JP2004069488A - MANUFACTURING METHOD FOR cDNA ARRAY - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing a cDNA array for improving the problems in the conventional method by immobilizing double-stranded DNAs, having only a desired sequence in the double-stranded DNAs amplified by a PCR method on a substrate. <P>SOLUTION: When a cDNA array is created on a carrier, the cDNA array is manufactured by a process for preparing at least two kinds of one-stranded cDNAs that have a known sequence and introduce a functional group for immobilization to the carrier to one end section (1), and a process for combining each one-stranded cDNA via a functional group for combination so that each of at least two types of one-stranded cDNAs is arranged; while each of an immobilization region for each one-stranded cDNA is separately arranged one another to manufacture the cDNA array, where the immobilization region is arranged in a given sequence (2). <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing a cDNA array useful for detecting and identifying a desired nucleotide sequence of a nucleic acid (DNA or RNA) of a virus, microorganism, animal, plant, human, or the like. In particular, the present invention relates to a method for preparing a cDNA array used for comparing the expression level at each stage or comparing the expression level between tissues.
[0002]
[Prior art]
A cDNA array in which cDNAs are arranged in an array has been used in many research applications since it was developed at Stanford University in 1995 (Science, 270, p467, (1995)). In this method, cDNA is synthesized from extracted mRNA, amplified by PCR, and then supplied to a substrate by micropipetting and immobilized to produce a cDNA array. The cDNA to be immobilized on the substrate is selected from a stock DNA solution called a cDNA library in which various cDNAs are covered, and is supplied onto the substrate. In the preparation of this library, amplification is performed by PCR on the basis of mRNA extracted from tissues or the like, and a part thereof is used.
[0003]
In the PCR method, usually two kinds of primers are designed, they bind to a template DNA, and the synthesis of the DNA sequence is repeatedly performed enzymatically starting from the site. Therefore, the synthesized DNA is a double-stranded DNA having these primer sequences at the ends, and is supplied to the substrate, and in a DNA array in which the DNA is bonded to the substrate surface by ionic bonding or UV link, the double-stranded DNA is used. Both DNAs in the strand will bind in the same place. In addition, in order for these bonds to be denatured into a single strand by denaturing a double strand on the substrate, a base moiety involved in the hybridization reaction is used for the binding.
[0004]
In such a conventional method, these cDNAs are supplied on a substrate by a micropipette, or a DNA solution is attached to a tip of a needle, called a pin method, and supplied on a glass substrate by stamping. Is done. At this time, the drop amount of the micropipette is limited to nanoliter.
[0005]
On the other hand, the ejection amount of the ink by the inkjet method is on the order of picoliter. In an ink-jet printer, it is also possible to mix inks of such a discharge amount by overprinting with accurate positioning.
[0006]
[Problems to be solved by the invention]
In conventional cDNA arrays, both strands contribute to the hybridization reaction because double-stranded DNA is immobilized on the same spot. As a result, not only the sequence to be evaluated, but also the hybrid formed with the complementary strand thereof has a fluorescence amount, so that there has been a problem that accurate evaluation may not be performed. In the case of cDNA, since a region forming a hybrid is long, there are hybrids due to partial binding to other genes, and a hybrid containing a mismatch that is not a perfect match becomes noise. In addition, the presence of the complementary strand at the same location doubles the noise. Therefore, the application to the case where such noise becomes a problem has been limited. Therefore, in order to quantitatively evaluate the expression level, a method of selectively fixing only one cDNA to be evaluated has been desired.
[0007]
Further, in order to ensure more precise quantification, it is considered that a method in which a nucleobase portion that affects hybridization is immobilized is not preferable. For that purpose, it is desired to fix cDNA at the end of a nucleobase that does not participate in hybridization.
[0008]
Further, in the micropipetting method, the sample amount of the DNA probe that can be dropped on the substrate as a reaction solution is limited to 5 nl. The spot diameter is 100-200 μm, and considering the accuracy of pipetting, it is difficult to arrange more than 10,000 kinds of probes in one inch square, and it is important to evaluate a large number of probes simultaneously. Therefore, there has been a demand for a method for immobilizing probes at a higher density.
[0009]
In view of the above problems, an object of the present invention is to provide a cDNA array that can achieve the above object by immobilizing only those having a desired sequence on a substrate among double-stranded DNAs amplified by a PCR method. It is to provide a method that can be manufactured. Another object of the present invention is to supply and fix a cDNA solution onto a substrate by an inkjet method in order to produce a high-density cDNA array.
[0010]
[Means for Solving the Problems]
The method for producing a cDNA array of the present invention that can achieve the above object is a method for producing a cDNA array on a carrier,
(1) a step of preparing two or more single-stranded cDNAs having a known sequence and having, at one end, a functional group for immobilization to the carrier,
(2) Each of the two or more single-stranded cDNAs is connected to each other via a functional group for binding such that the immobilized regions of the single-stranded cDNAs are separated from each other. Producing a cDNA array in which the immobilized region is arranged in a predetermined sequence,
This is a method for producing a cDNA array, characterized by having:
[0011]
The present invention also provides a method for producing a cDNA array on a carrier,
(1) Two or more types of double-stranded cDNA amplified by a PCR reaction using a set of a primer having a known sequence and having a functional group for immobilization to the carrier and a primer having no functional group Preparing a;
(2) supplying a solution of each of the two or more double-stranded cDNAs to a carrier such that the immobilized regions of each cDNA are arranged separately from each other;
(3) a step in which, of the double-stranded cDNA, only a single-stranded cDNA having a functional group introduced therein is immobilized on the carrier by a chemical bond via a binding functional group;
(4) a step of immersing the carrier in hot water and performing thermal denaturation to remove a complementary strand not bound to the carrier to obtain a single-stranded probe DNA;
And a method for preparing a cDNA array in which the immobilized region is arranged in a predetermined sequence.
[0012]
In the cDNA array obtained by the production method of the present invention, the cDNA immobilized in each immobilized region is in a single-stranded state, and the functional group for binding to the carrier introduced at the end of the sequence is used. And a carrier. With this bonding form, it is possible to solve the above-described problems in the case of binding to a carrier in a double-stranded state and the problem of using a part of the sequence for binding to the carrier.
[0013]
In the present invention, it is not necessary to previously separate the double-stranded DNA obtained by the PCR reaction into single-stranded DNA. By binding a functional group capable of binding to the carrier to one of the primers, only those having the functional group are immobilized on the substrate from the cDNA solution supplied on the carrier in a double-stranded state. Because. Such a method can be suitably used, for example, when a functional group such as an amino group is introduced into a terminal. In this case, the amino group contained in the base moiety is blocked by formation of a double strand with a complementary strand, and is not provided for binding to a carrier. Therefore, only the amino group at the terminal portion is selectively used for binding to the carrier, and then the complementary strand is separated by heat denaturation, and the single-stranded cDNA is easily bound at the end by single-stranded cDNA array. Can be formed.
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, as the cDNA immobilized on the immobilized region of the carrier, for example, a single-stranded polynucleotide having a function as a probe capable of recognizing a target DNA having a specific sequence to be detected is preferably used. it can.
[0015]
Probes can be specifically recognized by a specific target (target), and are often called ligands. Further, the probe includes an oligonucleotide, a polynucleotide, or another polymer that can be recognized by a specific target. The term `` probe '' refers to a molecule having a probe function such as an individual polynucleotide molecule, and a molecule having the same probe function such as a polynucleotide having the same sequence immobilized on a carrier surface in a dispersed state or the like. It may mean a group. Also, a probe is one that can bind to or become capable of binding to a target as part of a ligand-antiligand. Probes and targets can include bases as found in nature, or analogs thereof.
[0016]
A plurality of these probes, each of which is fixed on the carrier surface as an independent immobilized region, for example, a dot spot, is called a probe carrier, and those arranged at predetermined intervals are called a probe array. In the present invention, cDNA is used as a probe, and those arranged at high density correspond to a microarray.
[0017]
In the present invention, the functional group that can be used for binding the single-stranded cDNA to the carrier is preferably one that can bind to the carrier surface by a chemical reaction. Examples of such a functional group include an amino group, a thiol group, a carboxyl group, a hydroxyl group, an acid halide (haloformyl group; -COX), a halide (-X), an aziridine, a maleimide group, a succinimide group, and an isothiocyanate group. , A sulfonyl chloride group (—SO ₂ Cl), an aldehyde group (formyl group; —CHO), hydrazine, iodine acetamide, and the like, are preferably formed by introducing at least one organic functional group. These functional groups may be linked to the end of the cDNA via a spacer molecule of an appropriate length, such as an alkylene chain.
[0018]
Further, the surface of the carrier may be subjected to necessary treatment depending on the structure required for binding the probe to the carrier.
[0019]
On the other hand, the probe has a structure capable of binding to the surface of the carrier, and it is desirable that the probe be fixed on the carrier via the structure capable of binding. At this time, the structure of the probe that can be bonded to the carrier surface includes an amino group, a mercapto group, a carboxyl group, a hydroxyl group, an acid halide (haloformyl group; -COX), a halide (-X), an aziridine, a maleimide group, and a succinimide. It is preferably formed by a treatment for introducing an organic functional group such as a group, isothiocyanate, sulfonyl chloride (—SO ₂ Cl) group, aldehyde (formyl group; —CHO) group, hydrazine, and acetamide iodide. Further, depending on the type of the functional group for binding to the carrier on the probe side, the surface of the carrier may be subjected to necessary treatment.
[0020]
Preferred combinations of such a single-stranded cDNA side functional group and carrier surface treatment include:
(1) a combination in which a thiol group (-SH) is introduced into the single-stranded cDNA side, and the thiol group is reacted with a maleimide group introduced on the surface of the carrier to perform covalent immobilization;
(2) a combination in which an amino group is introduced into the single-stranded cDNA and reacted with an epoxy group introduced into the surface of the carrier to fix the amino acid by a covalent bond; and (3) an amino group is introduced into the single-stranded cDNA. And then reacting it with an aldehyde group introduced on the surface of the carrier to perform covalent immobilization, and (4) introducing an amino group on the single-stranded cDNA side and introducing it on the surface of the carrier. A combination of reacting with the N-hydroxysuccinimide group thus immobilized by a covalent bond. As the carrier in each of the embodiments (1) to (4), a glass substrate can be suitably used from the viewpoints of securing availability and easiness of surface treatment. When a carrier having a surface made of a material that can be surface-treated with a silane coupling agent such as glass is used as the carrier, the carrier surface is reacted with the silane coupling agent, and then the EMCS (N- (6- A method in which a maleimide group is introduced using a maleimidocaprooxy / succinimide) reagent can be suitably used.
[0021]
As the carrier, those having a surface capable of binding to a functional group for binding to a modified carrier at the end of cDNA can be used, and a surface for immobilization is formed from glass, plastic, metal, etc. Shaped materials can be suitably used. Further, the carrier may be entirely composed of a material forming a surface for immobilization. When gold is used as the metal, bonding to a thiol group as a functional group of cDNA enables direct bonding to gold.
[0022]
Various spotting and dot formation methods can be used to supply the single-stranded cDNA to the carrier. Among them, a method of discharging a discharge liquid containing the single-stranded cDNA to a predetermined position on the carrier using an inkjet method is used. preferable. In this case, the length of the single-stranded cDNA that can be suitably applied is preferably a length required for a desired analysis and not more than 5000 bases. The lower limit preferably has a minimum length that can be synthesized by the PCR method.
[0023]
The concentration of the single-stranded cDNA in the ejection liquid is preferably 500 μM or less, more preferably 5 to 10 μM. Those having a concentration range and a base length give a good sequence for forming an array by an ink-jet method, and a higher-density array can be produced.
[0024]
When a single-stranded cDNA having a length exceeding 5000 bases is used, a cDNA array can be prepared by using a pin method or a micropipette method other than the inkjet method.
[0025]
However, at present, the surface treatment of the glass plate is relatively easy, so the glass plate will be described as an example. One of them is a binding reaction between a maleimide group on the glass surface and an SH group at the end of DNA.
[0026]
The liquid medium constituting the ejection liquid used when the single-stranded cDNA is applied to the carrier by the ink-jet method has an effect on the single-stranded cDNA when mixed with the single-stranded cDNA and when ejected. What is necessary is just to provide a discharge liquid which is not substantially given and which can discharge normally. As such a liquid medium, water can be mentioned as a preferable one, and if necessary, at least one of additives such as glycerin, urea, thiodiglycol or ethylene glycol, isopropyl alcohol and acetylene alcohol is added. Can be used. The concentration of these additives is preferably, for example, in the range of 0.01% by weight (wt) to 40% by weight. As a preferred liquid composition for preparing a liquid for ejection, 5 to 10% by mass of urea, 5 to 10% by mass of glycerin, 5 to 10% by mass of thiodiglycol, and acetylene alcohol in water as a liquid medium. A liquid composition containing 0.02 to 5% by mass, more preferably 0.5 to 1% by mass, is suitably used.
[0027]
In consideration of the suitability for ejection by the ink jet method, the ejected liquid preferably has physical properties such that the viscosity is 1 to 15 cps, the surface tension is 30 dyn / cm or more, and the ink satisfies the suitability for inkjet. When the viscosity is 1 to 5 cps and the surface tension is 30 to 50 dyn / cm, the impact position on the solid phase becomes appropriate, and it can be used particularly preferably.
[0028]
Furthermore, when the single-stranded cDNA has a thiol group at one end, it is preferable that the ejection liquid contains a thiol group protecting agent. In this case, the concentration of the thiol group protecting agent may be within the range of the liquid characteristics that allow the resulting solution to be ejected by the inkjet method since the solution obtained by dissolving the probe is ejected by the inkjet method. For example, when a probe solution is ejected from a bubble jet head, it is desirable to add a thiol-based protecting agent so as to satisfy the above-mentioned viscosity range and surface tension value as characteristics of the liquid.
[0029]
Components other than the thiol group protecting agent in the case of using the thiol group protecting agent are not particularly limited, but examples of the liquid composition that satisfies the properties as the ejection solution for bubble jet include glycerin, urea, and glycerin described above. At least one of thiodiglycol, ethylene glycol and acetylene alcohol can be used as an additive.
[0030]
As the thiol group protecting agent, dithiothreitol (DTT), dithioerythritol and 2-mercaptoethanol (β-mercaptoethanol) are preferable. The concentration of DTT may be 10 μM to 10 mM, and the concentration of β-mercaptoethanol may be 100 μM to 50 mM.
[0031]
More specifically, from the viewpoint of the concentration of the single-stranded cDNA contained in the liquid for ejection, the concentration of the thiol-group protecting agent is set as a thiol-group protecting agent when the probe concentration is 100 μmol / L or less. When dithiothreitol is used, the concentration of the protective agent is preferably in the range of 0.1 mmol / L to 10 mmol / L, and more preferably in the range of 0.1 mmol / L to 2 mmol / L. When 2-mercaptoethanol is used as a protective agent at the same concentration of single-stranded cDNA in the same ejection liquid, the concentration of the thiol-based protective agent is preferably in the range of 1 mmol / L to 50 mmol / L, and further, Is preferably in the range of 1 mmol / L to 20 mmol / L.
[0032]
On the other hand, the introduction of the SH group into the single-stranded cDNA is performed by synthesizing a primer using 5′-Thiol-Modifier C6 (manufactured by Glen Research) on an automatic DNA synthesizer, and performing PCR using an unmodified primer set. Perform the reaction to obtain cDNA.
[0033]
When an aldehyde group, an epoxy group, or a succinimide group is introduced toward the surface of the carrier, cDNA having an amino group at the end participates in the binding. As a primer for this, as in the case of the SH group, 5′- Automatic synthesis can be performed using Amino-Modifier C6 (manufactured by Glen Research).
[0034]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples.
Example 1 cDNA array of p53 gene 1) Preparation of cDNA array (glass substrate treatment)
As a glass substrate for preparing cDNA, a substrate having a maleimide group on the surface was prepared according to the method described in JP-A-11-187900.
(Preparation of cDNA having terminal thiol group)
Each exon of the p53 gene of human genomic DNA (Cosmo Bio Inc.) was amplified by the PCR method, and PCR products of 269, 181, 171 and 229 bp were obtained for exons 5, 6, 7 and 8, respectively.
[0035]
The nucleotide sequences of the primers for each exon used in the PCR reaction are as follows. A thiol group, which is a functional group for binding to a substrate, is bound to the 5 'end of each antisense side.
E5S: 5'-TGTTCACTTGTGCCCTGAACT-3 '(exon 5-sense) (SEQ ID NO: 1);
E5A: 5'-SH-TGAGGAATCAGAGGCCTGG-3 '(exon 5-antisense) (SEQ ID NO: 2);
E6S: 5'-GCCTCTGATCTCCTACTGAT-3 '(exon 6-sense) (SEQ ID NO: 3);
E6A: 5'-SH-TTAACCCTCCTCCCCAGAGA-3 '(exon 6-antisense) (SEQ ID NO: 4);
E7S: 5'-ACTGGCCTCATCTTGGGCCT-3 '(exon 7-sense) (SEQ ID NO: 5);
E7A: 5'-SH-TGTGCAGGGTGGCAAGTGGC-3 '(exon 7-antisense) (SEQ ID NO: 6);
E8S: 5'-TAAATGGGACAGGTAGGACC-3 '(exon 8-sense) (SEQ ID NO: 7);
E8A: 5'-SH-TCCACCGCTTCTTGTCCTGC-3 '(exon 8-antisense) (SEQ ID NO: 8).
[0036]
For the reaction, a cycle of 94 ° C. (30 seconds) to 60 ° C. (45 seconds) was performed 40 times using a PCR reaction solution (50 μl) containing 10 to 25 ng of genomic DNA and 0.4 μM of each primer.
[0037]
As a result of examining the amplified product of the PCR reaction by gel electrophoresis, in addition to a DNA band of an expected length, a slight band was observed at the position of the dimer.
(Printing cDNA by inkjet method)
According to the method described in JP-A-11-187900, the cDNA solution of each of the four types of exons was filled in place of ink and printed. Thereafter, the reaction between the thiol and the maleimide group was completed by leaving the mixture at room temperature for one hour, and the substrate was immersed in warm water at 80 ° C. to remove unreacted cDNA. By this step, the cDNA on the sense side without a thiol group could not be bound to the substrate and was washed away, and only the cDNA on the antisense side bound on the substrate at the 5 'end.
2) cDNA array evaluation (adjustment of cDNA sample)
Rhodamine-labeled single-stranded DNA was obtained by PCR based on the exon 7 gene obtained by the above reaction. That is, a PCR reaction was performed using a primer on the sense side of exon 7, and during the reaction, deoxy UTP (FluoroRed: Amersham Pharmacia Biotech) labeled with tetramethylrhodamine was introduced. In the PCR reaction, a cycle of 96 ° C, 30 seconds to 50 ° C, 30 seconds to 60 ° C, and 4 minutes was performed 25 times. Thereafter, unreacted dye was removed by gel filtration to obtain single-stranded DNA of exon 7 labeled with tetramethylrhodamine.
(Blocking reaction)
After blocking the cDNA array with 2% bovine serum albumin, a hybridization reaction was performed as follows.
(Hybridization reaction)
The cDNA array was immersed in a labeled single-stranded DNA solution (containing a final concentration of 100 mM NaCl) obtained by the PCR reaction, and left at 65 ° C. for 2 hours. Thereafter, the substrate was lightly washed and observed with a microscope. Fluorescence was detected only in the exon 7 cDNA portion.
[0038]
Example 2
(DTT added during PCR reaction)
In the preparation of cDNA having a thiol group at the end in Example 1, DTT (dithiothreitol) was added to the PCR reaction solution to a concentration of 1 mM to carry out the reaction. After completion of the reaction, the PCR reaction product was confirmed by gel electrophoresis. As a result, the dimer band observed in Example 1 had completely disappeared. In addition, as a result of the hybridization reaction using this sample, the fluorescence intensity of the exon 7 part was increased by 20% or more as compared with Example 1.
Example 3
(Detection of mismatch)
A cDNA array was prepared in the same manner as in Example 1. A blocking reaction was performed on the CDNA array in the same manner as in Example 1, and then a hybridization reaction was performed with the following samples.
1) Preparation of labeled target DNA A labeled DNA having a normal sequence of the p53 gene and a length of 18 bases was prepared. The sequence is as shown below, with rhodamine bound to the 5 'end.
[0039]
No. ^{15 '} Cy3-ATGAACCGGAGGCCCATC ^3' (SEQ ID NO: 9)
In addition, a labeled DNA having a sequence that differs from the above sequence by one base was synthesized.
[0040]
No. ^{25 '} Cy5-ATGAACC A GAGGCCCATC ^3' (SEQ ID NO: 10)
These two kinds of labeled oligonucleotides were mixed and used for a hybridization reaction.
2) Hybridization reaction The above-mentioned labeled oligonucleotide solution (containing 100 mM NaCl) was put in a bag containing a DNA array substrate, heated at 70 ° C for 30 minutes first, then the temperature of the incubator was lowered to 40 ° C, and the reaction was allowed to proceed overnight. .
3) Detection The fluorescence of each spot was observed using a fluorescence microscope. Cy3 fluorescence was observed in the exon 7 region. Only one sequence was found to form a hybrid. It is considered that a single base mismatch has been discriminated. No fluorescence was observed at the other exon cDNA spots.
Example 4 and Comparative Example 1) Preparation of cDNA Array A commercially available polylysine-coated slide glass was subjected to PCR using the same primers as in Example 1 to obtain a cDNA solution. However, this primer is a commonly used primer and does not have a thiol group at the end.
[0041]
Each spot corresponding to four types of exons was obtained by the inkjet method in the same manner as in Example 1, and treated at 70 to 80 ° C. for 1 hour. Thereafter, 120 kJ of UV was applied to immobilize the cDNA. The slide glass is immersed in a succinic acid solution (70 mM succinic anhydride, 0.1 M sodium borate (pH 8.0), 1-methyl-2-pyrrolidinone) for 10 to 20 minutes with occasional shaking. It was immersed in boiling pure water for 2 minutes, dehydrated with ethanol, and dried at room temperature.
2) Hybridization reaction A hybridization reaction was performed using two types of labeled oligonucleotides as in Example 3. The conditions are the same as in the third embodiment.
3) Detection The fluorescence of each spot was observed using a fluorescence microscope. The strongest fluorescence of Cy3 was observed at exon 7, but the intensity was about half the amount of fluorescence obtained in Example 3. In exon 7, fluorescence of Cy5 as well as fluorescence of Cy3 was observed to the same extent, indicating that mismatches were not distinguished. In addition, the fluorescence of Cy3 and Cy5 was observed also in the spots of other exons, and the contrast between the spots was generally poor and the S / N ratio was poor.
[0042]
【The invention's effect】
By producing a single-stranded cDNA array in which only the desired strands were immobilized among the double-stranded DNAs constituting the cDNA, a higher fluorescence amount could be obtained as compared with a conventional double-stranded DNA having immobilized double strands. . In addition, the background due to adsorption to other spots was reduced, and a hybridization result with a high S / N ratio could be obtained. Furthermore, since only the ends are fixed, it has become possible to detect a single base mismatch, which was difficult with the conventional method.
[0043]
[Sequence list]

Claims (24)

A method for producing a cDNA array on a carrier, comprising:
(1) a step of preparing two or more single-stranded cDNAs having a known sequence and having, at one end, a functional group for immobilization to the carrier,
(2) Each of the two or more single-stranded cDNAs is connected to each other via a functional group for binding such that the immobilized region of each single-stranded cDNA is arranged separately from each other. Producing a cDNA array in which the immobilized region is arranged in a predetermined sequence,
A method for producing a cDNA array, comprising: The single-stranded cDNA is prepared by a PCR reaction using a set of a primer having the functional group bonded to the end and a primer having no functional group, so that the single-stranded cDNA has the functional group only at one end. The production method according to claim 1. The method according to any one of claims 1 to 3, wherein the single-stranded cDNA is immobilized on the carrier by a chemical reaction between the functional group and the surface of the carrier. A method for producing a cDNA array on a carrier, comprising:
(1) Two or more types of double-stranded cDNA amplified by a PCR reaction using a set of a primer having a known sequence and having a functional group for immobilization to the carrier and a primer having no functional group Preparing a;
(2) supplying a solution of each of the two or more double-stranded cDNAs to a carrier such that the immobilized regions of each cDNA are arranged separately from each other;
(3) a step in which, of the double-stranded cDNA, only a single-stranded cDNA having a functional group introduced therein is immobilized on the carrier by a chemical bond via a binding functional group;
(4) a step of immersing the carrier in hot water and performing thermal denaturation to remove a complementary strand not bound to the carrier to obtain a single-stranded probe DNA;
A method for producing a cDNA array in which said immobilized regions are arranged in a predetermined sequence. The method according to claim 4, wherein the functional group is a thiol group. The production method according to claim 5, wherein the solution for the PCR reaction contains the thiol group protecting agent. The method according to claim 6, wherein the thiol group protecting agent is DTT (dithiothreitol). The method according to claim 7, wherein the concentration of the DTT is 10 µM to 10 mM. The method according to claim 6, wherein the protective agent is β-mercaptoethanol. The method according to claim 9, wherein the concentration of β-mercaptoethanol is 100 μM to 50 mM. The carrier has a surface having a maleimide group, and the single-stranded cDNA is modified at its 5 ′ end with a thiol group, and the single-stranded cDNA is converted by the reaction between the maleimide group and the thiol group. The method according to any one of claims 5 to 10, which is immobilized on a carrier by a covalent bond. The method according to claim 11, wherein the carrier is a glass substrate. The method according to claim 11, wherein the glass surface has a maleimide group introduced by an EMCS (N- (6-maleimidocaproyloxy) succinimide) reagent after a reaction with a silane coupling agent. The method according to claim 1, wherein the carrier is a metal. The method according to claim 1, wherein the carrier is gold, and the functional group is a thiol group. The method according to claim 1, wherein the functional group is an amino group. The carrier has a surface having an epoxy group, and the single-stranded cDNA is modified at its 5 ′ end with an amino group, and the single-stranded cDNA is converted by the reaction between the epoxy group and the amino group. 17. The method according to claim 16, wherein the method is covalently immobilized on a carrier. The carrier has a surface having an aldehyde group, and the single-stranded cDNA is modified at its 5 ′ end with an amino group, and the reaction between the aldehyde group and the amino group converts the single-stranded cDNA into a single-stranded cDNA. 17. The method according to claim 16, wherein the method is covalently immobilized on a carrier. The carrier has a surface having an N-hydroxysuccinimide group, and the single-stranded cDNA is modified at its 5 ′ end with an amino group, and is reacted by the N-hydroxysuccinimide group with the amino group. 17. The method according to claim 16, wherein the single-stranded cDNA is immobilized on the carrier by a covalent bond. 20. The method according to claim 1, wherein the supply of the single-stranded cDNA to the carrier is performed by applying a discharge liquid containing the single-stranded cDNA to a predetermined position of the carrier by an inkjet method. Production method. 21. The method according to claim 20, wherein the ink jet method is based on a bubble jet method. 22. The method according to claim 20, wherein the concentration of the single-stranded cDNA in the ejection liquid is 300 μM or less. The production method according to claim 22, wherein the concentration of the single-stranded cDNA in the ejection liquid is 5 to 10 µM. The method according to any one of claims 1 to 23, wherein the length of the single-stranded cDNA is 5000 bases or less.