JP2007330104A - Method for elongating dna chain and array for elongating dna chain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for elongating a DNA chain with high elongating reaction efficiency of the DNA chain on a substrate. <P>SOLUTION: The method for elongating the DNA chain is carried out as follows. A primer for elongating the DNA is immobilized on a substrate surface having a hydrophilic polymer component and a functional group for immobilizing the DNA chain on the surface and a solution containing a nucleic acid chain to be a template having a desired sequence, a nucleotide monomer and a DNA elongation enzyme is fed to the substrate surface. A treatment, as necessary, is carried out at a temperature for thermally denaturing the DNA chain and an elongating reaction of the primer DNA chain is then conducted at a temperature for carrying out an annealing treatment. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a DNA chain extension method and a DNA chain extension array in which a primer DNA chain is immobilized on a predetermined substrate surface to extend the DNA chain.

  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.

Non-Patent Document 2 describes a hybrid property with a predetermined DNA strand and a PCR-like environment using a DNA microarray in which DNA fragments are immobilized on the surface using poly (methyl methacrylate) instead of a glass substrate. The thermostability below has been evaluated, and a description suggesting the possibility of a device that can be applied to a novel PCR technique has been made.
Adessi, Celine et al, “Solid phase DNA amplification: Characterisation of primer attachment and amplification mechanisms”, Nucleic Acids Research, 2000, Vol. 20, No. 20, e87 Fixe, F. et al, “Functionalization of poly (methyl methacrylate) (PMMA) as a substrate for DNA microarrays”, Nucleic Acids Research, 2004, January 12, Vol. 32, No.1, e9

  The present inventors conducted a DNA chain extension reaction by the MPEC method (Multiple Primer Extension on a Chip) using various DNA microarrays represented by Non-Patent Documents 1 and 2, and the like. When the heat denaturation treatment is performed after extending the DNA strand, the primer fixed to the DNA microarray substrate is removed together with the template DNA, and a substrate with a hydrophilic polymer substance on the surface is used. Thus, this problem was solved and the present invention was completed.

  In the DNA chain extension method according to the present invention, a sample containing a template DNA fragment having a desired sequence is introduced by immobilizing a primer for DNA extension on a substrate having a hydrophilic polymer and a functional group for immobilizing DNA on the surface. The reaction system thus obtained is raised to a temperature at which the DNA strand is thermally denatured as necessary, and then subjected to an elongation reaction of the DNA strand at an annealing treatment temperature.

That is, the present invention
(1) Immobilization of a primer for DNA extension on a substrate surface having a hydrophilic polymer component and a functional group for immobilizing a DNA chain on the surface, and a nucleic acid chain, nucleotide monomer, and DNA extension serving as a template having a desired sequence A solution containing an enzyme is supplied to the surface of the substrate, and if necessary, a DNA strand is heated up to a temperature for heat denaturation, and then the primer DNA strand is extended at an annealing temperature. DNA chain extension method,
(2) The DNA chain elongation method according to (1), wherein the hydrophilic polymer and the functional group for immobilizing the DNA strand form a matrix entangled in a network,
(3) In the DNA chain extension method according to (1), the functional group for immobilizing the DNA chain is a functional group that reacts with an amino group, an amino group is introduced into the 5 ′ end of the primer DNA chain, and the amino group A DNA chain extending method, wherein a primer is immobilized by reacting and binding with a functional group;
(4) The DNA chain extension method according to (3), wherein the functional group that reacts with an amino group is an active ester group,
(5) The method for extending a DNA chain according to (1), wherein the nucleic acid chain used as the template is DNA or RNA,
(6) The DNA chain extension method according to (1), wherein the nucleic acid chain as the template is a cDNA fragment obtained by treating a predetermined RNA with a reverse transcriptase,
(7) In the DNA chain extension method described in (1), a solution containing a nucleic acid chain, a nucleotide monomer, and a DNA extension enzyme as a template having a desired sequence is preliminarily raised to a temperature at which the DNA chain is thermally denatured. A DNA chain extending method characterized by
(8) In the DNA chain extension method according to (1), the DNA chain extension reaction is performed by gradually increasing the temperature of the reaction system from the annealing temperature to the heat denaturation temperature. A DNA chain extension method,
(9) In the DNA strand extension method according to (1), the DNA strand extension reaction is performed by changing the temperature of the reaction system to a predetermined temperature between the annealing treatment temperature and the heat denaturation treatment temperature. A DNA chain extending method characterized by being performed,
(10) In the DNA chain extension method according to (1), the primer is a DNA fragment chain in which one base of a base sequence including a characteristic sequence of a predetermined gene of interest is replaced with another base. DNA chain extension method,
(11) The DNA chain extension method according to (1), wherein the primer comprises a predetermined number of base sequences, and each primer is a DNA fragment having the respective sequences of all combinations. Method,
(12) The DNA chain extension method according to (1), wherein at least one of the nucleotide monomers is labeled,
(13) The DNA chain extension method according to (1), wherein the substrate is made of silicon, glass, metal, graphite, or a plastic material,
(14) The method for extending a DNA strand according to (1), wherein the substrate has a glass slide shape, a microtiter plate shape or a container shape,
(15) a DNA chain extension array in which a primer for DNA extension is fixed on the surface of a substrate having a hydrophilic polymer component and a functional group for fixing the DNA chain on the surface;
(16) The DNA chain according to (15), wherein the hydrophilic polymer and the functional group for immobilizing the DNA chain form a matrix entangled in a mesh shape. Expansion array,
(17) The DNA chain extension array according to (15), wherein the substrate is made of silicon, glass, metal, graphite, or a plastic material,
(18) The DNA chain extension microarray according to (15), wherein the primer is composed of 5 to 50 base sequences,
(19) The DNA chain extension array according to (15), wherein the primer is covalently bonded to a functional group for immobilizing the DNA strand on the substrate surface and immobilized on the substrate surface. An array for DNA chain elongation,
(20) In the DNA chain extension array according to (15), the base is a substrate, the substrate is provided with a plurality of spots in a fixed section, and a primer is fixed to each spot. An array for extending a DNA chain,
(21) The DNA chain extension array according to (15), wherein the substrate is a microtiter plate-like substrate having a plurality of wells, and a primer is immobilized on the well surface. For arrays,
It is.

  According to the present invention, the efficiency of the elongation reaction of DNA strands on the DNA microarray is high, and after the reaction is performed to make double strands, the single-stranded DNA is left on the substrate of the DNA microarray by heat denaturation treatment. become able to.

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

(First embodiment)
FIG. 1 is a flowchart showing the procedure of the DNA strand amplification method as the first embodiment. In this DNA strand amplification method, a primer for DNA extension (hereinafter referred to as “primer”) is immobilized on a substrate having a hydrophilic polymer and a functional group for immobilizing DNA on the surface (step S10), and a desired sequence. The reaction system in which a sample containing a nucleic acid chain, a nucleotide monomer, and a DNA extender as a template having a DNA is introduced is raised to a temperature at which the DNA chain is thermally denatured as necessary (step S30), and the temperature of this reaction system is increased. The temperature is lowered to the annealing temperature (step S40), the DNA strand is elongated in the reaction system (step S50), the DNA strand is thermally denatured (step S60), and the DNA strand is annealed as necessary. The extension reaction and the heat denaturation treatment are performed (repeating step S40 to step S60).
After preparing a solution containing a nucleic acid chain, a nucleotide monomer and a DNA extension enzyme as a template, the solution may be supplied to the substrate surface after heating until heat denaturation treatment. In this case, the solution was supplied to the substrate surface. It is not necessary to raise the temperature to the heat denaturation temperature immediately after.

In step S10, as shown in FIG. 2, the primer 14 for DNA extension is fixed to the surface of the substrate 12.

  The substrate used here has a hydrophilic polymer component and a functional group for immobilizing the DNA strand on the surface. The functional group that immobilizes the hydrophilic polymer component and the DNA strand forms a matrix that is naturally united by cross-linking the hydrophilic polymer component with each other, and the functional group that immobilizes the DNA strand within the matrix is uniform. It is desirable to adopt the structure introduced in By forming the matrix, the hydrophilic environment on the substrate surface can be kept uniform, and variations in the DNA elongation reaction environment can be suppressed.

  The hydrophilic polymer component plays a role of providing an environment suitable for the DNA chain elongation reaction of the DNA elongation enzyme by inhibiting nonspecific adsorption of the DNA strand and inhibiting the adsorption of the DNA elongation enzyme in the DNA chain elongation reaction. .

  Next, functional groups for immobilizing DNA strands are described.

  In immobilizing the primer in the DNA chain extension method of the present invention, a functional group for immobilization is introduced at the 5 'end of the primer DNA chain, and the DNA chain is immobilized and bonded to the functional group. In this case, examples of the functional group to be introduced at the end of the DNA chain include an amino group, a thiol group, an active ester group, and biotin. However, introduction of an amino group is preferable because of ease of introduction and binding stability. There is a functional group that reacts with an amino group as a functional group for immobilizing the DNA strand. Examples of the functional group that reacts with an amino group include an aldehyde group and an active ester group, and an active ester group is preferred. The active ester group is a carboxylic acid obtained by activating a carboxyl group of a carboxylic acid and having a leaving group via C═O. Examples of the activated carboxylic acid derivatives include carboxylic acids such as acrylic acid, methacrylic acid, crotonic acid, maleic acid, and fumaric acid, which are acid anhydrides, acid halides, active esters, activated amides. The compound converted into is mentioned. Carboxylic acid-derived groups are activated groups derived from such compounds, for example, active ester groups such as p-nitrophenyl and N-hydroxysuccinimide groups, and groups such as halogen such as -Cl and -F. The thing which has.

  The functional group for immobilizing the DNA chain may be present in the main chain or side chain of the hydrophilic polymer structure.

  Examples of the hydrophilic polymer used in the present invention include a polymer substance having a hydrophilic group in the main chain or side chain, and any of polyalkylene oxide, polyethylene oxide, polypropylene oxide, polyacrylamide, and copolymers thereof. It is preferable that these are included in the structure. In particular, polyacrylamide can be formed into a network structure to form a polyacrylamide gel having a matrix by adding a crosslinking component such as NN′-methylenebisacrylamide or three-dimensionally crosslinking with a photocrosslinkable compound.

  The shape of the substrate is not particularly limited, but examples include a substrate typified by a slide glass, a microtiter plate having a plurality of wells, and a container used for PCR. For the production of the array, a glass-like substrate or a microtiter plate is suitable.

Next, the method for immobilizing the primer on the surface of the substrate will be described by taking as an example the case where the substrate shape is a substrate and the functional group for immobilizing the DNA strand is a functional group that reacts with an amino group.
For example, (i) among functional groups that react with amino groups present on the surface of a substrate or contained in a hydrophilic polymer, a functional group that reacts with at least some of the amino groups reacts with a primer and is covalently bonded. Then, the primer is immobilized on the surface of the substrate, and (ii) the functional group that reacts with the amino group on the substrate surface other than the one where the primer is immobilized is deactivated, that is, reacts with the remaining amino group. By inactivating the functional group, the primer can be immobilized on the surface of the substrate. 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. Some of the functional groups that are present on the surface of the substrate or react with amino groups contained in the hydrophilic polymer react with the primer to form a covalent bond 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.

  In addition, as shown in the above step (ii), after the washing, the functional group reacting with the amino group on the substrate surface other than the immobilized primer is deactivated with an alkali compound or a compound having a primary amino group. It can be carried out.

  As described above, the DNA microarray 10 having the primer 14 immobilized on the surface of the substrate 12 as shown in FIG. 2 is obtained.

  Returning to FIG. 1, in step S20, a sample including a DNA fragment template DNA fragment 16 to be annealed to the primer 14 immobilized on the surface of the substrate 12 of the DNA microarray 10 obtained in step S10 and a nucleotide monomer are introduced. .

  The reaction system consisting of the introduced sample includes nucleotide monomers (dATP, dCTP, dGTP, dTTP, etc.) in the presence of DNA elongation enzymes such as Taq DNA polymerase, Tth DNA polymerase, and Pfu DNA polymerase, which are DNA polymerases derived from thermostable bacteria. ) Containing MPEC).

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.

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 is applied. DNA can be detected by reacting with labeled alkaline phosphatase or alkaline phosphatase and nitroblue tetrazolium (NBT) in 5-bromo-4-chloro-3-indolyl phosphate (BCIP) solution for several hours. .

  In step S30, the temperature of the reaction system into which the sample has been introduced is increased to a temperature equal to or 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, in step S40, 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 50 ° 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 (FIG. 3).

In step S50, control is performed so that the temperature of the reaction system that has performed the annealing process is gradually increased from the annealing process temperature to the thermal denaturation process temperature. Alternatively, the temperature of the reaction system is controlled to change to a predetermined temperature between the annealing temperature and the heat denaturation temperature, for example, 65 ° C to 75 ° C. By controlling the temperature of the reaction system in this way, an elongation reaction of the DNA strand by the MPEC method occurs, that is, a DNA fragment complementary to the template DNA fragment 16 is formed on the substrate, and a double strand of DNA is obtained. (FIG. 4).

  Returning to FIG. 1, in step S60, the reaction system after the extension reaction is maintained at the heat denaturation temperature of the DNA strand, for example, 90 ° C. to 95 ° C. By this heat denaturation treatment, the template DNA fragment 16 (FIG. 4) is detached from the double-stranded DNA on the surface of the substrate 12, and the single-stranded DNA fragment 18 remains (FIG. 5).

  In step S70, it is determined whether or not the next extension reaction is performed. This determination may be performed automatically by designating the number of times of reaction using a temperature adjusting device (thermocycler) or the like and determining whether or not the number has been reached, or by the operator's judgment every time.

  When this determination result is YES, that is, when the next extension reaction is performed, the process returns to step S40, and the annealing process (step S40) -extension reaction (step S50) -thermal denaturation process (step S60) is repeated, and the unreacted primer A DNA extension reaction is carried out. As a result of repeating this cycle 1 to 50 times, DNA strand amplification by the MPEC method is performed on the substrate.

  If the determination result in step S70 is NO, that is, if the next extension reaction is not performed, the process proceeds to step S80, the reaction solution is discarded, and the DNA microarray is washed with, for example, a 0.1 wt% SDS solution. And exit.

  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.

(Second embodiment)
FIG. 6 is a flowchart showing the procedure of the DNA chain extension method as the second embodiment. In FIG. 6, parts similar to those in FIG. 1 are denoted by the same reference numerals as those in FIG.

  In this DNA chain extension method, a primer for DNA extension (hereinafter referred to as “primer”) is immobilized on a substrate having a hydrophilic polymer component and a functional group that reacts with an amino group on the surface (step S10), and is desired. The reaction system into which a sample containing a nucleic acid chain, DNA or RNA fragment, nucleotide monomer and DNA extender enzyme as a template having a sequence to be introduced is raised to a temperature at which the DNA chain is thermally denatured (step S30). The temperature is lowered to the annealing temperature (step S40), and a DNA chain extension reaction is performed in this reaction system (step S50).

  The heat denaturation treatment in step 30 is performed when the sample solution containing DNA or RNA fragment, nucleotide monomer, and DNA extension enzyme as a template in advance is heated at the heat denaturation treatment temperature before sample introduction (step 20). This heat denaturation treatment can be omitted.

  As described above, in step S20, a sample including a nucleic acid chain serving as a template for DNA extension to be annealed to a primer immobilized on the surface of the substrate is introduced, and this sample and the reaction system into which this sample is introduced are The same as described above can be applied.

  In addition, after the extension reaction in step S50, the reaction solution is discarded in step S55, and the DNA microarray is washed with, for example, a 0.1 wt% SDS solution, and the process ends.

  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, in step S10, a DNA fragment that is mismatched by 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. In step S20, a sample is introduced using the DNA strand containing the characteristic sequence as a template DNA fragment, heat denaturation is performed in step S30, and annealing is performed in step S40. In step S50, the extension reaction occurs only on the primer that has formed a double strand with the template DNA strand fragment by the annealing treatment.

  Further, as described above, by including the labeled nucleotide monomer in the sample introduced in step S20, the DNA fragment obtained by the extension reaction from the primer is labeled, and remains on the substrate after washing in step S55. Since the spot containing the fluorescent DNA fragment is detected and the sequence of the primer immobilized on each array is known, the sequence of the primer immobilized on the detected spot can be known. 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.

  That is, in step S10, each of the primers is immobilized on each spot of the microarray. At this time, the sequence of the primer immobilized on each spot is known. In step S20, a sample is introduced using a DNA fragment of an unknown base sequence as a template DNA fragment, heat denaturation is performed in step S30, and annealing is performed in step S40. In step S50, the extension reaction occurs only on the primer that has formed a double strand with the template DNA strand fragment by the annealing treatment.

  Further, as described above, by including the labeled nucleotide monomer in the sample introduced in step S20, the DNA fragment obtained by the extension reaction from the primer is labeled, and remains on the substrate after washing in step S55. Since the spot containing the fluorescent DNA fragment is detected and the sequence of the primer immobilized on each array is known, the sequence of the primer immobilized on the detected spot can be known. Thereby, the base sequence of the primer in which the extension reaction has occurred is known.

  Here, as shown in FIG. 7, after detecting the spot where the extension reaction occurred on the substrate 41, each sequence of the random sequence group 45 of the primers immobilized on the detected spot was taken out, and the sequence of An ordering sequence group 47 is obtained by shifting the order before and after so as to overlap each other. In accordance with the order shown in the ordered sequence group 47, the first base of each primer is read, and the last base is read based on the base sequence obtained by reading the entire base sequence. The base sequence 49 of the template DNA fragment possessed 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)
FIG. 8 shows a conventional method for preparing and analyzing an expression profile of a sample into which a label is introduced as a gene expression profile sample.

  Total RNA (total RNA) is extracted from the biological tissue according to the purpose. Furthermore, cDNA is synthesized from the total RNA by reverse transcription (first strand synthesis). In the reverse transcription reaction, 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, after synthesizing cDNA by reverse transcription reaction, double-stranded cDNA (transcription template DNA fragment) is synthesized (second-strand synthesis) and used as a template for anti-antibody Amplify the sense strand RNA (aRNA). 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, 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 (eg, DNASIS® Array; manufactured by Hitachi Software Engineering).
Although FIG. 8 shows the synthesis of aRNA, 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, after performing first strand synthesis to obtain cDNA from total RNA, bases corresponding to the characteristic sequence of the gene to be analyzed are formed on the surface of the substrate in the same manner as in step S10 of FIG. Using a DNA fragment having a sequence as a primer, each primer is immobilized on one spot of a microarray, and cDNA obtained by first strand synthesis (or aRNA obtained by amplification if necessary) is a template DNA fragment Can be used to identify 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, in step S10, each of the primers is immobilized on each spot of the microarray. At this time, the sequence of the primer immobilized on each spot is known. In step S20, a sample is introduced using the cDNA synthesized from the predetermined total RNA obtained as described above as a template DNA fragment, heat denaturation is performed in step S30, and annealing is performed in step S40. Do. In step S50, the extension reaction occurs only on the primer that has formed a double strand with the template DNA strand fragment by the annealing treatment.

  Further, as described above, by including the labeled nucleotide monomer in the sample introduced in step S20, the DNA fragment obtained by the extension reaction from the primer is labeled, and remains on the substrate after washing in step S55. Since the spot containing the fluorescent DNA fragment is detected and the sequence of the primer immobilized on each array is known, the sequence of the primer immobilized on the detected spot can be known. 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;

  Pharmaceutical and food manufacturing processes such as microbiological limit tests and microbiological tests in food and drink water, and clinical examinations in the dental field such as detection of caries and periodontal disease related bacteria and opportunistic infections, and food factory / kitchen facility environment Environmental inspection in water quality inspections such as inspections, beverages / public baths, well water, etc., and prevention of infectious diseases / food poisoning, hygiene such as hygiene management of company employees, and identification of general bacteria including resistant bacteria, hepatitis virus, Helicobacter pylori, 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 live oysters), influenza virus, fungi and mold, and the like.

(Experimental example 1)
The primer is immobilized on the surface of each substrate of the glass substrate corresponding to the present embodiment and the aldehyde substrate corresponding to the conventional substrate by the following method, and DNA strand extension reaction is performed on each substrate, and the primer The DNA chain elongation reaction was detected.

(Creation of glass substrate)
Commercially available CodeLink ^™ Activated Slides (Amersham Bioscience, GE Healthcare) were used. This substrate has a layer having a three-dimensional structure of polyacrylamide as a hydrophilic polymer on the surface of a glass substrate and an active ester group.

(Creation of aldehyde substrate)
A solution prepared by dissolving γ-aminopropyltriethoxysilane as an aminoalkylsilane in methanol at a concentration of 5% by volume (V / V%) on a commercially available slide glass substrate was prepared as an amino group introduction treatment solution. After 2 hours of immersion in the substrate, the substrate was taken out of the solution, immersed in ultrapure water and allowed to stand, and then the substrate was taken out and dried. Glutaraldehyde was dissolved in PBS (-) at a concentration of 2% by volume (V / V%) to prepare a glutaraldehyde solution, and the substrate treated with aminoalkylsilane was immersed in the glutaraldehyde solution for 4 hours. After standing, the substrate was taken out, 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 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 chain elongation reaction)
As a template DNA fragment to be introduced in step S20 of FIG. 6, 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.

  Subsequently, the thermal denaturation process of step S30, the annealing process of step S40, and the extension reaction of step S50 of FIG. The temperature was 95 ° C. for 1 minute (denaturation).

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

  In the glass substrate corresponding to the present embodiment, DNA strand elongation on the substrate was detected, whereas in the aldehyde substrate corresponding to the conventional substrate, DNA strand elongation is not detected.

(Experimental example 2)
Using the glass substrate from which DNA strand elongation was detected in Experimental Example 1, DNA strand elongation by the MPEC method was performed in 1 cycle, 5 cycles, 10 cycles, and 20 cycles for each of the 20-mer, 25-mer, and 30-mer probes. An amplification reaction was performed, and the fluorescence intensity was measured after the end of each cycle.

In addition, the thermal denaturation process of step S30 of FIG. 1, the annealing process of step S40, the extension reaction of step S50, and the thermal denaturation process of step S60 are each 95 ° C. for 5 minutes (denaturation; first time only) -95 ° C. for 1 minute (denaturation) -50 ° C for 3 minutes (annealing) -95 ° C for 1 minute (denaturation).
The results are shown below.

  Compared with “blank” (when no probe was used) as a control experiment, DNA strand amplification was detected with any length of probe.

It is a flowchart which shows the DNA strand amplification method which concerns on 1st embodiment of this invention. It is a figure which shows typically a part of DNA microarray used by said 1st embodiment. It is a figure which shows the state which annealed template DNA to the primer of the said DNA microarray. It is a figure which shows the state which performed the elongation reaction of the DNA strand on the said primer. It is a figure which shows the state which denatured the double strand obtained after the said extension reaction. It is a flowchart which shows the DNA chain | stretch extension method which concerns on 2nd embodiment of this invention. It is a figure which shows one example of application of said 2nd embodiment. It is a figure which shows the example which prepares the sample for the conventional gene expression profile.

Explanation of symbols

10 DNA microarray 12 Substrate 14 Primer 16 Template DNA fragment 18 Single-stranded DNA fragment 41 Substrate 43 Unknown base sequence 45 Primer random sequence group 47 Ordering sequence group 49 DNA fragment base sequence

Claims (21)

  Includes a nucleic acid chain, a nucleotide monomer, and a DNA extension enzyme as a template having a desired sequence by immobilizing a primer for DNA extension on the surface of a substrate having a hydrophilic polymer component and a functional group for immobilizing the DNA chain on the surface. A method for extending a DNA strand, comprising: supplying a solution to the substrate surface, performing a treatment at a temperature at which the DNA strand is thermally denatured, if necessary, and then subjecting the primer DNA strand to an elongation reaction at a temperature for annealing. .   2. The DNA chain elongation method according to claim 1, wherein the hydrophilic polymer and the functional group for immobilizing the DNA chain constitute a matrix intertwined in a network.   2. The method for extending a DNA strand according to claim 1, wherein the functional group for immobilizing the DNA strand is a functional group that reacts with an amino group, an amino group is introduced into the 5 ′ end of the primer DNA strand, and the amino group and the functional group are introduced. A DNA chain extending method, wherein a primer is immobilized by reacting and binding to.   The DNA chain extending method according to claim 3, wherein the functional group that reacts with an amino group is an active ester group. The DNA chain extending method according to claim 1, wherein the nucleic acid chain used as the template is DNA or RNA.   2. The DNA chain extending method according to claim 1, wherein the nucleic acid chain used as the template is a cDNA fragment obtained by treating a predetermined RNA with a reverse transcriptase.   2. The method for extending a DNA chain according to claim 1, wherein a solution containing a nucleic acid chain, a nucleotide monomer, and a DNA extending enzyme as a template having a desired sequence is preliminarily raised to a temperature at which the DNA chain is thermally denatured. A DNA chain extending method.   2. The DNA chain extension method according to claim 1, wherein the DNA chain extension reaction is performed by gradually increasing the temperature of the reaction system from the annealing temperature to the heat denaturation temperature. Chain extension method.   2. The DNA chain extension method according to claim 1, wherein the DNA chain extension reaction is performed by changing the temperature of the reaction system to a predetermined temperature between the annealing temperature and the heat denaturation temperature. A DNA chain extending method characterized by the above.   2. The DNA chain extension method according to claim 1, wherein the primer is a DNA fragment chain in which one base of a base sequence including a characteristic sequence of a predetermined gene of interest is replaced with another base. Method.   2. The DNA chain extending method according to claim 1, wherein the primer comprises a predetermined number of base sequences, and each primer is a DNA fragment having the respective sequences of all combinations.   2. The DNA chain extending method according to claim 1, wherein at least one of the nucleotide monomers is labeled.   2. The DNA chain extending method according to claim 1, wherein the substrate is made of silicon, glass, metal, graphite, or a plastic material.   2. The method for extending a DNA strand according to claim 1, wherein the substrate has a glass slide shape, a microtiter plate shape or a container shape.   An array for DNA chain extension, in which a primer for DNA extension is fixed on the surface of a substrate having a hydrophilic polymer component and a functional group for fixing the DNA chain on the surface.   16. The DNA chain extending array according to claim 15, wherein the hydrophilic polymer and the functional group for immobilizing the DNA chain form a matrix entangled in a mesh shape. .   The DNA chain extending array according to claim 15, wherein the substrate is made of silicon, glass, metal, graphite, or a plastic material.   16. The DNA chain extension microarray according to claim 15, wherein the primer comprises 5 to 50 nucleotide sequences.   16. The DNA chain extension array according to claim 15, wherein the primer is covalently bonded to a functional group for immobilizing the DNA strand on the substrate surface and immobilized on the substrate surface. Array.   16. The DNA chain extending array according to claim 15, wherein the substrate is a substrate, the substrate is provided with a plurality of spots in a fixed section, and a primer is immobilized on each spot. An array for extending a DNA strand.   16. The DNA chain extension array according to claim 15, wherein the substrate is a microtiter plate-like substrate having a plurality of wells, and a primer is immobilized on the well surface.

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