JP4916689B2 - DNA strand amplification method - Google Patents

Description

  The present invention relates to a DNA strand amplification method for extending a DNA strand by immobilizing a primer DNA strand on a predetermined plastic substrate surface.

  Non-Patent Document 1 discloses DNA amplification by solid-phase PCR (Polymerase Chain Reaction) using a DNA microarray in which a DNA strand as a primer is covalently bonded to the surface of a glass substrate modified with a predetermined amino-silane reagent. Techniques for performing are disclosed.

Adessi, Celine et al, "Solid phase DNA amplification: Characterisation of primer attachment and amplification mechanisms", Nucleic Acids Research, 2000, Vol. 20, No. 20, e87

  In the process of examining the DNA chain extension reaction by the MPEC method (Multiple Primer Extension on a Chip) using various DNA microarrays represented by Non-Patent Document 1, the present inventors put a predetermined polymer substance on the surface. By using the arranged substrate, the primer extension reaction proceeds and amplifies without hybridization, by hybridizing the template DNA strand to the primer, that is, by annealing and maintaining a constant temperature. As a result, the present invention has been completed.

That is, the present invention
(1) a first unit having a group derived from a phosphate ester constituting the hydrophilic part of the phospholipid and a second unit having a carboxylic acid derivative group in which an electron-withdrawing substituent is bonded to a carbonyl group; A DNA strand that serves as a primer for DNA extension is immobilized on a substrate having a polymer substance containing a template, a template DNA fragment or template RNA fragment having a desired sequence, an enzyme system for DNA strand extension, and a nucleotide monomer The liquid phase system into which the sample is introduced is raised to a temperature at which the DNA strand is thermally denatured (hereinafter referred to as “thermal denaturation treatment temperature”),
The temperature of the liquid phase system is lowered to a temperature for annealing treatment (hereinafter referred to as “annealing temperature”), and the primer DNA strand immobilized on the substrate is extended and amplified while maintaining a constant treatment temperature. A DNA strand amplification method,
(2) In the DNA strand amplification method according to (1), a DNA strand amplification method characterized in that no washing treatment is performed before the DNA strand elongation reaction is performed after annealing in the liquid phase system,
(3) In the DNA strand amplification method according to (1), when the template is a DNA fragment, the DNA chain elongation enzyme system is either DNA polymerase or DNA ligase Method,
(4) In the DNA strand amplification method of (1), when the template is an RNA fragment, the enzyme system for DNA strand extension is either reverse transcriptase or a combination of DNA ligase and reverse transcriptase A DNA strand amplification method characterized by
(5) In the DNA strand amplification method of (1), the groups derived from the phosphate ester contained in the first unit of the substrate are phosphorylcholine group, phosphorylethanolamine group, phosphorylserine group, phosphorylinositol group, phosphorylglycerol. A DNA strand amplification method, wherein the group is any one of a group and a phosphatidyl phosphorylglycerol group,
(6) In the DNA strand amplification method according to (1),
A DNA strand amplification method, wherein any of the nucleotide monomers is labeled;
It is.

  In this way, by immobilizing a primer on a predetermined substrate and annealing the template DNA fragment to this primer, or by hybridizing the template RNA fragment, the DNA strand extension reaction can be carried out by maintaining a constant processing temperature. When the extension occurs to a certain length, the extension stops, the template DNA strand moves at any time with another primer, and then the extension reaction of the next primer occurs, and only the single-stranded DNA amplified and amplified by the primer is the substrate. Will remain on top.

  Conventionally, in order to amplify a DNA strand using an extension reaction using a primer, after the extension reaction, the temperature is increased to a heat denaturation temperature of about 95 ° C. to form a single-stranded DNA, and then the annealing temperature again. It was necessary to form another primer double strand and perform an extension reaction, the amplification efficiency of the gene was additive, and the amplification efficiency was very low.

  Further, conventionally, after the annealing treatment and before the extension reaction, a washing treatment for removing DNA fragments or RNA fragments that did not form a double strand was required, but DNA adsorbed nonspecifically on the substrate. It is considered that there is no fragment or RNA fragment, and the enzymatic reaction related to DNA chain elongation functions effectively, and the substrate is not required to be washed before the elongation reaction.

  In this DNA chain extension method, the group derived from the phosphate ester contained in the first unit of the substrate is phosphorylcholine group, phosphorylethanolamine group, phosphorylserine group, phosphorylinositol group, phosphorylglycerol group, phosphatidylphosphorylglycerol group. Can be either.

  Thus, by providing an environment similar to phospholipid on the surface of the substrate, the DNA chain elongation reaction occurring on the surface of the substrate is an environment close to the inside of the cell, and therefore the DNA chain elongation reaction proceeds efficiently. It is considered that gene amplification is possible only by the action of enzymes without thermal cycling.

In the above DNA strand amplification method,
The primer may be a DNA fragment in which one base of a base sequence including a characteristic sequence of a predetermined gene of interest is replaced with another base.

  As a result, the DNA strand amplification method of the present invention can be applied to single nucleotide polymorphism (SNP) analysis.

In the above DNA strand amplification method,
The primer comprises a predetermined number of base sequences, and each primer may be a DNA fragment having the respective sequences in all combinations.

  As a result, the DNA chain extension amplification reaction of the present invention can be applied to base sequence determination (SBH: sequencing by hybridization) analysis by hybridization.

In the DNA chain extension method,
The template DNA fragment may be a cDNA fragment obtained by treating a predetermined RNA with reverse transcriptase.

  As a result, the DNA chain elongation reaction of the present invention is applied to gene expression profile analysis, so that almost no laborious and time-consuming sample preparation is required in normal analysis, and from genomic DNA or total RNA. The reverse transcript cDNA can be used as a template DNA fragment and can be carried out by a simple operation.

  According to the present invention, a DNA strand extension reaction can be carried out only by maintaining a constant temperature below the heat denaturation temperature on the DNA microarray, and the DNA strand can be amplified without heat denaturation treatment. The elongated and amplified single-stranded DNA can be left.

The DNA chain elongation method according to the present invention is a carboxylic acid derivative comprising a first unit having a group derived from a phosphate ester constituting a hydrophilic part of a phospholipid and an electron-withdrawing substituent bonded to a carbonyl group. A primer DNA strand for DNA extension is immobilized on a substrate having a polymer substance containing a second unit having a group on the surface, and a template DNA fragment or template RNA fragment having a desired sequence, an enzyme system for DNA chain extension , And the liquid phase system in which the sample containing the nucleotide monomer is introduced, the DNA strand is raised to the heat denaturation temperature,
By reducing the temperature of the reaction system to the annealing temperature and maintaining a constant processing temperature, the elongation reaction and amplification of the DNA strand immobilized on the substrate are performed in the same liquid phase system.

  Hereinafter, embodiments of the present invention will be described in detail.

(First embodiment)
This DNA strand amplification method has a first unit having a group derived from a phosphate ester constituting the hydrophilic part of a phospholipid and a carboxylic acid derivative group in which an electron-withdrawing substituent is bonded to a carbonyl group. A DNA amplification primer (hereinafter referred to as “primer”) is immobilized on a substrate having a polymer substance containing the second unit on its surface, and a template DNA fragment or template RNA fragment having a desired sequence, DNA chain extension The temperature of the reaction system is increased to a temperature at which the DNA strand is thermally denatured (hereinafter referred to as “annealing temperature”). While maintaining the temperature, the DNA chain is elongated and amplified in the reaction system. Further, each of these treatments is performed in the same liquid phase system.

  On the surface of the substrate used here, there is a polymer substance containing a first unit having a group derived from a phosphate ester constituting the hydrophilic part of the phospholipid and a second unit having a carboxylic acid derivative group. It is supposed to be.

  A polymer substance having a first unit containing a group derived from a phosphate ester constituting the hydrophilic part of the phospholipid and a second unit containing a carboxylic acid-derived group suppresses nonspecific adsorption of DNA strands. It is a polymer having both properties and the property of immobilizing DNA strands. In particular, a group derived from a phosphate ester constituting the hydrophilic part of the phospholipid contained in the first unit plays a role in suppressing nonspecific adsorption of the template DNA fragment, and the carboxylic acid-derived group contained in the second unit. Serves to chemically immobilize the primer. That is, the primer is immobilized on the surface of the substrate by covalent bonding at the site of the carboxylic acid derivative group of the coating layer made of the polymer material.

The first unit is, for example, a (meth) acryloyloxyalkyl phosphorylcholine group such as a 2-methacryloyloxyethyl phosphorylcholine group, a 6-methacryloyloxyhexyl phosphorylcholine group;
(Meth) acryloyloxyalkoxyalkylphosphorylcholine groups such as 2-methacryloyloxyethoxyethyl phosphorylcholine group and 10-methacryloyloxyethoxynonylphosphorylcholine group;
Alkenylphosphorylcholine groups such as allylphosphorylcholine group, butenylphosphorylcholine group, hexenylphosphorylcholine group, octenylphosphorylcholine group, and decenylphosphorylcholine group;
And a phosphorylcholine group is included in these groups.

  Of these groups, 2-methacryloyloxyethyl phosphorylcholine is preferable. By adopting a structure in which the first unit has 2-methacryloyloxyethyl phosphorylcholine, nonspecific adsorption of the template DNA fragment on the substrate surface can be more reliably suppressed.

  In addition, although the example which is the phosphorylcholine group shown to following formula (a) as a basic skeleton was given here, this phosphorylcholine is phosphorylethanolamine group of following formula (b), phosphorylinositol group of following formula (c), following formula The phosphorylserine group in (d), the phosphorylglycerol group in the following formula (e), and the phosphate group such as the phosphatidylphosphorylglycerol group in the following formula (f) may be substituted (the same applies to the following).

  The carboxylic acid derivative is a carboxylic acid in which the carboxyl group of the carboxylic acid is activated and has a leaving group via C═O. Specifically, the carboxylic acid derivative is a compound in which a nucleophilic reaction is activated by bonding a group having higher electron withdrawing property than an alkoxyl group to a carbonyl group. The carboxylic acid derivative group is a compound having reactivity with an amino group, a thiol group, a hydroxyl group and the like.

More specifically, carboxylic acid derivatives include carboxyl groups such as acrylic acid, methacrylic acid, crotonic acid, maleic acid, and fumaric acid, which are carboxylic acids, in acid anhydrides, acid halides, active esters, and activated amides. Examples include converted compounds. Carboxylic acid-derived groups are activated groups derived from such compounds, for example, active ester groups such as p-nitrophenyl groups and N-hydroxysuccinimide groups;
-Halogen such as Cl, -F;
And the like.

  The carboxylic acid derivative group can be a group represented by the following formula (1).

（ただし、上記式（１）において、Ａは水酸基を除く脱離基である。）

(In the above formula (1), A is a leaving group excluding a hydroxyl group.)

  The monovalent group represented by the above formula (1) can be, for example, any group selected from the following formula (p) or formula (q).

(However, in the above formulas (p) and (q), R ¹ and R ² are each independently a monovalent organic group, which may be linear, branched, or cyclic. In Formula (p), R ¹ may be a divalent group that forms a ring with C. In Formula (q), R ² is a divalent group that forms a ring with N. It may be a group of

  Examples of the group represented by the formula (p) include groups represented by the following formulas (r), (s), and (w). Moreover, as group shown by the said formula (q), group shown by following formula (u) is mentioned, for example.

The group represented by the above formula (1) is, for example, a group derived from an acid anhydride represented by the following formula (r), formula (s) or the like;
A group derived from an acid halide represented by the following formula (t);
A group derived from an active ester represented by the following formula (u) or formula (w); or a group derived from an activated amide represented by the following formula (v).

  Of the carboxylic acid-derived groups, an active ester group is preferably used because of its excellent reactivity under mild conditions. The mild conditions include, for example, neutral or alkaline conditions, specifically pH 7.0 or more and 10.0 or less, more specifically pH 7.6 or more and 9.0 or less, and more specifically pH 8.0. It can be.

  In addition, the definition of “active ester group” as defined in this specification is not strictly defined. However, as a conventional technical expression, an electron withdrawing property having high acidity on the alcohol side of the ester group. An ester group having a group and activating a nucleophilic reaction, that is, an ester group having a high reaction activity, is commonly used in various chemical synthesis fields such as polymer chemistry and peptide synthesis. is there. In the field of peptide synthesis, as described in Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi, Noriaki Waki, “Basics and Experiments of Peptide Synthesis”, published in 1985, Maruzen, It is used as one of the methods for activating the C-terminus of amino acids or peptides.

  Actually, the ester group has an electron-attracting group on the alcohol side of the ester group and is activated more than the alkyl ester. The active ester group has reactivity with groups such as amino group, thiol group, and hydroxyl group. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc. Known as.

  Here, the case where the activated carboxylic acid derivative group in the polymer substance is an active ester group will be described as an example. Examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, 5-norbornene-2,3-dicarboximide group, and the like. A nitrophenyl group is preferably used.

  In the case of a substrate on which a primer is immobilized on the surface, a more specific combination of the first unit and the second unit includes, for example, a group containing a group derived from a phosphate ester that forms the hydrophilic part of the phospholipid. One unit may have a 2-methacryloyloxyethyl phosphorylcholine group and the active ester group may be a p-nitrophenyl group.

  In addition, the polymer substance used for the coating layer of the substrate of the present embodiment may contain other groups in addition to the group derived from the phosphate ester and the carboxylic acid derived group constituting the hydrophilic part of the phospholipid. The polymer substance can be a copolymer. Specifically, the polymer substance is preferably a copolymer containing a butyl methacrylate group. By so doing, the polymer substance can be appropriately hydrophobized, and the adsorptivity of the polymer substance to the substrate surface can be more suitably ensured.

  Specifically, the polymer substance is composed of a first monomer having a 2-methacryloyloxyethyl phosphorylcholine (MPC) group and a second monomer having a p-nitrophenyloxycarbonyl polyethylene glycol methacrylate (NPMA) group. , And a copolymer with a third monomer having a butyl metallate (BMA) group. These copolymers, poly (MPC-co-BMA-co-NPMA) (PMBN), are schematically represented by the following general formula (2).

  However, in the said General formula (2), a, b, and c are respectively independently a positive integer. In the general formula (2), the first to third monomers may be block copolymerized, or these monomers may be copolymerized randomly.

  The copolymer represented by the general formula (2) is more suitable for the balance between moderate hydrophobicity of the polymer substance, the property of suppressing nonspecific adsorption of the template DNA fragment, and the property of immobilizing the primer. It is an even better configuration. For this reason, by using such a copolymer, the surface of the substrate is more reliably coated with the polymer material, and nonspecific adsorption of the template DNA fragment onto the substrate coated with the polymer material is suppressed. However, the primer can be more reliably immobilized by covalent bonding and introduced onto the substrate.

  The copolymer represented by the general formula (2) can be obtained by mixing MPC, BMA, and NPMA monomers and using a known polymerization method such as radical polymerization. When the copolymer represented by the general formula (2) is prepared by radical polymerization, solution polymerization can be performed, for example, in an inert gas atmosphere such as Ar under a temperature condition of 30 ° C. to 90 ° C.

  The solvent used for the solution polymerization is appropriately selected. For example, alcohols such as methanol, ethanol and isopropanol, ethers such as diethyl ether, and organic solvents such as chloroform can be used alone or in combination. Specifically, a mixed solvent in which diethyl ether and chloroform are in a volume ratio of 8 to 2 can be obtained.

Moreover, what is used normally can be used as a radical polymerization initiator used for radical polymerization reaction. For example, azo initiators such as azobisisobutyronitrile (AIBN) and azobisvaleronitrile;
Oil-soluble organic peroxides such as lauroyl peroxide, benzoyl peroxide, t-butylperoxyneodecanoate, t-butylperoxypivalate;
Etc. are used.

  More specifically, polymerization can be carried out in Ar at 60 ° C. for about 2 to 6 hours using a mixed solvent of diethyl ether and chloroform in a volume ratio of 8 to 2 and AIBN.

  In this embodiment, an example in which the high molecular substance has a third unit containing a butyl methacrylate group has been described. However, the first unit containing a group derived from a phosphate ester constituting the hydrophilic part of the phospholipid and a carboxylic acid A polymer substance having a second unit containing an acid-derived group as a first polymer substance, and in addition to this, a first unit containing a group derived from a phosphate ester constituting the hydrophilic part of the phospholipid; A second polymer substance having a third unit containing a butyl methacrylate group may be included.

  The first unit of the first polymer substance and the first unit of the second polymer substance may have the same structure or different structures. In addition, when the first polymer substance includes a third unit containing a butyl methacrylate group, the third unit of the first polymer substance and the third unit of the second polymer substance have the same structure. There may be a different structure.

  Such a second polymer substance is used as a polymer that suppresses nonspecific adsorption of the template DNA fragment. As such a polymer, for example, an MPC polymer (manufactured by NOF Corporation) containing 30 mol% phosphorylcholine groups and 70 mol% butyl methacrylate groups can be used.

  In addition, when a high molecular substance consists of said 1st high molecular substance and 2nd high molecular substance, it can be set as the structure by which these high molecular substances are mixed. Since the polymer of each polymer substance can be dissolved in, for example, an ethanol solution, a mixed polymer can be easily obtained by mixing the respective polymer solutions.

  The substrate including the coating layer made of the polymer material as described above can be obtained by applying a liquid containing the polymer material to the surface of the substrate processed into a predetermined shape and drying it. Alternatively, the substrate may be immersed in a liquid containing a polymer substance and dried.

  In addition, when a plastic material is used as the substrate, flexibility in changing the shape and size is secured, and it is preferable from the viewpoint that it can be provided at a lower cost than that of a glass substrate. As such a plastic material, a thermoplastic resin can be used from the viewpoint of easy surface treatment and mass productivity.

As a thermoplastic resin, a thing with little fluorescence generation amount can be used. By using a resin with a small amount of fluorescence generation, the background in the detection reaction of the DNA strand can be lowered, and therefore the detection sensitivity can be further improved. Examples of the thermoplastic resin with a small amount of generated fluorescence include linear polyolefins such as polyethylene and polypropylene;
Cyclic polyolefin;
Fluorine-containing resin;
Etc. can be used. Among the above resins, saturated cyclic polyolefin is particularly excellent in heat resistance, chemical resistance, low fluorescence, transparency, and moldability, and therefore is suitable for optical analysis and is preferably used as a substrate material.

  Here, the saturated cyclic polyolefin refers to a saturated polymer obtained by hydrogenating a polymer having a cyclic olefin structure or a copolymer of a cyclic olefin and an α-olefin. Examples of the former are produced by hydrogenating norbornene monomers represented by, for example, norbornene, dicyclopentadiene, and tetracyclododecene, and polymers obtained by ring-opening polymerization of these alkyl-substituted products. It is a saturated polymer. The latter copolymer is an α-olefin such as ethylene, propylene, isopropylene, 1-butene, 3-methyl-1-butene, 1-pentene, 3-methyl-1-pentene, 1-hexene, 1-octene and the like. It is a saturated polymer produced by hydrogenating a random copolymer of cyclic olefin monomers. As the copolymer, a copolymer with ethylene is most preferable. These resins may be used alone, or two or more copolymers or a mixture may be used. Further, not only a saturated cyclic polyolefin obtained by ring-opening polymerization of a monomer having a cyclic olefin structure, but also a saturated cyclic polyolefin obtained by addition polymerization of a monomer having a cyclic olefin structure can be used.

  A substrate made of a plastic material including a polymer substance as described above can be obtained by applying a liquid containing the polymer substance to the surface of the substrate processed into a predetermined shape and drying it. Alternatively, the substrate may be immersed in a liquid containing a polymer substance and dried.

  When the substrate material is plastic, the shape is not limited to a plate shape, and may be a film shape or a sheet shape, for example. Specifically, the substrate can be a flexible plastic film. Moreover, the board | substrate may be comprised from one member and may be comprised from the some member.

Next, a method for immobilizing the primer on the surface of the substrate will be described.
For example, (i) among a plurality of active ester groups contained in a polymer substance on a substrate, at least a part of the active ester groups react with a primer to form a covalent bond, thereby fixing the primer on the substrate surface. And (ii) inactivating the active ester group on the substrate surface other than the one on which the primer is immobilized, that is, immobilizing the remaining active ester group, thereby immobilizing the primer on the surface of the substrate. it can. Hereinafter, each process will be described.

  In the step (i), when the primer to be annealed with the template DNA fragment is immobilized on the substrate, a method of spotting a liquid in which the primer is dissolved or dispersed is preferable. A part of the active ester group contained in the polymer substance reacts with the primer, and a covalent bond is formed between the primers.

  The liquid in which the primer is dissolved or dispersed can be, for example, neutral to alkaline, for example, pH 7.6 or higher.

  In addition, after the spotting, in order to remove the primer not immobilized on the substrate surface, the primer may be washed with pure water or a buffer solution.

  Further, as shown in the above step (ii), after the washing, the inactive treatment of the active ester on the surface of the plastic substrate other than the primer immobilized is performed with an alkali compound or a compound having a primary amino group.

  Examples of the alkali compound include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate, calcium hydroxide, magnesium hydroxide, sodium borate, lithium hydroxide, and potassium phosphate. it can.

  Examples of the compound having a primary amino group include glycine, 9-amino aquadine, aminobutanol, 4-aminobutyric acid, aminocaprylic acid, aminoethanol, 5-amino2,3-dihydro-1,4-pentanol, aminoethanethiol Hydrochloride, aminoethanethiolsulfuric acid, 2- (2-aminoethylamino) ethanol, 2-aminoethyl dihydrogen phosphate, aminoethyl hydrogensulfate, 4- (2-aminoethyl) morpholine, 5-aminofluorescein, 6- Aminohexanoic acid, aminohexyl cellulose, p-aminohippuric acid, 2-amino-2-hydroxymethyl-1,3-propanediol, 5-aminoisophthalic acid, aminomethane, aminophenol, 2-aminooctane, 2-amino Octanoic acid, 1-amino-2-propanol, 3-amino-1-propyl Lopanol, 3-aminopropene, 3-aminopropionitrile, aminopyridine, 11-aminoundecanoic acid, aminosalicylic acid, aminoquinoline, 4-aminophthalonitrile, 3-aminophthalimide, p-aminopropiophenone, aminophenylacetic acid , Aminonaphthalene and the like can be used. Of these, aminoethanol and glycine are preferably used.

  Moreover, it is preferable to introduce an amino group into the primer to be immobilized on the substrate in order to increase the reactivity with the active ester group. Since the amino group is excellent in reactivity with the active ester group, the primer can be efficiently and firmly immobilized on the surface of the substrate by using the primer having the amino group introduced. The amino group may be introduced at the end of the molecular chain or at the side chain of the primer, but it can be annealed with the complementary template DNA fragment more efficiently if it is introduced at the end of the molecular chain. From this point of view, it is preferable.

  As described above, a DNA microarray in which a primer is immobilized on the surface of the substrate is obtained.

  A DNA strand extending template DNA fragment to be annealed to the primer immobilized on the surface of the DNA microarray substrate obtained as described above, a DNA strand extending enzyme system, and a sample containing nucleotide monomers are introduced. Further, the same effect can be obtained by using a template RNA fragment instead of a DNA fragment as a template used for DNA amplification.

  As a reaction system comprising the introduced sample, when the template is a DNA fragment, the enzyme system for DNA chain extension uses either DNA polymerase or DNA ligase, and when the template is an RNA fragment. The DNA chain elongation enzyme system comprises a buffer for MPEC containing nucleotide monomers (dATP, dCTP, dGTP, dTTP, etc.) in the presence of either reverse transcriptase or a combination of DNA ligase and reverse transcriptase. Can be used.

  Among DNA polymerases, Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, and the like, which are DNA polymerases derived from heat-resistant bacteria, can also be used.

In addition, at least one of these nucleotide monomers can be labeled. For example, by using Cy3-dUTP fluorescently labeled at position 3 of the base of dTTP as a nucleotide monomer, Cy3-dUTP is inserted at a position on the extension (primer) side corresponding to adenine (A) of the template DNA fragment. Thereby, the DNA fragment formed from the primer in which the extension reaction has occurred is fluorescently stained with Cy3-dUTP, and this DNA fragment can be detected.
By introducing

Other nucleotide monomers may be labeled, and a plurality of types of nucleotide monomers may be labeled. In addition to also label Methods of introduction of the phosphor, a method of introducing light absorbing method of radiolabeled ^{(P 32 -ATP, P 32 -dATP} ), by a method of non-radioactive labels such as enzyme labels DNA The strand can be detected.

  In this enzyme labeling method, a nucleic acid (for example, biotin-dUTP, DIG-dUTP) bound to biotin (biotin) or digoxigenin (DIG: steroidal natural product) is used to extend the primer, and then fluorescence. DNA can be detected by labeling or treating with alkaline phosphatase or alkaline phosphatase and reacting in nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for several hours. it can.

First, the temperature of the reaction system into which the sample has been introduced is raised to a temperature higher than the heat denaturation temperature (Tm) of the DNA strand, for example, 90 ° C. to 95 ° C. By this heat denaturation treatment, a template DNA fragment or primer having a folded structure found in a self-complementary strand or the like becomes a linear single strand.

  Subsequently, the temperature of the reaction system is lowered to a temperature at which the primer and the template DNA fragment anneal (annealing temperature), for example, 4 ° C. to 65 ° C., preferably 35 ° C. to 65 ° C. By this annealing treatment, a primer having a sequence complementary to a part of the template DNA fragment and the template DNA fragment become double stranded. The reaction system is maintained at a constant temperature without washing, and a DNA strand extension reaction and an amplification reaction are performed.

  Here, conventionally, a washing process for removing a DNA fragment (or RNA fragment) that did not form a double strand was required after the annealing process and before the extension reaction. Since there is no non-specifically adsorbed DNA fragment (or RNA fragment) and the substrate surface environment is suitable for an enzymatic reaction related to DNA chain elongation, no substrate washing treatment is required. In this way, the same liquid phase system from the sample introduction to the DNA chain extension reaction, that is, the reaction system can be used as it is.

  The temperature of the reaction system that has been annealed is controlled so as to maintain a constant temperature. Alternatively, the temperature of the reaction system is maintained at a predetermined temperature between the annealing temperature and the heat denaturation temperature, for example, a predetermined temperature in the range of 35 ° C to 75 ° C. The holding time at a constant temperature is preferably 30 minutes to 4 hours. By keeping the temperature of the reaction system constant in this way, a DNA chain elongation reaction by the MPEC method occurs. When the DNA chain proceeds to a certain length, the template DNA fragment is converted to a primer that has not yet been extended as needed. Then, a primer extension reaction occurs. In this way, DNA strands can be amplified without repeating a heat cycle as in the prior art.

  Here, an example in which a heat-resistant DNA polymerase is used for a template DNA fragment is shown, but there is no particular limitation as long as it is an enzyme that synthesizes a new DNA strand using a DNA strand as a template. Examples of such a DNA polymerase include Pol I type DNA polymerase (E. coli DNA polymerase I, Klenow fragment, etc.), α type DNA polymerase (Pyrococcus furiosus-derived DNA polymerase, VENT DNA polymerase, KOD DNA polymerase, DEEP VENT DNA polymerase) and Non-α non-pol I type DNA polymerase (DNA polymerase described in WO 97/24444 pamphlet) and the like.

  It is possible to perform DNA chain extension and amplification using DNA ligase instead of DNA polymerase.

  In addition, when a template RNA fragment is used, a primer on the substrate is allowed to act directly on the template RNA fragment, and the DNA strand can be extended to the primer side using reverse transcriptase. Even if reverse transcriptase is allowed to act on the template RNA fragment to synthesize cDNA (complementary DNA) once (first strand synthesis) and then DNA polymerase or DNA ligase is allowed to act, DNA strand extension and It is possible to amplify.

  In order to amplify the DNA strand, it is preferable to provide a plurality of spots in a certain section on the substrate, immobilize a primer on each spot, and form a microarray.

  Moreover, the length of the DNA amplification primer to be immobilized on the surface of the substrate can be arbitrarily determined according to the purpose and application, and can be set to 5 to 50 bases, for example.

  After the extension reaction, the reaction solution is discarded, and the DNA microarray is washed with, for example, a 0.1 wt% SDS solution to finish.

  Hereinafter, application examples of the DNA chain extension method of the present embodiment will be described.

(SNP analysis)
When a sequence complementary to a base sequence including a characteristic sequence of a predetermined gene of interest is used as a perfect match sequence, SNP analysis can be performed by replacing one base of this perfect match sequence with another base. Become. In addition, it is preferable to use a primer having a length of 25 bases or less.

  That is, a DNA fragment that has a mismatch of one base with a characteristic sequence of a certain gene of interest is immobilized on the surface of the substrate as a primer. At this time, for example, by using a microarray of 384 holes (256 spots for each hole) and 96 holes (1024 spots for each hole), the sequence of the primer immobilized on each spot is grasped. A sample is introduced using the DNA strand containing this characteristic sequence as a template DNA fragment, heat denaturation treatment is performed, and annealing treatment is performed. By maintaining the annealing temperature, an extension reaction occurs only for a primer that can form a double strand with a template DNA strand fragment, and amplification is performed by an extension reaction occurring as needed.

  In addition, by including the labeled nucleotide monomer in the sample introduced as described above, the DNA fragment obtained by the extension reaction from the primer is labeled, and after the reaction, the fluorescent DNA remaining on the substrate is washed. By detecting spots containing fragments and knowing the sequences of the primers immobilized on each array, it is possible to know the sequences of the primers immobilized on the detected spots. As a result, the base sequence of the primer in which the extension reaction has occurred can be known, and single nucleotide polymorphisms for the template DNA fragment containing the characteristic sequence of the gene of interest can be analyzed.

(SBH analysis)
Primers having a predetermined number, for example, 6 to 10 mer, preferably 8 mer base sequences, all combinations, for example, when 8 mer primers are used, prepare 4 ⁸ = 65536 kinds of primers that are all combinations, The base sequence determination (SBH) analysis can be performed by immobilizing the primers in one spot of the microarray.

  That is, each of the above primers is immobilized on each spot of the microarray. At this time, the sequence of the primer immobilized on each spot is known. A sample is introduced using a DNA fragment of an unknown base sequence as a template DNA fragment, heat denaturation treatment is performed, and annealing treatment is performed. By maintaining the annealing temperature, only the primers that can form double strands with the template DNA strand fragments are extended and amplified.

  In addition, as described above, by including the labeled nucleotide monomer in the sample, the DNA fragment obtained by the extension reaction from the primer is labeled, and after the DNA chain extension amplification, washing is performed, and the fluorescence remaining on the substrate Since the spots containing the DNA fragments are detected and the sequences of the primers immobilized on each array are known, the sequences of the primers immobilized on the detected spots can be known. Thereby, the base sequence of the primer in which the extension reaction has occurred is known.

  Here, after detecting a spot where an extension reaction has occurred on the substrate, each sequence of the random sequence group of primers immobilized on the detected spot is taken out and overlapped by shifting by one base before and after the sequence. To obtain an ordered array group. A template having an unknown base sequence based on the base sequence obtained by reading the first base of each primer and reading the entire base sequence for the last primer according to the order shown in this ordered sequence group The base sequence of the DNA fragment can be determined.

  In addition, when a primer is 6 base length, it is necessary to prepare 4096 types of primers, and when a primer is 7 base length, it is necessary to prepare 16384 types of primers. These primers can be used for searching relatively small molecules of 20 mer to 50 mer such as unknown function (nc) RNA and micro (mi) RNA, and gene sequence analysis.

  When the primer is 8 bases long, 65536 types of primers need to be prepared, and when the primer is 9 bases long and 10 bases long, 262144 and 1048576 types of primers need to be prepared, respectively. These primers can be used for normal gene sequence analysis.

(Gene expression profile analysis)
First, a conventional method for preparing a sample and analyzing an expression profile is shown.

  Total RNA (total RNA) is extracted from the biological tissue according to the purpose. Furthermore, cDNA is synthesized from the total RNA by a reaction using reverse transcriptase (first strand synthesis). In the reaction using reverse transcriptase, a mixture of dNTP and aminoallyl dUTP in a certain ratio is added to synthesize cDNA incorporating aminoallyl dUTP.

  If the amount of total RNA obtained from the tissue is small, cDNA is synthesized by reverse transcription reaction, then double-stranded cDNA (transcription template DNA fragment) is synthesized (second-strand synthesis), and it is used as a template. RNA polymerase (aRNA) is amplified by the action of RNA polymerase. By adding a mixture of dNTP and aminoallyl dUTP at a certain ratio during the RNA amplification, aRNA incorporating aminoallyl dUTP can be synthesized.

Subsequently, the above cDNA or antisense aminoallyl RNA (aRNA) is ethanol precipitated and then dissolved in 0.2 M sodium carbonate buffer (NaHCO ₃ —Na ₂ CO ₃ (pH 9.0)). Fluorescent dye (Cy3 or Cy5) dissolved in DMSO is added and coupled with aminoallyl dUTP incorporated in cDNA or aRNA according to a conventional method to fluorescently label cDNA or aRNA. After removing the free fluorescent dye by conventional purification using a gel filtration column or a filter, for aRNA, further fragmentation buffer (final concentration 0.04M triaminomethane acetate (pH 8.1), 0.1M potassium acetate, 0.03M magnesium acetate) is added and reacted at 95 ° C. for 15 minutes, and then rapidly cooled with crushed ice to fragment aRNA. After confirming fragmentation by a method such as gel electrophoresis, the solution is purified and concentrated with a gel filtration filter.

Next, hybridization of the prepared cDNA or aRNA and a microarray primer is performed. Hybridization buffer (final concentration 5 × SSC, 0.5 (v / v)% SDS, 4 × Denhardt's solution) and formamide is mixed with cDNA or aRNA as appropriate, and mixed with DNA microarray at 45 ° C. to 60 ° C. Hybridize overnight. After hybridization, after washing with 2 × SSC-0.1 (v / v)% SDS solution, 2 × SSC solution, and 1 × SSC solution for 5 minutes each time, a fluorescence reader (for example, CRBIO (r) IIe; Hitachi The image is scanned using software engineering, and the signal intensity is quantified using analysis software (for example, DNASIS (r) Array; manufactured by Hitachi Software Engineering).
Although the synthesis of aRNA is shown here, this cDNA may be directly analyzed if the amount of cDNA obtained in the intermediate step is sufficient for analysis.

  In this method, since a complicated pretreatment process is performed, setting conditions such as condition selection increase for each sample, and it takes at least 5 days to 1 week for all processes in terms of rapid genetic testing. It was a problem.

  Therefore, in this embodiment, a DNA fragment having a base sequence corresponding to the characteristic sequence of the gene to be analyzed is used as a primer on the surface of the substrate, and each primer is immobilized on one spot of the microarray. . Therefore, after performing first strand synthesis to obtain cDNA by acting reverse transcriptase on total RNA, cDNA obtained by first strand synthesis (or aRNA obtained by amplification if necessary) is used as a template DNA fragment. By using the DNA polymerase or DNA ligase to perform an extension reaction on the primer side on the substrate, it becomes possible to specify the expressed gene.

  In addition, as described above, using the total RNA as a template, a primer having a predetermined sequence provided on the substrate is hybridized, and a reverse transcriptase is allowed to act on the primer side on the substrate. It is possible to carry out the reaction, and this also makes it possible to specify the expressed gene.

  That is, it can be seen whether or not a probe having a base sequence complementary to a characteristic sequence of a known gene is present in a template DNA fragment obtained from total RNA derived from a specific cell.

  That is, taking the example of using cDNA for total RNA as a template DNA fragment, each of the above primers is immobilized on each spot of the microarray. At this time, the sequence of the primer immobilized on each spot is known. Samples are introduced using cDNA synthesized from the predetermined total RNA obtained as described above as a template DNA fragment, heat denaturation treatment is performed, and annealing treatment is performed. By maintaining the annealing temperature, an extension reaction occurs and is amplified only by a primer that can form a double strand with the template DNA strand fragment.

  In addition, by including the labeled nucleotide monomer in the sample introduced as described above, the DNA fragment obtained by the extension reaction from the primer is labeled, and the fluorescent DNA remaining on the substrate by washing after the extension amplification reaction By detecting spots containing fragments and knowing the sequences of the primers immobilized on each array, it is possible to know the sequences of the primers immobilized on the detected spots. As a result, the base sequence of the primer in which the extension reaction has occurred can be known, and from which gene the total RNA is expressed can be analyzed, that is, the gene expression profile can be analyzed.

  According to this method, gene expression profile analysis can be carried out using genomic DNA or reverse transcript cDNA from total RNA as a template DNA fragment, with almost no laborious and time-consuming sample preparation in normal analysis. It becomes possible to carry out by a simple operation.

Besides the above uses,
Application to basic tools for gene analysis such as microsatellite analysis, chromosomal aberration analysis (CGH), and unknown function (nc) RNA search;

Gene expression analysis chip for each organ / disease using these tools, mutagenicity test kit (environmental hormone), genetically modified food test kit, mitochondrial gene sequence analysis kit, analysis kit for parentage testing / crime investigation, congenital Disease analysis kit, chromosome / gene abnormality analysis kit, genetic diagnosis (pre-implantation / prenatal) kit, drug reaction-related gene polymorphism analysis kit, lipid metabolism-related gene polymorphism analysis kit, otolaryngology / ophthalmology gene polymorphism analysis Application to custom chips by use such as kits;

  Application to diagnostic / clinical custom chips such as cancer prognosis prediction chips, pharmaceutical development (clinical / drug discovery) chips, health food development chips;

Drug and food manufacturing processes such as microbiological limit tests and microbiological tests in food and drink water, clinical examinations in the dental field such as detection of caries and periodontal disease-related bacteria, opportunistic infections, and food factory / kitchen facility environment Environmental inspections in water quality inspections such as inspections, drinking and public baths, well water, and prevention of infectious diseases and food poisoning, hygiene such as hygiene management of company employees, and identification of general bacteria including resistant bacteria, hepatitis virus, Helicobacter pylori, Examples include application to microbe identification test kits such as clinical tests for hepatitis chlamydia, AIDS virus, SARS virus, West Nile virus, norovirus (food poisoning derived from raw oysters), influenza virus, fungi and mold.

(Experimental example 1)
The primer is immobilized on the surface of each substrate of a plastic and glass substrate corresponding to this embodiment and an aldehyde substrate corresponding to a conventional substrate by the following method, and DNA strand amplification reaction is performed on each substrate. The DNA strand amplification reaction of the primer was detected.

(Manufacture of plastic substrates)
Saturated cyclic polyolefin resin (hydrogenated ring-opening polymer of 5-methyl-2-norbornene (MFR (Melt flow rate): 21 g / 10 min, hydrogenation rate: substantially 100%, heat distortion temperature 123 ° C.)) A glass substrate was obtained by injection molding, and the substrate was 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate-p-nitrophenyloxycarbonylpolyethylene glycol methacrylate (NPMA) copolymer (each group was 25% by mol). : 74: 1) was immersed in a 0.5 wt% ethanol solution to introduce a polymer substance having a phosphorylcholine group and an active ester group on the substrate surface to obtain a plastic substrate.

(Manufacture of glass substrates)
An ordinary glass substrate is coated with 0.5 weight of 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate-p-nitrophenyloxycarbonylpolyethylene glycol methacrylate (NPMA) copolymer (each group is 25: 74: 1 in mol%). A polymer substrate having a phosphorylcholine group and an active ester group was introduced onto the substrate surface by dipping in a% ethanol solution to obtain a glass substrate.

(Manufacture of aldehyde substrates)
Saturated cyclic polyolefin resin 5-methyl-2-norbornene ring-opening polymer hydrogenated product (MFR: 21 g / 10 min, hydrogenation rate: substantially 100%, heat distortion temperature: 123 ° C.), injection A slide glass substrate was obtained by molding. The molded product was subjected to a hydrophilic treatment on the surface by low-temperature oxygen plasma treatment. Next, a solution prepared by dissolving γ-aminopropyltriethoxysilane as an aminoalkylsilane in methanol at a concentration of 5% is prepared as an amino group introduction treatment solution. After being immersed in this solution for 2 hours, the substrate is mounted. The substrate was taken out from the solution, immersed in ultrapure water and allowed to stand, and then the substrate was taken out and dried. Glutaraldehyde is dissolved in PBS (-) at a concentration of 2% to prepare a glutaraldehyde solution. The substrate treated with aminoalkylsilane is immersed in the glutaraldehyde solution and left for 4 hours, and then the substrate is taken out. And then immersed in ultrapure water, washed and dried. Thereby, an aldehyde substrate having an aldehyde group on the surface was obtained.

(Primer fixation)
Oligo DNA (20 base chain) whose 5 ′ end was modified with an amino group was dissolved using 0.25 M carbonate buffer (pH 9.0) to prepare a 10 μM oligo DNA solution. This solution was spotted on the surface of a plastic substrate, a glass substrate, and an aldehyde substrate using a spotter (Marks-I manufactured by Hitachi Software Engineering Co., Ltd.) with a 100 μm diameter cross cut pin. Each substrate on which oligo DNA was spotted was immersed overnight in a sealed container (10 cm x 15 cm x 3 cm) moistened with 200 µl of 0.25 M phosphate buffer (pH 8.5) to immobilize the oligo DNA (primer). Made it.

(DNA strand amplification reaction)
As a template DNA fragment to be introduced, a predetermined 50-mer DNA fragment whose 5 ′ end was previously labeled with Cy5 was used, and the reaction system was such that the concentration of the template DNA fragment was 100 pM. In addition, DNA polymerase (Ex Taq manufactured by Takara Bio Inc.) was used as an enzyme system for DNA chain elongation.

  Subsequently, heat denaturation treatment was performed at 95 ° C. for 5 minutes, and then annealing treatment and extension amplification reaction were performed at 37 ° C. The extension amplification reaction times were 30 minutes, 60 minutes, 90 minutes, 120 minutes, and 150 minutes.

  Hereinafter, the result of measuring the fluorescence intensity derived from Cy3-dUTP in the DNA chain amplification reaction performed on each substrate with Cy3-dUTP included in the sample is shown below.

  In the plastic substrate and the glass substrate corresponding to the present embodiment, the signal is strengthened by extending the extension amplification time, and DNA strand extension amplification occurs on the substrate. On the other hand, the aldehyde corresponding to the conventional substrate It is considered that DNA strand elongation does not occur on the substrate. In addition, in the plastic substrate and the glass substrate corresponding to the present embodiment, it is possible to obtain a detectable signal without performing a heat cycle.

Claims (5)

A DNA chain that serves as a primer for DNA elongation on a substrate having a polymer substance on the surface thereof, which includes a first unit having a 2-methacryloyloxyethyl phosphorylcholine group and a second unit having a p-nitrophenyloxycarbonylpolyethylene glycol methacrylate group A liquid phase system in which a sample containing a template DNA fragment or template RNA fragment having a desired sequence, a DNA chain elongation enzyme system, and a nucleotide monomer is introduced, is subjected to heat denaturation of the DNA chain (hereinafter, To "heat denaturation temperature")
The temperature of the liquid phase system is lowered to a temperature for annealing treatment (hereinafter referred to as “annealing temperature”), and the primer DNA strand immobilized on the substrate is extended and amplified while maintaining a constant treatment temperature. A DNA strand amplification method. In the DNA strand amplification method according to claim 1,
A DNA strand amplification method characterized in that no washing treatment is involved in the liquid phase system before the annealing treatment and the DNA strand elongation reaction. In the DNA strand amplification method according to claim 1,
When the template is a DNA fragment, the DNA chain extending enzyme system is one of DNA polymerase and DNA ligase. In the DNA strand amplification method according to claim 1,
When the template is an RNA fragment, the DNA chain extending enzyme system is any one of reverse transcriptase or a combination of DNA ligase and reverse transcriptase. In the DNA strand amplification method according to claim 1,
A DNA strand amplification method, wherein any one of the nucleotide monomers is labeled.