the introduction density of DNA strands can be easily controlled.

The inventors of the present invention have intensively studied to solve the above-mentioned problems, and by binding a DNA strand to a substrate through a polymer and interposing a hydrophilic DNA linker between the polymer and the probe DNA, It has been found that the selectivity and efficiency of hybridization can be dramatically improved. In addition, the present inventors have found that the introduction density of DNA strands can be controlled and that an infinite number of types of probe DNA sequences can be provided by using at least one type of linker DNA in one polymer main chain. It came to be completed.
That is, the present invention includes the following.
[1] A DNA chip in which a DNA strand is fixed on a substrate surface via a polymer, the DNA strand comprising a single-stranded probe DNA portion and a double-stranded linker DNA portion,
The DNA chip, wherein the DNA chain is bonded to the polymer on the linker DNA portion side, and the polymer is fixed to at least one surface of the substrate through a substrate binding group.
[2] The DNA chip according to [1], wherein the linker DNA portion is bonded to the polymer by a covalent bond.
[3] The DNA chip according to [1] or [2], wherein the linker DNA portion has 10 to 150 base pairs.
[4] The DNA chip according to any one of [1] to [3], wherein the polymer has a polymer main chain composed of a hydrophobic constituent unit.
[5] The DNA chip according to any one of [1] to [4], wherein the polymer has a functional group.
[6] The DNA chip according to [5], wherein the functional group includes an anionic functional group.
[7] The DNA chip according to [6], wherein an anionic functional group is contained in an amount of 50% or more based on the total number of functional groups.
[8] The DNA chip according to any one of [1] to [7], wherein the polymer is coated on a substrate via a sulfur atom in the substrate binding group.
[9] A method for detecting a target DNA comprising the following steps:
(1) A step of bringing the target DNA into contact with the surface to which the polymer of the DNA chip surface according to any one of claims 1 to 8 is bound (step 1),
(2) A step of detecting the target DNA hybridized to the probe DNA portion (step 2).
[10] The method according to [9], wherein in the step 1, the polymer in the DNA chip has an anionic functional group, and the target DNA is contacted in a basic aqueous solution.
[11] The method according to [9] or [10], wherein the step 1 is performed at a salt strength of 1 M or less.
[12] The method according to any one of [9] to [11], wherein the step 1 is performed in a temperature range of 25 to 50 ° C.

  According to the present invention, a DNA strand is bound to a substrate through a polymer, and a hydrophilic DNA linker is interposed between the polymer and the probe DNA, thereby improving the selectivity and efficiency of hybridization. Can do. In addition, the introduction density of DNA strands can be controlled. Furthermore, by using at least one kind of linker DNA in one polymer main chain, it is possible to provide an unlimited number of kinds of probe DNA sequences. In addition, according to the present invention, the substrate can be easily returned to the state before use by performing dehybridization after using the DNA chip, so that the DNA chip can be reused.

  The DNA chip according to the present invention is a DNA chip in which a DNA strand is fixed on a substrate surface via a polymer, and the DNA strand is composed of a single-stranded probe DNA portion and a double-stranded linker DNA portion. The DNA strand binds to the polymer on the linker DNA portion side, and the polymer is fixed to at least one surface of the substrate via a substrate binding group.

DNA strand The DNA strand of the DNA chip of the present invention is a combination of long-chain DNA and short-chain DNA having a sequence complementary to the sequence of the linker moiety in the sequence of the long-chain DNA. The single-stranded probe DNA portion is a site that hybridizes with the target DNA, and the double-stranded linker DNA portion is bonded to the polymer as a linker. The linker DNA moiety is preferably bound to the polymer by a covalent bond.

  For the number of base pairs in the linker DNA portion, for example, the lower limit is preferably 10 bases (mer) or more, more preferably 12 bases (mer) or more, more preferably 15 bases (mer) or more. The upper limit of the number of base pairs is preferably in the range of 150 bases (mer) or less, more preferably 100 bases (mer) or less, more preferably 40 bases (mer) or less.

When the linker DNA portion has a length in the above range, the spatial freedom of the probe DNA portion can be ensured, and hybridization can be performed efficiently. In addition, when the number of base pairs is in the above range, melting of base pairs does not occur during use at room temperature (for example, about 20 ° C.). When the number of base pairs composed only of T / A is 10 mer, the melting temperature (Tm) is 27 ° C., 12 mer is 38 ° C., and 15 mer is 50 ° C. (however, the salt strength is 0. 3M). Moreover, Tm rises by replacing with G / C.
Furthermore, when it has the length of the said range, a certain amount or more negative charge can be hold | maintained at a linker part. Although DNA usually has a negative charge, in the present invention, since the linker DNA portion is double-stranded, the charge is theoretically twice as large as that of normal single-stranded DNA. For this reason, when the polymer has an anionic functional group, the linker DNA has the number of base pairs in the above range, so that the repulsion of charges is further increased, and it is possible to more effectively prevent the DNA strand from falling on the substrate. it can. In addition, non-specific adsorption can be further eliminated and hybridization selectivity can be improved.

The base sequence of the linker DNA portion is not particularly limited, but the G / C content in the sequence where the long DNA and the short DNA are bound is preferably 13% or more, more preferably 13% or more and 26% or less. It is preferable. When the G / C content is in the above range, the hybridization between the long DNA and the short DNA can be performed specifically.
Moreover, it is preferable that the base pairs at both ends of the sequence to which the long DNA and the short DNA are bound are G / C or C / G. By setting it as such an arrangement | sequence, the shift | offset | difference of a coupling | bonding of long-chain DNA and short-chain DNA can be prevented.

  Although the number of base pairs in the probe DNA portion is not limited depending on the length of the target DNA, it is usually preferably in the range of, for example, 6 bases (mer) to 100 bases (mer).

Polymer The polymer used in the present invention has a DNA chain and a substrate binding group bonded to the polymer main chain. Further, a functional group may be bonded to the polymer main chain. The polymer main chain and the functional group will be specifically described.

<Polymer main chain>
In the polymer used in the present invention, the main chain is preferably hydrophobic. As a hydrophobic polymer main chain, it is preferable that it has a structural unit by a carbon-carbon bond, for example. Examples of the structural unit composed of such a carbon-carbon bond include those represented by the following formula (I).

In the formula (I), R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, a functional group, a DNA chain or a substrate binding group. As the structural unit, the same or different units can be continuously formed to form a polymer.

  In this invention, the polymer may have other structural units other than the structural unit by the said carbon-carbon bond.

As other structural units of the main chain, for example, an alkyleneoxy group such as an ethyleneoxy group — (C ₂ O) _n — (n is an integer) [— (C _m O) —, where m is an integer of 1 to 10. To express. And the like. In the other structural units, the same side chains as those of R ¹ , R ² , R ³ and R ⁴ can be bonded.

  The content ratio of the structural unit due to the carbon-carbon bond in the polymer main chain and other structural units (constituent unit due to the carbon-carbon bond: other structural unit) is preferably in the polymer main chain, preferably from 50:50 to 100: 0 (mol / g-polymer), more preferably 80:20 to 100: 0 (mol / g-polymer) or more, more preferably 90:10 to 100: 0 (mol / g-polymer) or more, particularly Preferably it is 100: 0 (mol / g-polymer).

  When the structural unit by the carbon-carbon bond is in the above range, the polymer main chain is hydrophobic and the DNA is hydrophilic. Therefore, when performing hybridization in an aqueous solution, It can be exposed to the aqueous solution side. For this reason, hybridization can be performed with high efficiency.

<Functional group>
The polymer used in the present invention may be referred to simply as a functional group remaining without being bonded to these other than the functional group used for bonding to the DNA strand and bonding to the substrate binding group (hereinafter simply referred to as “functional group”). ). When the functional group is present, the selectivity for hybridization with the target DNA can be improved by repulsion between the polymer main chain and the DNA chain.

  The type of functional group on the polymer is such that the functional group has a role of binding the DNA strand and the compound that induces the substrate binding group to the polymer. It depends on the type. For example, examples of the functional group include an anionic functional group, a nonionic functional group, and a cationic functional group. Among these, from the viewpoint of improving selectivity, an anionic functional group or a nonionic functional group is preferably contained, and an anionic functional group is more preferred. Further, from the viewpoint of hybridization efficiency, a cationic functional group or a nonionic functional group is preferably contained, and a cationic functional group is more preferable. For detection of single-base substitution sequences such as SNPs, an anionic functional group or a nonionic functional group is preferably contained, and an anionic functional group is more preferred.

Anionic functional group As the anionic functional group, any functional group may be used as long as the pH is near neutral and alkaline and negatively charged.

Specific examples of such anionic functional groups include a carboxyl group (—COOH group), a sulfonic acid group (—SO ₃ H group), a sulfinic acid group (—SO ₂ H group), and a phosphoric acid group. (—OPO ₃ H ₂ group (or its monoalkyl ester group, its dialkyl ester group)), sulfate group (—OSO ₃ H group), phenolic hydroxyl group and the like.

  Of these, a carboxyl group is preferred. In particular, for binding to the DNA strand, it is preferable to use a carboxyl group when the end of the DNA strand is an amino group.

  These anionic functional groups have their salts, for example, alkali metal salts (for example, Na, K salts), ammonium salts (for example, ammonia, methylamine, etc.), if the pH is near neutral and alkaline and negatively charged. Salt of dimethylamine or the like).

Nonionic Functional Group Examples of the nonionic functional group include groups that do not exhibit ionicity in a neutral or alkaline aqueous solution, such as an alkyl group, a hydroxyl group, and an alkyloxy group.

Cationic functional group Examples of the cationic functional group include -NH ₃ ⁺ , -NH ₂ (CH ₃ ) ⁺ , -NH (CH ₃ ) ₂ ⁺ , -N (CH ₃ ) ₃ ^{+, and the} like. And alkaline and positively charged groups.

  When the polymer contains a remaining functional group, the anionic functional group is preferably 50% or more, more preferably 80% or more, more preferably 90% or more, particularly with respect to the number of moles of all remaining functional groups. Preferably, all functional groups are anionic functional groups.

  Moreover, when it contains a functional group, the abundance ratio of the anionic functional group and the DNA chain in the polymer [anionic functional group: DNA chain (mol / g-polymer)] is preferably 50: 1 to 500: 1, more preferably in the range of 100: 1 to 300: 1. By making the presence rate of the DNA strand sparse, the spatial freedom of the probe DNA can be secured, so that the hybridization efficiency can be improved.

  When a functional group other than an anionic functional group is included as a functional group, a nonionic functional group or a cationic functional group is exemplified, but a nonionic functional group is more preferable in order to maintain repulsion with the DNA strand. .

DNA usually has an anionic property, but since the linker DNA portion of the DNA strand has a double-stranded structure, it has an anionic charge twice that of the single-stranded structure. When the polymer portion has an anionic functional group, the repulsion between the linker DNA portion and the anionic functional group of the polymer increases synergistically, and the DNA strand can be substantially prevented from falling. Further, in specific adsorption and nonspecific adsorption, the ability to eliminate nonspecific adsorption due to electrostatic repulsion is further enhanced.
That is, by imparting anionicity to the polymer, the hybridization selectivity can be significantly improved.

  The weight average molecular weight of the polymer used in the present invention varies depending on the size and density of the DNA chip used and is not limited. Usually, the weight average molecular weight (Mw) measured by GPC (gel permeation chromatography) is 50,000 to 150. It is preferably in the range of 10,000.

<Polymer production method>
The polymer that can be used in the present invention can be produced by a commercially available product or a (co) polymerization of monomers by a known method. The functional group present in the monomer may be used for bonding with a DNA chain or a compound that induces a substrate binding group, and may remain without being used for bonding to become a functional group of the polymer.

An anionic monomer such as a polymer having an anionic functional group can be obtained by polymerization of a monomer containing an anionic monomer.
Examples of such anionic monomers include (meth) acrylic acid, crotonic acid, itaconic acid, maleic acid, monoalkyl itaconate (eg monomethyl itaconate, monoethyl itaconate), monoalkyl maleate (eg maleic acid) Monomethyl, monoethyl maleate, etc.), monomers having a carboxyl group such as citraconic acid;

  Vinyl sulfonic acid, styrene sulfonic acid, vinyl benzyl sulfonic acid, acryloyloxyalkyl sulfonic acid (for example, acryloyloxymethyl sulfonic acid, acryloyloxyethyl sulfonic acid, acryloyloxypropyl sulfonic acid, etc.), methacryloyloxyalkyl sulfonic acid (for example, methacryloyl) Oxymethylsulfonic acid, methacryloyloxyethylsulfonic acid, methacryloyloxypropylsulfonic acid, etc.), acrylamide alkylsulfonic acid (eg 2-acrylamido-2-methylethanesulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, 2- Acrylamide-2-methylbutanesulfonic acid), methacrylamide alkylsulfonic acid (eg 2-methacrylamide-2-methylethanesulfonic acid) 2-methacrylamide-2-methylpropanesulfonic acid, a monomer having a sulfonic acid group such as a 2-methacrylamide-2-methyl butanoic acid);

Examples include vinyl sulfinic acid, vinyl sulfuric acid, vinyl phosphoric acid, styrene phosphoric acid, vinyl phenol, glutamic acid, and aspartic acid.
Among these, a monomer having a carboxyl group is desirable.

When the binding site of the DNA chain or substrate binding group to the polymer is an amino group, the carboxyl group, aldehyde group, isocyanate group (—NCO), isothiocyanate in the polymer is used for the binding to the amino group. It is preferred that a narto group (-NCS) is present.
For this reason, it is preferable to use the monomer which has a carboxyl group as an anionic monomer. In the case of using a monomer having a sulfonic acid group, a sulfinic acid group, a sulfuric acid group, a phosphoric acid group, etc., a monomer having a carboxyl group, an aldehyde group, an isocyanate group (—NCO) or an isothiocyanate group (—NCS) It is preferable to copolymerize (a monomer for bonding).
As a monomer which has an aldehyde group, the monomer whose carboxyl group of the monomer which has the said carboxyl group is an aldehyde group is mentioned.
Examples of the monomer having an isocyanate group include 2-isocyanate ethyl (meth) acrylate.
When these bonding monomers are copolymerized with an anionic monomer, the content of the bonding monomers may be a ratio corresponding to the number of DNA chains to be bonded and the number of substrate binding groups, as will be described later.

Polymers having an anionic functional group Among the polymers having an anionic functional group obtained by polymerizing such an anionic monomer, specific examples of polymers having a carboxyl group are shown. The DNA strand and the substrate binding group can be bound to a carboxyl group in the polymer.

  Examples of the polymer having a carboxyl group that can be used in the present invention include poly (meth) acrylic acid, polycrotonic acid, polyitaconic acid, polymaleic acid, monomethyl polyitaconate, monoethyl polyitaconate, monomethyl polymaleate, monoethyl maleate, poly Examples thereof include polymers having a carboxyl group such as citraconic acid.

  These acids may be salts of alkali metals such as Na and K or ammonium ions.

  In the present invention, a monomer having an anionic functional group may be copolymerized with a monomer having a nonionic functional group and a monomer having a cationic functional group.

Nonionic monomer Examples of the nonionic monomer include amides such as acrylamide and methacrylamide;

  Methyl acrylate, methyl methacrylate, vinyl acetate, allyl acetate, allyl acetoacetate, vinyl trimethyl acetate, vinyl formic acid, vinyl hexanoate, vinyl laurate, vinyl methacrylate, vinyl octoate, vinyl palmitate, vinyl pivalate, propion Vinyl acetate, vinyl stearate, mono-2- (methacryloyloxy) ethyl hexahydrophthalate, mono-2- (methacryloyloxy) ethyl phthalate, vinyl benzoate, p-vinylbenzoic acid, vinyl butyrate, vinyl caprate, capron Vinyl acid, vinyl crotonate, vinyl decanoate, vinyl cinnamate, allyl butyrate allyl benzoate, allyl n-butyrate, allyl n-caprate, allyl n-caproate, allyl enanthate, allyl heptanoate, isophthalic acid Allyl, allyl isothiocyanate, Valeric acid allyl esters such as n- valerate allyl;

  Ketones such as vinyl methyl ketone;

  Ethers such as vinyl butyl ether, allyl ether, allyl ethyl ether, allyl butyl ether, vinyl ethyl ether, allyl n-decanoate;

  Alcohols such as vinyl alcohol and allyl alcohol;

  Halides such as vinyl chloride, allyl chloride, methacryloyl chloride, vinyl chloroacetate, acryloyl chloride, allyl bromide, allyl iodide, allyl chloroacetate, allyl chloroformate, allyl chloroformate;

  Styrene, allylbenzene, 4-methacryloxy-2-hydroxybenzophenone, vinyl toluene, allyl benzyl ether, 4-allyl-2,6-dimethoxyphenol, allyl allyl, 4-allyl-1,2-dimethoxybenzene, etc. An aromatic compound having a benzene ring;

  3-silanes such as 3-methacryloxypropyltrimethoxysilane, vinyltrichlorosilane, allylchlorodimethylsilane, allylchloromethyldimethylsilane;

  Cyanides such as methacrylonitrile, vinylacetonitrile, acrylonitrile, allyl cyanoacetate, allyl cyanide;

  Cycloalkane derivatives such as 2-allylcyclohexanone, 1-allylcyclohexanol, allylcyclopentane;

  Others, vinyl anthracene, vinyl sulfone, allyl alcohol propoxylate, allyl-L-cysteine, allyl ethylene, allyl glycidyl ether, allyl trifluoroacetic acid, allyl cyclopentadienyl nickel, allyl diethyl phosphonoacetate, allyl diphenyl phosphine, allyl diphenyl Examples thereof include phosphine oxide and allyl disulfide.

  Of these, styrene, allylbenzene, and the like can be preferably used as a monomer that provides a hydrophobic nonionic polymer.

Cationic monomer Examples of the cationic monomer include primary amines such as allylamine, 3-acrylamide-N, N-dimethylpropylamine, allylcyclohexylamine, and 3-methacrylamide-N-dimethylpropylamine;

  Secondary amines such as methylallylamine;

  Tertiary amines such as N-allyldiethylamine and N-allyldimethylamine;

  Quaternary ammonium such as allyltriethylammonium, (3-acrylamidopropyl) trimethylammonium chloride, vinyltrimethylammonium bromide, 3- (methacryloylamino) propyltrimethylammonium chloride, ethyltrimethylammonium methacrylate, diallyldimethylammonium and the like can be mentioned.

  When the polymer has a functional group, the amount of the anionic monomer used is preferably 50 mol% or more, more preferably 80 mol% or more, more preferably 90 mol% or more, particularly with respect to the total number of moles of monomers. Preferably, all the monomers are anionic monomers. Moreover, when it contains monomers other than an anionic monomer, it is preferable that it is a nonionic monomer.

  Polymerization of the monomer can be performed by a known method, for example, in the presence or absence of a solvent, and a polymerization initiator can be added as necessary to polymerize the monomer. For the polymerization reaction, methods such as random (co) polymerization, block (co) polymerization, and graft (co) polymerization can be employed.

  The solvent is not limited as long as it dissolves the monomer. For example, THF, methanol, DMF, DMSO and the like can be used.

  Examples of the polymerization initiator include 2,2′-azobis (isobutyronitrile) (AIBN), 1,1′-azobis (cyclohexane-1-carbonitrile), 2,2′-azobis (2-methylbutyro). Nitrile) or the like can be used. In addition to such azo compounds, peroxides, organometallic compounds, and the like can also be used.

  The polymerization varies depending on the kind of the monomer and is not limited. Usually, for example, the polymerization can be performed in a temperature range of about room temperature to about 100 ° C. for about 1 to 72 hours.

Substrate There is no limitation on the substrate that can be used in the present invention. For example, any of a metal, an inorganic material, and an organic material can be used. Among these, a metal substrate can be preferably used.

  Examples of the metal substrate include gold, silver, chromium, gallium, nickel, and the like. Of these, gold is preferably used.

  Examples of the inorganic material substrate include a glass substrate.

  Examples of the organic material substrate include a silicon substrate, polyvinyl chloride, and paper.

  The shape of the substrate is not limited, and examples thereof include a plate shape, a tubular shape, and a spherical shape. Among these, a plate-like shape can be preferably used.

Method for Producing DNA Chip The DNA chip according to the present invention can be produced by introducing a DNA chain and a substrate binding group into a polymer main chain, and contacting and immobilizing the polymer on the substrate surface.

  The method for producing such a DNA chip is not limited. For example, (1) First, a short chain DNA (single strand) and a compound that induces a substrate binding group are bound to a polymer, and then the resulting short chain is obtained. Examples include a method of obtaining a DNA chip by binding a DNA binding polymer to a substrate and further binding (hybridizing) long DNA (single strand) to short DNA.

(2) First, a solution in which long-chain DNA and target DNA are mixed is prepared and dropped onto a short-chain DNA conjugate polymer-immobilized substrate. The temperature is higher than the melting temperature of the long DNA and the short DNA (Tm of hybridization of the target sequence portion of the long DNA). Hybridization of the target sequence portion of long-chain DNA is performed in a homogeneous system, and then surface hybridization of long-chain DNA and short-chain DNA is performed by drastically lowering the temperature (near the Tm of long-chain and short-chain DNA). By doing so, the target DNA can be present on the substrate surface without dissociating the hybridization of the target sequence portion of the long-chain DNA.
Furthermore, the long DNA having the probe DNA portion, the short DNA, and the completely complementary strand sequence is first hybridized with the target DNA, and then the short DNA bonded to the polymer and the completely complementary strand sequence portion Can also be applied to gene expression analysis.
The specific example of a manufacturing method is shown below.

Short DNA
It is preferable that the short-chain DNA has a group that can be bonded by reacting with a functional group in the polymer at a terminal bonded to the polymer. For example, when the functional group in the polymer has a carboxyl group that is an anionic functional group, or a functional group such as an aldehyde group, an isocyanate group, or an isothiocyanate group, the end of the short-chain DNA is aminated (—NH ₂ ). It is preferable that The terminal amino group may be directly bonded to the DNA chain, or may be bonded to the DNA chain via an alkyl group having 1 to 20 carbon atoms. A commercially available product can be used as such a terminally aminated DNA.

  In addition, in the short-chain DNA having an amino group at the terminal, a group serving as a spacer may be added if necessary. By introducing a spacer, it is possible to bind the short-chain DNA to the polymer with high yield. For example, N- (trifluoroacetylcaproyloxy) succinimide ester (TFCS) is reacted with DNA aminated at the end in a neutral (pH 7.0) solvent at a temperature of −10 to 10 ° C., Furthermore, in order to deprotect the trifluoroacetyl group of TFCS, the reaction was carried out at room temperature while gradually adjusting the pH of the solution to be alkaline (pH 8), whereby a spacer having a hydrazino group (terminal amino group) was introduced. Strand DNA can be obtained.

The compound substrate bonding group for deriving the substrate bonding group is a group for bonding a polymer to the substrate, and has a functional group for bonding to the substrate and a functional group for bonding to the polymer. The functional group for bonding to the substrate varies depending on the type of the substrate and is not limited. For example, a group having a sulfur atom can be preferably used. Examples of the group having a sulfur atom include a thiol group, a pyridyldithio group, a disulfide group, a benzenethiol group, a pyridinethiol group, and an alkanethiol group. The polymer is preferably coated on the substrate via sulfur atoms in the substrate binding group.

When the functional group in the polymer is an anionic functional group such as a carboxyl group or an acidic functional group such as a sulfonic acid group, the group to be bonded to the polymer in the substrate binding group is an amino group as in the short-chain DNA. A group (—NH ₂ ) and a hydrazide group are preferred.
Examples of the compound that induces such a substrate binding group include 3 (2-pyridyldithio) propionol hydrazide (PDPH), 3-[(2-aminoethyl) dithio] propionic acid hydrochloride (AEDP), and the like. Can be mentioned.

Bonding of polymer with short-chain DNA, compound that induces substrate binding group Short-chain DNA or compound that induces substrate-binding group, and polymer are bonded to the functional group of the polymer and short-chain DNA or substrate binding. There are no limitations depending on the type of functional group the group has, but for example, the carboxyl group, aldehyde group, isocyanate group, or isothiocyanate group, which is an anionic functional group, is present in the polymer before binding, short-chain DNA, substrate When the functional group possessed by the bonding group is an amino group (—NH ₂ ), it can be bonded by a conventional amidation reaction.

  Specifically, for example, the polymer and the short-chain DNA can be reacted at room temperature in the presence of a salt in a solvent.

As the solvent, a known phosphate buffer,
3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO),
3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS),
2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES),
2-morpholinoethanesulfonic acid monohydrate (MES),
3-morpholinopropane sulfonic acid (MOPS),
2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO),
Piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES),
Piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) dehydrate (POPSO),
N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS),
2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO),
N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and the like can be used. Examples of the salt include NaCl and KCl.

  The amount of short-chain DNA used depends on the number of functional groups remaining, but is preferably 50: 1 to 500: 1 with respect to the number of functional groups in the polymer (number of functional groups: number of moles of short-chain DNA). More preferably, it is in the range of 100: 1 to 300: 1.

  In addition, for example, a compound that induces a substrate binding group and a polymer can be reacted in a solvent in the presence of a base at room temperature. As the solvent, the same buffer as described above can be used, and as the salt, the same salt as described above can be used.

  The amount of the compound that induces the substrate binding group is preferably based on the number of functional groups in the polymer (functional group: the number of moles of the compound that induces the substrate binding group), although it depends on the number of functional groups that remain. Is preferably in the range of 5: 1 to 100: 1, more preferably 5: 1 to 50: 1.

The method for binding the polymer to the substrate surface is not limited depending on the type of substrate and the type of functional group possessed by the substrate binding group. For example, a short-chain DNA and a substrate binding group are formed on a metal substrate surface. The polymer can be fixed to the metal substrate by dissolving the polymer in an acidic buffer solution, dropping the buffer solution at a desired position on the metal substrate at room temperature, and allowing it to stand for a certain period of time.

  The standing time (fixing time) of the polymer is preferably 0.5 to 48 hours, more preferably 1 to 48 hours. When the immobilization time is increased, the amount of the polymer immobilized on the substrate can be increased, and by increasing the amount of the polymer immobilized, the hybridization amount and the selectivity can be improved.

  More specifically, the compound having a thiol group can be bonded to the metal substrate according to the method described in, for example, J. Am. Chem. Soc. 1998, 120, 9787-9792. In addition, for example, a method for bonding a compound having a pyridyldithio group to a metal substrate can be performed in accordance with the method described in the document.

In the present invention, since a compound that induces a plurality of substrate binding groups is bonded to the polymer, bonding to the substrate surface can be performed in a self-organized manner. Therefore, compared to the binding of single-molecule DNA strands, the DNA strands can be bound to the substrate with a higher degree of accuracy in arrangement of the DNA strands and in a very simple manner.
“Self-organizing” means that a certain arrangement is automatically formed.

Hybridization of long-chain DNA By hybridizing a long-chain DNA having a sequence complementary to the short-chain DNA and having a sequence complementary to the desired target DNA to the short-chain DNA portion bound to the polymer The DNA chip of the present invention can be obtained. Hybridization can be performed by a known method. For example, the following method is mentioned as a hybridization method.

(I) Solid surface hybridization
(i) The DNA to be used is dissolved in the following hybridization buffer (1) or (2) and annealed at 90 to 100 ° C. for 10 to 15 minutes (to solve by annealing the DNA self-complementary strand). After annealing, it is immersed in ice water and rapidly cooled (inhibition of re-self-complementary strand formation).
(ii) The target DNA solution is dropped on the probe DNA-immobilized substrate and incubated at a temperature of Tm or higher (usually around Tm) for 15 minutes to 24 hours.
(Hybridization buffer)
(1) 10-50 mM Tris-HCl / NaCl 0.1-0.5 M / 1 mM MgCl ₂ , pH 8.0
(2) 10-50 mM Tris-HCl / NaCl 0.1-0.5 M / 1 mM EDTA, pH 8.0
(II) Substrate cleaning The substrate is cleaned for about 5 to 20 minutes with the following cleaning buffer (3) or (4).
(Washing buffer)
(3) 10~50 mM Tris-HCl / NaCl 0.1~0.5 M / 1 mM MgCl 2 / 0.1~0.4% SDS, pH 8.0
(4) 10-50 mM Tris-HCl / NaCl 0.1-0.5 M / 1 mM EDTA / 0.1-0.4% SDS, pH 8.0
Among the above, a combination of (1) a hybridization buffer and (3) a washing buffer is preferable.

In the present invention, a long DNA having a sequence complementary to the target DNA is bound to the desired short DNA by hybridization at a desired position, and the long DNA is used for hybridization of the target DNA. A DNA chip corresponding to various types of target DNA can be manufactured very simply.
In addition, since the DNA strand is fixed to the substrate surface via the polymer, that is, the DNA strand is bound to the polymer surface in the form of a polymer coating, the DNA strand and the substrate surface are directly connected. Raction does not occur. For this reason, since the fall of a DNA strand can be suppressed, the selectivity and efficiency of hybridization can be improved. In particular, since a metal substrate such as gold and a DNA strand have a large interaction and the DNA strand easily falls, it is effective when a metal substrate is used on the substrate surface.

  In the DNA chip according to the present invention, since the DNA strand is bound to the polymer and the polymer is bound to the substrate, the introduction density of the DNA strand into the polymer can be controlled. For this reason, it is possible to easily control the introduction density of DNA strands onto the substrate surface. It is possible to easily control the introduction density of the DNA strands into the polymer by adjusting the introduction amount of the functional groups in the polymer and the reaction amount between the functional groups and the DNA strands.

  In addition, the DNA chip according to the present invention is capable of detaching long-chain DNA from the substrate by maintaining the substrate at a high temperature or performing an alkali treatment after the completion of hybridization, and is a polymer-immobilized short-chain DNA. Can remain on the surface of the substrate without being detached while being bonded to the polymer. For this reason, it is possible to reuse the same substrate or to newly bind long-chain DNA and change the sequence to determine the sequence of another target DNA.

  On the other hand, in the DNA chip of the present invention, the DNA strand is bound to the polymer, and the polymer can be bound to the substrate by a plurality of substrate binding groups, so that the separation including the DNA strand from the substrate is effectively suppressed. You can also.

  In the present invention, when the polymer is hydrophobic, the polymer main chain is hydrophobic and the DNA is hydrophilic. Therefore, when performing hybridization in an aqueous solution, only the DNA is exposed to the aqueous solution side on the substrate surface. be able to. For this reason, hybridization can be performed with high efficiency.

  Furthermore, when the polymer portion has an anionic functional group, the repulsion between the linker DNA portion and the anionic functional group of the polymer increases synergistically, contributing to prevention of the DNA strand from falling. For this reason, in specific adsorption and non-specific adsorption, the ability to eliminate non-specific adsorption due to electrostatic repulsion is further enhanced. That is, when the polymer has an anionic property, the selectivity of hybridization can be remarkably improved.

Target DNA Detection Method The DNA chip according to the present invention can be used for detection of target DNA having complementarity with the probe portion of the solidified DNA strand. Examples of the target DNA include labeled DNA fragments and RNA fragments. By hybridization with the target DNA, gene expression monitoring, nucleotide sequence determination, mutation analysis, polymorphism analysis, and the like can be performed. In particular, since the DNA chip according to the present invention is excellent in hybridization selectivity and efficiency, it is extremely effective for polymorphism analysis such as SNPs.

The method for detecting a target DNA according to the present invention comprises the following steps:
(1) A step of bringing the target DNA into contact with the surface to which the polymer on the surface of the DNA chip is bound (step 1),
(2) A step of detecting the target DNA hybridized to the probe DNA portion (step 2).

Contact with the target DNA can be performed in the same manner as in the hybridization method and the like.
In the step 1, the polymer preferably has an anionic functional group, and in this case, it is preferably performed under basic conditions. When hybridization is performed under basic conditions, the negative charge of the anionic functional group can be improved when the polymer has an anionic functional group.

In Step 1, the salt strength is preferably 0.1 to 1.0 M when the linker DNA is in the range of 12 to 40 bases and the probe DNA is in the range of 6 to 100 bases, for example. More preferably, it is 0.2 to 0.4M, and particularly preferably 0.25 to 0.35M.
When the salt strength is within the above range, the negative charge of the anionic functional group can be improved particularly when the polymer has an anionic functional group. For this reason, it is possible to improve hybridization selectivity and efficiency.

  Further, the step 1 is preferably performed in a temperature range of 25 to 50 ° C, more preferably 35 to 45 ° C. When it is within the above temperature range, the selectivity of hybridization can be improved.

  The detection of the target DNA can be performed by a conventional method. For example, a method of measuring the fluorescence intensity attached to the target DNA, an electrochemical measurement (cyclic voltammetry, impedance measurement) using an intercalator, or the like can be employed. In addition, the DNA chip of the present invention can rapidly measure the amount of binding of a specific target DNA by interlocking with the crystal resonator method (QCM), the surface plasmon method (SPR), or the like. In the case of QCM, the binding amount of the target DNA can be determined from the change in frequency, and in the case of SPR, from the change in resonance angle.

FIG. 1 is an example schematically showing a DNA chip or the like of the present invention.
As shown in FIG. 1- (1), for example, a short chain DNA 2 and a polymer 4 having a substrate binding group 5 are bonded to a substrate 6. By hybridizing the long DNA 3 to such a short DNA binding substrate, the DNA chip 1 of the present invention is obtained as shown in FIG. That is, in the DNA chip 1, the DNA strand 10 having the probe DNA portion 7 and the linker DNA portion 8 is bonded to the polymer 4. The polymer 4 is bonded to the substrate 6 by the substrate bonding group 5. As shown in FIG. 1- (3), the target DNA 10 can be detected by hybridizing the target DNA 10 to the DNA chip 1.

  The present invention will be described below with reference to examples, but the present invention is not limited to these examples. In the specification, “room temperature” means a temperature in the range of 15 to 25 ° C. (preferably 20 ° C.).

[DNA and reagents used in Examples]
The following synthetic DNA was purchased from Greiner.
(1) Short DNA (hereinafter referred to as “P1-DNA”) : 3′-TA-ATT-CTT-TTT-TTT-TTT-TTG-5 ′ (SEQ ID NO: 1)
(3 'terminal amination, 20 mer),
(2) Long-chain DNA (hereinafter referred to as “P2-DNA”) :
5'-GAA-AAA-AAA-AAA-AAC-TT-GAC-GAG-GGG-CGC-ACC-GG-3 '(SEQ ID NO: 2)
(3 'terminal FITC (fluorescein isothiocyanate) or unmodified, 34 mer. The sequence complementary to p1-DNA was incorporated.)
(3) Target DNA:
Full complement (5 'end FITC, 17 mer):
(tG-DNA): 3'-CTG-CTC-CCC-GCG-TGG-CC-5 '(SEQ ID NO: 3)
Single nucleotide substitution (SNPs) sequence (5 'terminal FITC, 17 mer):
(tC-DNA): 3'-CTG-CTC-CCC-CCG-TGG-CC-5 '(SEQ ID NO: 4)
(tT-DNA): 3'-CTG-CTC-CCC-TCG-TGG-CC-5 '(SEQ ID NO: 5)
(tA-DNA): 3'-CTG-CTC-CCC-ACG-TGG-CC-5 '(SEQ ID NO: 6)
Non-complementary strand :
3′-GGC-ACG-GAG-CAC-ACT-GG-5 ′ (SEQ ID NO: 7)
(5'-end FITC conversion, GC content is the same as complete complementary DNA. 17 mer).
(4) Polyacrylic acid (PAA) (Mw: 1,080,000) (Product No. 18,129-3, manufactured by Aldrich)
(5) 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) (Product No. [16,146-2], manufactured by Aldrich)
(6) 3- (2-pyridyldithio) propionyl hydrazide (PDPH) (Product No. [22301], manufactured by Pierce)
(7) N- (ε-trifluoroacetylcaproyloxy) succinimide ester (TFCS) (Product No. [22299], manufactured by Pierce)

[Example 1]
Preparation of hydrazide short DNA
TFCS dissolved in dimethyl sulfoxide (DMSO) and P1-DNA dissolved in 0.1 M phosphate buffer / 0.15 M NaCl (pH 7.0) so that the final concentration in DMSO is 10% by weight Over 5 minutes. The total volume was 1 mL. (Molar ratio; TFCS: P1-DNA = 10: 1)
This was reacted in an eggplant flask for 2 hours at 4 ° C. After the reaction was completed, unreacted TFCS was removed by ultrafiltration.

  Next, in order to deprotect the trifluoroacetyl group of TFCS, the reaction solution was gradually replaced with 0.1 M phosphate buffer / 0.15 M NaCl (pH 8.0), and the reaction solution was stirred at room temperature for 1 hour. did. Thereafter, ultrafiltration and lyophilization were performed to obtain a product (hydrazide short chain DNA). The reaction scheme is shown in FIG.

Preparation and characterization of DNA (P-1) conjugated polyacrylic acid (PAA) 0.1 M-phosphate buffer containing hydrazide short DNA (0.21 μmol), PAA (42 mol), EDC (420 mol) / 1 It was dripped at what was melt | dissolved in M-NaCl (pH 8.0). The total volume was 10 ml. (Molar ratio: PAA carboxyl group: ssDNA = 200: 1)
This was allowed to react at room temperature for 3 days in an eggplant flask. After the reaction, unreacted hydrazide short-chain DNA and EDC were removed by ultrafiltration, and lyophilized to obtain P1-DNA modified polyacrylic acid (hereinafter referred to as “P1-DNA-PAA”).

  P1-DNA-PAA, EDC, and PDPH were dissolved in 0.1 M phosphate buffer / 1 M NaCl (pH 8.0) (molar ratio; PAA carboxyl group: PDPH = 10: 1), and this was added to the eggplant flask. For 1 day at room temperature. After the reaction, unreacted PDPH and EDC were removed by ultrafiltration and lyophilized to obtain polyacrylic acid (P1-DNA-PDPH-PAA) modified with short-chain DNA and PDPH. The reaction scheme is shown in FIG.

Determination of introduction amount of short-chain DNA and substrate binding group
The determination of the number of moles per unit mass of short-chain DNA and PDPH in P1-DNA-PDPH-PAA was calculated from the results of ultraviolet light absorption spectrum measurement as described below. The spectrophotometer used was DU 7400 (Beckman).
When the molar absorption count is ε [L / mol · cm], the molar concentration is C _M [mol / L], and the length of the spectroscopic measurement cell is l [cm], The relationship is established.
A = ε × C _M × l (1)
Since the molar extinction coefficient has the following relationship between the sample and the concentration, ε = (1 / C _M l) log ₁₀ (I ₀ / I) (2)
Here, l represents the thickness of the absorption layer, I ₀ represents the intensity of incident light, and I represents the light intensity after passing through the absorption layer.
When the pyridyl disulfide group of PDPH is reduced, pyridine-2-thione is eliminated. The amount of PDPH introduced was determined by measuring the absorbance at 343 nm, which is the absorption wavelength of this substance (FIG. 4). The PDPH molar extinction coefficient is ε = 0.08 × 10 ³ [M ⁻¹ cm ⁻¹ ].
If the number of moles per unit mass is a [mol / mg-solid] and the weight concentration is Cw [mg-solid / mL], the length of the spectroscopic measurement cell used this time is l = 1cm. Can be simplified as: A = 8.08 × 10 ⁶ a × C _W (3)
A was determined from the relationship between the short-chain DNA of polyacrylic acid side chain and the absorption spectrum of PDPH and the formula (3). For quantification of short-chain DNA, a was determined in the same manner as PDPH from the absorption spectrum at 260 nm, which is the absorption wavelength of short-chain DNA. * A was determined for short DNA as 1 OD = 33 μg.

As a result, PDPH was 2.56 [AU / mg-solid] from the equation (3) and the result of the absorption spectrum measurement at 343 nm (0.31). Therefore, a = 307 [nmol / mg-solid] was determined from the relationship of 2.56 = 8.08 × 10 ⁶ × a.
Since the short-chain DNA is 1 OD = 33 μg, it is 2.32 [OD / mg-solid] from the result of the absorption spectrum measurement at 260 nm (0.29). Since the molecular weight of the short-chain DNA was 6130, it was determined that a = 20 [nmol / mg-solid].
From these results, it was confirmed that the PDPH and short-chain DNA contents of P1-DNA-PDPH-PAA were 307 [nmol / mg-solid] and 20 [nmol / mg-solid], respectively.

Production of Gold Substrate A gold substrate was produced using a sputtering apparatus. The slide glass was immersed in methanol and ultrasonically cleaned for 15 minutes, and then sufficiently washed with ultrapure water. The slide glass was introduced into the sputtering apparatus, and the glass was masked and sputtered onto the slide glass substrate in a sequence of 2.5 minutes of chromium and 10 minutes of gold in order of 2 mm in diameter.

Immobilization of P1-DNA-PDPH-PAA on a gold substrate (Preparation of P1-DNA immobilization substrate)
Immediately before the experiment, the gold substrate was immersed in 5N nitric acid heated for 1 minute and washed with ultrapure water three times before use. P1-DNA-PDPH-PAA (0.4 mg / ml) dissolved in 50 mM MES (2-morpholinoethanesulfonic acid monohydrate) buffer / 1 M NaCl (pH 5.5) was added to the gold part of the washed substrate. 2 μL was added dropwise.
The substrate was left at room temperature for 2 hours. After immobilization, the substrate was washed with 10 mM Tris-HCl / 0.3 M NaCl / 1 mM MgCl ₂ /0.1% SDS (pH 8.0) (hereinafter referred to as “cleaning buffer”) and ultrapure water. After cleaning, the substrate was subjected to removal of moisture using an aeroduster (Futaba) and dried to obtain a P1-DNA-immobilized substrate (P1-DNA-PDPH-PAA substrate).

Hybridization between P1-DNA and P2-DNA (Preparation of P2-DNA immobilized substrate)
Hybridization of P1-DNA and P2-DNA on the substrate surface was performed.
P2-DNA (100 nM) having a 5'-terminal FITC was dissolved in 10 mM Tris-HCl / 0.3 M NaCl / 1 mM MgCl ₂ (pH 8.0), and 2 μL was dropped onto the P1-DNA-immobilized substrate. . In order to prevent evaporation of the solution from the substrate, the substrate was allowed to stand at 35 ° C. for 30 minutes in a container with sufficient moisture for hybridization.

After hybridization, the substrate was washed 3 times with washing buffer for 5 minutes to remove nonspecific adsorbate, and P2-DNA hybridized to P1-DNA, P2-DNA immobilized substrate (P1P2 -DNA-PDPH-PAA substrate).
As a control, the target DNA (3′-CTG-CTC-CCC-GCG-TGG-CCC-CTG-CAC-CAG-CAG-CTC-C-5 ′ (SEQ ID NO: 8) (the 5′-end was converted to FITC) ( 34mer): no complementary portion to P1-DNA) (100 nM) was dropped onto a gold substrate. These hybridizations were confirmed using a ScanArray (Perkin Elmer) at a scanning speed of 50 μm / min.
As shown in FIG. 5, the fluorescence intensities of P2-DNA and control sequence DNA were 224 AU and 1.64 AU at 35 ° C., respectively.
When these values were normalized with the value of P2-DNA as 100%, the control sequence was 0.73%.

[Example 2]
Hybridization with target DNA (temperature 30 ° C)
To the P2-DNA immobilization substrate (P1P2-DNA-PDPH-PAA substrate) produced in Example 1, 2 μL each of completely complementary strand DNA, SNPs sequence (tC-DNA) and non-complementary strand DNA was dropped. All solutions were 10 mM Tris-HCl / NaCl 0.3 M / 1 mM MgCl ₂ , pH 8.0 to a concentration of 100 nM. In order to prevent evaporation of the solution from the substrate, the substrate was allowed to stand at 30 ° C. for 30 minutes in a container with sufficient moisture for hybridization.
After hybridization, the substrate was washed 3 times with a washing buffer for 5 minutes.
The fluorescence intensity of each target DNA was measured in the same manner as in Example 1 using ScanArray (Perkin Elmer). The result is shown in FIG.

Example 3
Hybridization with target DNA (temperature 35 ° C)
Hybridization was performed in the same manner as in Example 2 except that the hybridization temperature was set to 35 ° C. in Example 2.
The fluorescence intensity of each target DNA was measured in the same manner as in Example 1 using ScanArray (Perkin Elmer). The result is shown in FIG.
As shown in FIG. 6 and FIG. 7, the fluorescence intensity is 8.3 AU, 4.7 when the hybridization temperature is 30 ° C. with respect to the completely complementary strand DNA, single nucleotide substitution (SNPs) sequence (tC-DNA), and non-complementary strand DNA. AU, 2.3 AU, and at 35 ° C., they were 145 AU, 24.5 AU, 20 A.U.

As a control experiment, the complementary strand DNA was dropped, incubated and washed on the P-1 DNA-immobilized substrate prepared in Example 1 under the same conditions. From this substrate, 8.1 AU of fluorescence was observed.
When these fluorescence intensities (F.I.) are normalized using signals of completely complementary strand DNA, the SNPs sequence is 56%, 16.8%, and the non-complementary strand is 27.5% at 30 ° C. and 35 ° C., respectively. The value of 13.7% and the control experiment was 5.5%.

  From the above results, it was confirmed that hybridization occurred selectively at both the hybridization temperature of 30 ° C. and 35 ° C. Further, when these comparisons were made, when the hybridization temperature was 35 ° C., It was confirmed that the selectivity was further improved. Moreover, from these results, it was confirmed that complete complementary strand DNA can be detected in a sequence-specific manner even with a single base difference.

Example 4
Salt strength 0.4 M
TG-DNA (3′-CTG-CTC-CCC-GCG-TGG-CC-5 ′) (SEQ ID NO: 3) (“A” in FIG. 8) of the completely complementary strand,
TC-DNA (3′-CTG-CTC-CCC-CCG-TGG-CC-5 ′) (SEQ ID NO: 4) (“B” in FIG. 8) of the single nucleotide substitution (SNPs) sequence, tT-DNA ( 3′-CTG-CTC-CCC-TCG-TGG-CC-5 ′) (SEQ ID NO: 5) (“C” in FIG. 8), tA-DNA (3′-CTG-CTC-CCC-ACG-TGG- CC-5 ′) (SEQ ID NO: 6) (“D” in FIG. 8), and the non-complementary strand DNA (3′-GGC-ACG-GAG-CAC-ACT-GG-5 ′) (SEQ ID NO: 7 ) (“E” in FIG. 8) (all 5 ′ terminal FITC) were dissolved in 10 mM Tris-HCl / NaCl 0.3 M / 1 mM MgCl ₂ , pH 8.0, respectively (concentration 100 nM), and contained NaCl. Solutions with a salt strength of 0.4M were prepared.

These were dropped on the P2-DNA-immobilized substrate (P1P2-DNA-PDPH-PAA substrate) prepared in Example 1 and incubated at 35 ° C. for 30 minutes for hybridization. After washing, FITC fluorescence was observed with the same scan array as in Example 1.
FIG. 8 shows a graph of fluorescence intensity.

As a result, the fluorescence intensities of tG-DNA (fully complementary strand DNA), tC-DNA, tT-DNA, tA-DNA, and non-complementary strand DNA were 65.6 AU, 36 AU, 28.1 AU, 29.9 AU, 6 It was AU. When these were normalized using the fluorescence intensity of tG-DNA, they were 100, 54.8, 42.8, 45.5, and 9.1%, respectively.
The fluorescence intensity of the fully complementary strand DNA sequence was the highest, and the difference in fluorescence intensity sufficient for qualitative diagnosis of other target DNAs was at most 54%, confirming SNPs sequence recognition ability.

Example 5
Salt strength 0.3 M
In Example 4, hybridization and washing were performed in the same manner as in Example 4 except that the salt strength was 0.3M. Further, the fluorescence intensity was measured in the same manner as in Example 4.
FIG. 9 shows a graph of fluorescence intensity.

As a result, tG-DNA (completely complementary strand (“A” in FIG. 9)), tC-DNA (“B” in FIG. 9), tT-DNA (“C” in FIG. 9), tA-DNA (FIG. 9). The fluorescence intensity of the non-complementary strand DNA (“E” in FIG. 9) was 61.3 AU, 9.4 AU, 5.8 AU, 3.6 AU, and 7.1 AU, respectively. When these were normalized using the fluorescence intensity of tG-DNA, they were 100, 15.3, 9.4, 5.9, and 11.5%, respectively.
There is a difference in fluorescence intensity that is sufficient for qualitative diagnosis, with the fluorescence intensity of the fully complementary DNA sequence being the highest and the other target DNA being at most 9.4%.
By reducing the salt strength, the negative charge of the anionic functional group on the polymer increases, and it is speculated that the difference in the SNPs sequence was recognized and eliminated more strictly.

FIG. 1 is a schematic view showing an example of the DNA chip of the present invention. FIG. 1- (1) shows an example of a short-chain DNA-immobilized substrate in which a polymer on which short-chain DNA is immobilized is immobilized on a substrate. 1- (2) shows the DNA chip of the present invention, and FIG. 1- (3) shows an example in which the target DNA is hybridized to the DNA chip of the present invention. FIG. 2 is a diagram showing an example of a reaction scheme for hydrazide converting DNA ends. FIG. 3 is a diagram showing an example of a reaction scheme for introducing a short-chain DNA and a compound that induces a substrate binding group into a polymer. FIG. 4 is a diagram showing the relationship between the absorbance and the absorption wavelength in a polymer having a short-chain DNA and a substrate binding group. MES / NaCl (0.5 M) / dithiosereitol (DTT) (0.7 mg / ml) was used as a solvent, and the concentration of the short DNA and the polymer to which the substrate binding group was bound was 0.125 mg / ml. . FIG. 5 shows the fluorescence intensity (FI (AU)) when long-chain DNA is hybridized to a polymer having short-chain DNA and a substrate binding group, and the fluorescence intensity (FI) of the control (the polymer not hybridized). (AU)). Normalized F.I. (%) is the ratio of the fluorescence intensity when normalized using the fluorescence intensity when long-chain DNA is hybridized. FIG. 6 shows fluorescence intensity when hybridization is performed by bringing a complete complementary strand DNA, a single nucleotide substitution (SNP) sequence DNA, and a non-complementary strand DNA into contact with a long DNA-immobilized substrate at a temperature of 30 ° C. as target DNA. It is a figure which shows (FI (AU)). Normalized F.I. (%) is the ratio of the fluorescence intensity when normalized using the fluorescence intensity when hybridizing completely complementary strand DNA. FIG. 7 shows the fluorescence intensity when fully complementary strand DNA, single nucleotide substitution (SNP) sequence DNA and non-complementary strand DNA are brought into contact with a long DNA-immobilized substrate at 35 ° C. as the target DNA and hybridized. It is a figure which shows (FI (AU)). Normalized F.I. (%) is the ratio of the fluorescence intensity when normalized using the fluorescence intensity when hybridizing completely complementary strand DNA. FIG. 8 shows examples of target DNAs including completely complementary strand DNA (A), single nucleotide substitution (SNP) sequence DNA (tC-DNA, tT-DNA, tA-DNA) (B, C, D) and non-complementary strand DNA ( It is a figure which shows the fluorescence intensity (FI (AU)) when contacting E) with a long-chain DNA fixed board | substrate at the density | concentration of salt intensity | strength 0.4M, and hybridizing. Normalized F.I. (%) is the ratio of the fluorescence intensity when normalized using the fluorescence intensity when hybridizing completely complementary strand DNA. FIG. 9 shows as target DNAs, complete complementary strand DNA (A), single nucleotide substitution (SNP) sequence DNA (tC-DNA, tT-DNA, tA-DNA) (B, C, D) and non-complementary strand DNA ( It is a figure which shows the fluorescence intensity (FI (AU)) when contacting E) and making it contact with a long-chain DNA fixed board | substrate with the density | concentration of 0.3 M of salt intensity | strength. Normalized F.I. (%) is the ratio of the fluorescence intensity when normalized using the fluorescence intensity when hybridizing completely complementary strand DNA.

Explanation of symbols

1 DNA chip 2 Short DNA
3 Long DNA
4 Polymer 5 Substrate binding group 6 Substrate 7 Probe DNA
8 Linker DNA
9 DNA strand 10 Target DNA

Claims (12)

A DNA chip having a DNA strand immobilized on a substrate surface via a polymer, the DNA strand comprising a single-stranded probe DNA portion and a double-stranded linker DNA portion;
The DNA strand binds to the polymer on the linker DNA portion side, and the polymer is coated on the substrate surface by being fixed to at least one surface of the substrate through a plurality of substrate binding groups,
The number of base pairs of the linker DNA portion is in the range of 10 to 150 bases,
The polymer includes a polymer main chain composed of hydrophobic structural units,
The DNA chip, wherein the polymer has a functional group, and the ratio of the number of anionic functional groups in the functional group is 50% or more.   The DNA chip according to claim 1, wherein the linker DNA portion is bonded to the polymer by a covalent bond.   The DNA chip according to claim 1 or 2, wherein the linker DNA portion has 10 to 150 base pairs. In the polymer, the hydrophobic structural unit is represented by the following formula (I):

Wherein R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, a functional group, a DNA chain or a substrate binding group. A DNA chip according to any one of the above.   The weight average molecular weight of the polymer measured by gel permeation chromatography is in the range of 50,000 to 1,500,000, and the constituent unit of the formula (I) in the polymer main chain is 50 to 100% (mol / g-polymer). 5) The DNA chip according to any one of claims 1 to 4, which is contained.   The anionic functional group is selected from the group consisting of a carboxyl group, a sulfonic acid group, a sulfinic acid group, a phosphoric acid group, a sulfuric acid group, a phenolic hydroxyl group, an alkali metal salt thereof, and an ammonium salt thereof. The DNA chip according to any one of 1 to 5. Said linker DNA segment is (polymer backbone) -CONH -, (polymer backbone) -CONH- alkylene group or a (polymer backbone) carbon atoms _{1~20 -CONH- (CH 2) 5 -CO} -NH - _{(CH 2) 7} - a, covalently attached to the polymer, DNA chip according to any one of claims 1 to 6.   The DNA chip according to any one of claims 1 to 7, wherein the polymer is coated on a substrate through a sulfur atom in the substrate binding group. A target DNA detection method comprising the following steps:
(1) A step of bringing the target DNA into contact with the surface to which the polymer on the surface of the DNA chip according to any one of claims 1 to 8 is bound (step 1),
(2) A step of detecting the target DNA hybridized to the probe DNA portion (step 2).   The method according to claim 9, wherein, in the step 1, the polymer in the DNA chip has an anionic functional group, and the target DNA is contacted in a basic aqueous solution.   The method according to claim 9 or 10, wherein the step 1 is performed at a salt strength of 1M or less.   The method according to any one of claims 9 to 11, wherein the step 1 is performed in a temperature range of 25 to 50 ° C.

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