JP2006174788A - Method for elongating dna strand, method for amplifying dna strand, and microarray for elongating dna strand - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To make it possible to leave a single-stranded DNA on a plastic substrate by annealing treatment, after forming double strands by conducting elongation reaction of a DNA strand on a DNA chip. <P>SOLUTION: The method for elongating the DNA strand comprises fixing a primer for DNA elongation to the substrate which has a layer comprising a hydrophilic polymer and a functional group reactive with an amino group on its surface, heating a reaction system into which a sample containing a template DNA fragment having a desired sequence and a nucleotide monomer are introduced up to a temperature at which the DNA strand is thermally denaturated, and cooling the reaction system down to a temperature at which the annealing treatment is conducted, so as to conduct the elongation reaction of the DNA strand in the reaction system. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a DNA chain extension method and a DNA chain amplification method for extending a DNA chain by immobilizing a primer DNA chain on a predetermined plastic substrate surface, and a DNA chain extension microarray used in these methods.

  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 inventors of the present invention performed 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. After bonding, that is, annealing treatment to elongate the DNA strand, heat denaturation treatment found that the primer immobilized on the DNA microarray substrate was dropped together with the template DNA. By using the arranged substrate, this problem was solved and the present invention was completed.

In the DNA chain extension method according to the present invention, a primer for DNA extension is immobilized on a layer comprising a hydrophilic polymer and a substrate having a functional group that reacts with an amino group on the surface,
The reaction system in which the sample containing the template DNA fragment having the desired sequence is introduced is raised to a temperature at which the DNA strand is thermally denatured,
Lowering the temperature of the reaction system to the annealing temperature,
It is characterized in that a DNA chain elongation reaction is performed in the reaction system.

  As described above, when a primer is immobilized on a predetermined substrate, a template DNA fragment is annealed to this primer, and a DNA chain extension reaction is performed, it becomes double-stranded on the substrate by heat denaturation treatment after the reaction. The template DNA fragment is removed from the existing DNA, and single-stranded DNA extended from the primer remains on the substrate.

In the above DNA chain extension 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.

  This makes it possible to apply the DNA chain elongation method of the present invention to single nucleotide polymorphism (SNP) analysis.

  In the DNA chain extension method, the primer may be composed of a predetermined number of base sequences, and each primer may be a DNA fragment having each sequence in all combinations.

  Thereby, the DNA chain elongation 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 a 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.

  In addition, the DNA strand amplification method according to the present invention comprises a template having a desired sequence by immobilizing a primer for DNA amplification on a substrate comprising a hydrophilic polymer layer and a functional group that reacts with an amino group on the surface. The reaction system in which the sample containing the DNA fragment is introduced is raised to a temperature at which the DNA strand is thermally denatured, the temperature of the reaction system is lowered to a temperature at which the annealing treatment is performed, and a DNA strand elongation reaction is performed in the reaction system. And a DNA strand annealing treatment, an extension reaction, and a heat denaturation treatment as necessary.

  As described above, when a primer is immobilized on a predetermined substrate, a template DNA fragment is annealed to this primer, and a DNA chain extension reaction is performed, it becomes double-stranded on the substrate by heat denaturation treatment after the reaction. The template DNA fragment is removed from the existing DNA, and the single-stranded DNA extended from the primer remains on the substrate. The template DNA fragment forms a double strand with the unreacted primer in the next annealing treatment, and the next DNA strand elongation is performed. By repeating this, a DNA strand amplification reaction by the MPEC method can be performed.

  The DNA chain extension microarray according to the present invention includes a layer made of a hydrophilic polymer, a substrate having a functional group that reacts with an amino group on the surface, and a primer for DNA amplification immobilized on the surface of the substrate. Is included.

  As described above, when a primer is immobilized on a predetermined substrate, a template DNA fragment is annealed to this primer, and a DNA chain extension reaction is performed, it becomes double-stranded on the substrate by heat denaturation treatment after the reaction. The template DNA fragment is removed from the existing DNA, and single-stranded DNA extended from the primer remains on the substrate.

  According to the present invention, it becomes possible to leave single-stranded DNA on the substrate of the DNA microarray by heat denaturation treatment after carrying out DNA strand extension reaction on the DNA microarray to make it into double strand.

  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 amplification (hereinafter referred to as “primer”) is immobilized on a layer comprising a hydrophilic polymer and a substrate having a functional group that reacts with an amino group on the surface (step S10). Then, the reaction system in which the sample containing the template DNA fragment having the desired sequence and the nucleotide monomer is introduced is raised to a temperature at which the DNA strand is thermally denatured (step S30), and the temperature of this reaction system is lowered to the temperature for annealing treatment ( Step S40), a DNA strand extension reaction is performed in the reaction system (Step S50), a DNA strand is thermally denatured (Step S60), and a DNA strand annealing treatment, an extension reaction and a heat denaturation treatment are performed as necessary. (Repeating step S40 to step S60).
In step S10, as shown in FIG. 2, the DNA amplification primer 14 is fixed to the surface of the substrate 12.

The substrate used here has a layer made of a hydrophilic polymer and a functional group that reacts with an amino group on the surface. This makes it possible to have both the property of suppressing nonspecific adsorption of DNA strands and the property of immobilizing DNA strands.
A layer made of a hydrophilic polymer mainly serves to suppress nonspecific adsorption of DNA strands, and a functional group that reacts with an amino group serves to chemically immobilize a primer. In particular, the primer is immobilized on the surface of the substrate by covalent bonding at the site of a functional group that reacts with an amino group.

  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. Particularly preferred is a polyacrylamide gel having a network structure in which polyacrylamide is three-dimensionally crosslinked with a photocrosslinkable compound or the like.

Examples of the functional group that reacts with the amino group used in the present invention 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.
As the active ester group, for example, p-nitrophenyl ester group, N-hydroxysuccinimide ester group, succinimide ester group, phthalimide ester group, 5-norbornene-2,3-dicarboximide ester group and the like are preferable. , P-nitrophenyl ester group or N-hydroxysuccinimide ester group is more preferable.
The functional group that reacts with the amino group may be introduced directly to the substrate surface, or may be included in the main chain or side chain of the hydrophilic polymer structure.

Next, a method for immobilizing the primer on the surface of the substrate will be described.
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 substrate surface, and then (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 groups. 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. Do.

  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 functional group that reacts with the amino group. Since the amino group is excellent in reactivity with the functional group that reacts with the amino group, the primer can be efficiently and firmly immobilized on the surface of the substrate by using the primer into which the amino group is 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, 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 DNA amplification 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 sample containing nucleotide monomers are introduced. .

  The reaction system comprising the introduced sample contains nucleotide monomers (dATP, dCTP, dGTP, dTTP, etc.) in the presence of Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, etc., which are DNA polymerases derived from thermostable bacteria. An MPEC buffer is provided.

  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.

  In addition, 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, for example, 5 to 50 bases.

(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 layer comprising a hydrophilic polymer and a substrate having a functional group that reacts with an amino group on the surface (step S10). Then, the reaction system in which the sample containing the template DNA fragment having the desired sequence and the nucleotide monomer is introduced is raised to a temperature at which the DNA strand is thermally denatured (step S30), and the temperature of this reaction system is lowered to the temperature for annealing treatment ( In step S40), a DNA chain elongation reaction is performed in this reaction system (step S50).

  As described above, in step S20, a sample containing a template DNA fragment for DNA extension to be annealed to a primer immobilized on the surface of the substrate is introduced, and the sample and the reaction system into which this sample is introduced are described above. The same ones 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 target gene 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.

(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, 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 adjusting 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 time, a fluorescence reader (for example, CRBIO® 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 by organ / disease using these tools, mutagenicity test kit (environmental hormone), genetically modified food test kit, mitochondrial gene sequence analysis kit, analysis kit for paternity / 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 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 commercially available slide glass substrate prepared by dissolving γ-aminopropyltriethoxysilane as an aminoalkylsilane in methanol at a concentration of 5% was prepared as an amino group-introducing treatment solution, and immersed in this solution for 2 hours. Thereafter, the substrate was taken out of the solution, immersed in ultrapure water, allowed to stand, and then 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 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 in step S30, the annealing process in step S40, and the extension reaction in step S50 in FIG. 6 are performed at 95 ° C. for 5 minutes (denaturation) —95 ° C. for 1 minute (denaturation) —50 ° C. for 3 minutes (annealing) — 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 (modified)
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.

(Experimental example 3)
As shown in FIG. 9, 5 types of probes (21 mer) were designed for the purpose of SNP typing using the 21st base C from the 5 'position of 5'-Cy5-AAGGCGGGAGGGAGCGCAATCCGGGAGTTTACAAATGGACAAACTTCTAT-3' as the mutation site. And immobilized on a glass substrate. Using this microarray, SNP typing (extension reaction) by MPEC (multiple primer extension on a chip) reaction is performed.

This SNP typing is performed at 95 ° C. (1 mM) in the presence of DNA polymerase (0.05 U / ml), Cy3-dUTP (0.05 mM), dATP, dCTP, dGTP (each 0.2 mM) in 50 μl of PCR buffer. Min) with a temperature cycle of −50 ° C. (3 min).
As a result of the extension reaction, only the perfect match sequence probe having a sequence complementary to the target is extended using the 50-mer DNA fragment as a target, and as a result, fluorescence is developed.

(Experimental example 4)
The MPEC reaction of Experimental Example 1 is applied to the detection of p53 tumor suppressor gene.
This p53 gene abnormality is frequently required in colorectal cancer and lung cancer, and if there is an abnormality (single nucleotide polymorphism: SNP) in the p53 gene, the anticancer agent becomes less effective and cell death of cancer cells Cancer cells will proliferate without causing (apoptosis).

  Therefore, SNP typing (extension reaction) using normal p53 gene and cancer cell p53 gene is performed. It is known that there are mutations at the 993th and 1069th positions of the p53 gene, and the probe was designed so that the base at this position was the 3 'end and the Tm value was almost constant, and immobilized on the substrate. A total of 16 probes 5'-ATGGGCGGCATGAACN-3 ', 5'-TGAGGATGGGCCTCCN-3', 5'-GGAACAGCTTTGAGGTGCN-3 ', 5'-CCAGGACAGGCACAAACAN-3' (N = A, G, C, T) Used to perform SNP typing by MPEC reaction.

(Experimental example 5)
A simulation experiment of base sequence determination analysis by the SBH method is performed.
As shown in FIG. 10, an 8-base probe (total of 8 mer) shifted toward the 3 ′ end by one base so that the base sequence of a 50-base target whose 3 ′ end is labeled with Cy5 can be determined. SBH by MPEC reaction is performed using a microarray in which 50) is fixed on a glass substrate. 50 probes of 8 bases (8mer) in which a mismatch was set at one base at the 3 ′ end of each probe were also added and immobilized on the substrate. The base sequence is determined using this SBH simulation array.

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. It is a figure which shows another example of application of said 2nd embodiment. It is a figure which shows another example of application of said 2nd embodiment.

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 (16)

A primer containing DNA template and nucleotide monomer having a desired sequence was introduced by immobilizing a primer for DNA extension on a layer composed of a hydrophilic polymer and a substrate having a functional group that reacts with an amino group on the surface. A DNA chain extension method comprising raising a reaction system to a temperature at which a DNA strand is thermally denatured, lowering the temperature of the reaction system to a temperature for annealing, and performing a DNA chain extension reaction in the reaction system.   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 in which one base of a base sequence including a characteristic sequence of a predetermined gene of interest is replaced with another base. .   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 extension method according to claim 1, wherein the template DNA fragment is a cDNA fragment obtained by treating predetermined RNA with a reverse transcriptase.   2. The DNA chain extension method according to claim 1, wherein at least one nucleotide monomer contained in the sample introduced into the reaction system is labeled.   The DNA chain extension method according to claim 1, wherein the primer is covalently bonded to a functional group that reacts with an amino group on the substrate surface and immobilized on the surface of the substrate. Method.   9. The DNA chain extending method according to claim 1 or 8, wherein the functional group that reacts with an amino group is an active ester group.   2. The DNA chain extending method according to claim 1, wherein the substrate is made of silicon, glass, metal, graphite, or a plastic material.   A reaction system in which a sample containing a template DNA fragment having a desired sequence is introduced by immobilizing a primer for DNA amplification on a substrate comprising a hydrophilic polymer layer and a functional group that reacts with an amino group on the surface. The temperature of the DNA strand is raised to a temperature for heat denaturation, the temperature of the reaction system is lowered to a temperature for annealing treatment, an elongation reaction of the DNA strand is performed in the reaction system, a heat denaturation treatment of the DNA strand is performed, and if necessary And a DNA strand amplification method comprising performing annealing treatment, elongation reaction and heat denaturation treatment of the DNA strand.   A microarray for extending a DNA chain, comprising: a layer comprising a hydrophilic polymer; a substrate having a functional group that reacts with an amino group on the surface; and a primer for DNA amplification immobilized on the surface of the substrate.   13. The DNA chain extension microarray according to claim 12, wherein the substrate is made of silicon, glass, metal, graphite, or a plastic material.   The microarray for DNA chain extension according to claim 12, wherein the primer comprises 5 to 50 nucleotide sequences.   13. The DNA strand extension microarray according to claim 12, wherein the primer is covalently bonded to a functional group that reacts with an amino group on the surface of the substrate and immobilized on the surface of the substrate. Microarray for extension.   13. The DNA chain extension microarray according to claim 12, wherein the substrate is provided with a plurality of spots in a fixed section, and a primer is fixed to each spot. Microarray.

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