JP2001330608A - Method for producing nucleic acid chain-immobilized carrier - Google Patents

Abstract

(57) Abstract: The present invention provides a method for producing a DNA array simply and at low cost. SOLUTION: Using a nucleic acid probe template strand immobilized on a template substrate, synthesizing a nucleic acid probe strand along the template strand,
By immobilizing the synthesized probe on another array substrate using an electric field, a nucleic acid chain immobilized array is manufactured simply and at low cost. By forming an array substrate with electrodes, a DNA array capable of electrical DNA detection is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a nucleic acid chain-immobilized carrier having a large number of nucleic acid probes having a predetermined base sequence implanted on a substrate.

[0002]

2. Description of the Related Art Recently, genetic testing techniques using DNA arrays have attracted attention (Beattie et al. 1993, Fodor et a.
l. 1991, Khrapko et al. 1989, Southern et al. 199
Four). This DNA array is composed of a carrier in which 10 ¹ to 10 ⁵ types of DNA probes having different sequences are immobilized on the surface of a glass substrate or a silicon substrate of several cm square. It has greatly contributed. The outline of the principle is as follows.

[0003] First, by reacting the DNA probe with a sample gene labeled with a fluorescent dye or a radioisotope (RI) on a DNA array, the DNA probe has a sequence complementary to the base sequence of the DNA probe. Sample gene,
Binding to DNA probe. Thus, when the sample gene has a sequence complementary to the DNA probe on the array, a signal derived from the label is obtained at a specific site on the array. Therefore, if the sequence and position of the immobilized DNA probe are known in advance, the base sequence present in the sample gene can be easily examined. Also, DNA
If an array is used, a lot of information on the nucleotide sequence can be obtained by performing only one test, so that it is expected to be used not only as a simple gene detection technique but also as a sequencing technique (Pease et al. 1994, Parinov et al.
1996).

On the other hand, regarding immobilization of DNA probes on an array, (1) a method of sequentially extending the DNA strand of a DNA probe on an array for every unit nucleotide (US Pat. No. 5,889,
165) and (2) a method of immobilizing a DNA probe synthesized in advance on an array (US Pat. No. 5,807,522). The former method uses photolithography technology to immobilize DNA probes with different arrangements in a 1/2 inch square array at intervals of 100 mm every 20 × 20 μm, so about 4 × 10 ⁵ types of probes (Chee et al. 1996). With photolithography technology, patterning on the order of 0.1 μm is now possible, and there is a possibility that further integration of probes will progress in the future. In addition, the latter method has problems such as the need to prepare various types of probes in advance and the integration degree of the probes is lower than that of the former method (every 60 × 60 μm at 120 μm intervals). Since the probe can be immobilized three-dimensionally in the reactor, the reaction efficiency is excellent (G
uschin et al. 1997). In addition, a method of immobilizing a probe three-dimensionally on porous silicon instead of gel has been reported (Beattie et al. 1995).

[0005] As described above, the DNA array has the advantage that a plurality of information can be obtained in one test, but the production thereof requires complicated reaction control. It was very expensive.

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and its object is to
It is an object of the present invention to improve a conventional DNA array preparation technology and to provide a nucleic acid chain-immobilized carrier excellent in cost, simplicity, and sensitivity.

[0007]

According to the present invention, in order to achieve the above object, a second nucleic acid strand to be a nucleic acid probe chain is synthesized using a first nucleic acid strand fixed on a first substrate as a so-called template. Then, by fixing the synthesized second nucleic acid chain on another second substrate by using an electric field, a nucleic acid chain-immobilized carrier was produced.

That is, the present invention provides a method for producing a nucleic acid chain-immobilized carrier having a second nucleic acid chain having a predetermined base sequence immobilized on a second substrate, comprising: Providing a first nucleic acid chain-immobilized carrier on which a first nucleic acid chain having a base sequence complementary to a base sequence of a nucleic acid chain is immobilized, and the first nucleic acid chain-immobilized carrier in a nucleic acid synthesis solution. Synthesizing a second nucleic acid strand having a sequence complementary thereto along with the first nucleic acid strand; and arranging the second substrate facing the first nucleic acid strand side of the first substrate. Applying an electric field from the second substrate to the first substrate to cause the second nucleic acid chain synthesized along the first nucleic acid chain to migrate to the second substrate surface, And a step of immobilizing the substrate.

In the present invention, a pair of electrodes is used as the means for applying the electric field. This electrode is disposed and used outside the first substrate and the second substrate. Or,
By forming the first substrate and / or the second substrate with an electrode material, the substrate and the electrode may be integrated into a substrate electrode.

In the present invention, there are a plurality of nucleic acid probe chains, which may have the same sequence.
Preferably, each has a different sequence. In that case,
When the substrate electrode is used, the surface of the substrate electrode is divided into a number of electrode regions by an insulating layer, and different nucleic acid probe chains are fixed to each electrode region. Alternatively, different probe chains can be immobilized on the same electrode.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

In the present invention, the second nucleic acid strand is RN
A, DNA, PNA (peptide nucleic acid) and any of these similar compounds may be used, but DNA is preferable. The first nucleic acid strand is not particularly limited, and a synthetic oligonucleotide, cDNA, RNA, PNA, methylphosphonate nucleic acid, or the like can be used.

In the first and second substrates of the present invention, the shape of the substrate is not limited to a plate. Further, the substrate material to be used is not particularly limited. For example, inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used. In addition, polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin Organic materials such as polycarbonate, polyamide, phenolic resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used. Furthermore, a membrane used for nucleic acid blotting, such as a nitrocellulose membrane or a PVDF membrane, can also be used. In addition, a substrate electrode that also serves as a substrate and an electrode can be formed by using an electrode material described below. In the case of such a substrate electrode, it is preferable that the surface of the substrate electrode is separated at the insulating layer region, and different nucleic acid chains are fixed to the respective separated electrode regions.

The electrode material is not particularly limited.
For example, gold, gold alloys, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium,
Metals such as tungsten and their alloys, carbon such as graphite and glassy carbon, and oxides and compounds thereof can be used. Further, various kinds of semiconductor devices such as a semiconductor compound such as silicon oxide, CCD, FET, and CMOS can be used.

When an electrode film is formed on an insulating substrate and a substrate electrode in which the substrate and the electrode are integrated is used, this electrode film
It can be produced by plating, printing, sputtering, vapor deposition and the like. When performing evaporation, resistance heating method, high frequency heating method,
The electrode film can be formed by an electron beam heating method. When sputtering is performed, an electrode film can be formed by direct current bipolar sputtering, bias sputtering, asymmetric alternating current sputtering, getter sputtering, high frequency sputtering, or the like. Further, an electropolymerized film such as polypyrrole and polyaniline and a conductive polymer can also be used.

In the present invention, the insulating material used for separating the electrode surface is not particularly limited, but is preferably a photopolymer or a photoresist material. As the resist material, a photoresist for light exposure, a photoresist for far ultraviolet, a photoresist for X-ray, and a photoresist for electron beam are used. Examples of the photoresist for light exposure include those whose main raw materials are cyclized rubber, polycinnamic acid, and novolak resin. Cyclic rubber, phenolic resin, polymethyl isopropenyl ketone (PMIPK), polymethyl methacrylate (PMMA), and the like are used for the deep ultraviolet photoresist. For the X-ray resist, COP, metal acrylate, and other substances described in Thin Film Handbook (Ohm) can be used. Further, for the electron beam resist, it is possible to use a substance described in the above-mentioned literature such as PMMA.

The resist used here has a thickness of 100.degree.
It is desirable that it is not less than 10 mm. By covering the electrodes with a photoresist and performing lithography, the area can be made constant. As a result, the amount of immobilized nucleic acid chains becomes uniform between the respective electrodes, enabling measurement with excellent reproducibility. Conventionally, the resist material is generally finally removed, but the substrate electrode can be used as a part of the electrode without removing the resist material. In this case, it is necessary to use a highly water-resistant substance for the resist material to be used. The insulating layer formed on the electrode can be made of a material other than the photoresist material. For example, Si, Ti, Al, Zn, Pb, C
oxides, nitrides, such as d, W, Mo, Cr, Ta, and Ni;
It is also possible to use carbide and other alloys. After forming a thin film from these materials by sputtering, vapor deposition, CVD, or the like, patterning of the electrode exposed portion is performed by photolithography, and the area is controlled to be constant.

When a substrate electrode is used as the substrate of the nucleic acid chain-immobilized carrier obtained by the method of the present invention, several electrode regions are formed on one element, and each of the probe regions has a different probe nucleic acid chain. Can be tested on several types of targets at once. In addition, it is also possible to test several samples at a time by configuring several electrode units on one element and immobilizing the same probe nucleic acid chain. In this case, using photolithography
A plurality of electrodes are patterned on a substrate in advance. At this time, it is effective to form a partition with an insulating film so that adjacent electrodes do not come into contact with each other. The height of the partition is desirably about 0.1 to 100 microns.

The method for preparing the first nucleic acid chain-immobilized carrier that acts as a so-called template, in which the first nucleic acid chain is immobilized on the surface of the first substrate, is not particularly limited. For example, a direct synthesis method by photolithography using an optically active reagent (Fodor et al. 1991), or a nucleic acid chain prepared in advance is dropped and immobilized on the first substrate by a method such as a micropipette or an ink jet. A method can be used. It is preferable that a spacer exists between the first nucleic acid strand to be immobilized and the first substrate.

Next, the first nucleic acid chain-immobilized carrier serving as a template for nucleic acid chain synthesis prepared as described above is used as an electrolyte solution containing primers, nucleic acid synthase, nucleotide monomers, electrolytes, etc. By immersing and reacting in a synthesis solution, a second nucleic acid strand having a sequence complementary to the first nucleic acid strand is synthesized. Next, a second substrate for immobilizing the second nucleic acid chain is prepared and placed in the electrolyte solution near or adjacent to the first substrate, preferably facing the first substrate. Although the distance between the first substrate and the second substrate varies depending on the length of the nucleic acid chain, the distance between the first substrate and the second substrate should be about 1 mm or less.
It is preferable to set the interval from m to 1 mm. Subsequently, the electrolyte solution is subjected to conditions for denaturing the double-stranded DNA, for example, the temperature condition is preferably set to 90 ° C. or higher. For the denaturation, an alkaline solution containing sodium oxide or the like, urea, or the like can be used. Denaturation can also be performed by applying a potential. At this time, a positive potential is applied to the second substrate for immobilizing the second nucleic acid strand, and a negative potential is applied to the first substrate so that an electric field is applied from the second substrate to the first substrate. At this time, both the first substrate and the second substrate need to be substrate electrodes. The potential gradient is desirably about 0.1 to 100 V / cm. Since the nucleic acid chain is generally negatively charged, this operation causes the second nucleic acid chain synthesized on the first substrate to migrate to and bond to the surface of the second substrate.

For the binding between the second nucleic acid strand and the surface of the second substrate, any means known to those skilled in the art including covalent bonding, affinity binding, electrostatic binding and the like can be used. That is,
The terminal of the second nucleic acid chain is modified with a reactive functional group such as an amino group, a carboxyl group, or a thiol group; a protein that forms a specific affinity pair such as biotin or avidin, and the nucleic acid chain is further immobilized. The surface of the second substrate to be converted is also modified with a molecule having a reactive functional group such as a silane coupling agent; a protein constituting a specific affinity pair such as biotin and avidin; By doing so, it is possible to fix more firmly. At that time, a suitable spacer between the substrate and the functional group, for example, polyacetylene, polypyrrole, polythiophene,
By introducing a conductive polymer such as polyaniline, an alkyl group, polyethylene, or the like, the detection efficiency can be improved. When gold is used as the substrate electrode, it is preferable that sulfur having a high affinity for gold is bonded to the end of the second nucleic acid chain.

Further, the first nucleic acid chain can be immobilized on a first substrate made of a material other than an electrode, for example, glass, silicon, silicon oxide, silicon nitride, synthetic polymer, nitrocellulose film, nylon film, or the like. It is. In this case, at the stage of transferring the second nucleic acid strand synthesized on the first substrate onto the second substrate, another electrode is prepared using the second substrate as an electrode, and the electrode is connected to the first nucleic acid chain. May be installed on the opposite side of the second substrate with the first substrate immobilized therebetween, and a potential may be applied between the first substrate and the second substrate. At this time, the second substrate is set to be positive.

The synthesized second nucleic acid chain can be immobilized on a second substrate made of a material other than an electrode, for example, glass, silicon, silicon oxide, silicon nitride, synthetic polymer, nitrocellulose film, nylon film and the like. is there. In this case, another electrode is prepared at the stage of transferring the nucleic acid chain synthesized on the first substrate composed of the electrode to the second substrate, and the other electrode is provided on the opposite side to the first substrate, and A potential may be applied between the first substrate and the first substrate. At this time, the first substrate is set to a negative value.

Further, by combining these, it is also possible to use a material other than the electrodes for both the first substrate and the second substrate. In this case, electrodes may be provided outside the first substrate and the second substrate, and a potential may be applied from the electrode outside the first substrate to the electrode outside the second substrate.
At this time, the outer electrode of the second substrate is set to be positive.

In the present invention, the nucleic acid synthetase used for synthesizing the second nucleic acid chain from the first nucleic acid chain is not particularly limited, and can be selected from various DNA synthases and RNA synthases.

When cDNA is used for the first nucleic acid strand, the nucleic acid strand can be synthesized using random primers. When an oligonucleotide is used for the first nucleic acid chain, a spacer molecule such as polybenzyl glutamate (PBLG) or polyethylene glycol can be bound to the terminal in advance. Furthermore, by inserting a common sequence into the template probe chain, it becomes possible to synthesize a nucleic acid chain using a primer complementary to the sequence.

The nucleic acid-immobilized carrier produced according to the present invention can be used for detecting various genes, and the gene to be detected is not particularly limited. For example, hepatitis virus (A, B, C, D, E, F, G), HIV, influenza virus, herpes group virus, adenovirus, human polyoma virus, human papilloma virus, human parvovirus, mumps virus, human Viral infections such as rotavirus, enterovirus, Japanese encephalitis virus, dengue virus, rubella virus, HTLV, etc., Staphylococcus aureus, hemolytic streptococci, pathogenic Escherichia coli, Vibrio parahaemolyticus, Helicobacter pylori, Campylobacter, cholera, Shigella. , Salmonella, Yersinia, Neisseria gonorrhoeae, Listeria monocytogenes, Leptospira, Legionella, Spirochetes, Mycoplasma pneumonia, Rickettsia,
It can be used for detecting bacterial infections such as chlamydia, malaria, dysentery amoeba, and pathogenic fungi, parasites, and fungi. In addition, hereditary diseases, retinoblastoma, Wilms tumor, familial polyposis of the colon, hereditary nonpolyposis colorectal cancer, neurofibromatosis, familial breast cancer, xeroderma pigmentosum, brain tumor, oral cancer, esophageal cancer, Tumors such as stomach cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, thyroid tumor, breast tumor, urinary tumor, male organ tumor, female organ tumor, skin tumor, bone / soft tissue tumor, leukemia, lymphoma, solid tumor, etc. It is possible to test for sexual diseases. Further, the present invention can be applied to polymorphism analysis of RFLP, SNPs, etc., analysis of nucleotide sequence, and the like. In addition to medical treatment, it can be applied to all foods requiring inspection, quarantine, medicine inspection, forensic medicine, agriculture, livestock, fishery, forestry, etc. Further, it can be used for detection of a gene amplified by PCR, SDA, NASBA method or the like. Furthermore, the target gene is a previously electrochemically active substance, a fluorescent substance such as FITC, rhodamine and acridine, an enzyme such as alkaline phosphatase, peroxidase and glucose oxidase, a hapten, a luminescent substance, an antibody, an antigen, and a gold colloid. It is also possible to label with colloid particles, metals, metal ions, and metal chelates with trisbipyridine, trisphenanthroline, hexaamine and the like.

In the gene detection using the nucleic acid-immobilized carrier obtained by the present invention, the sample to be used is not particularly limited, and examples thereof include blood, serum, leukocytes, urine, stool, semen, saliva, tissue, cultured cells, sputum Etc. can be used. The nucleic acid components are extracted from these sample samples. The extraction method is not particularly limited, and a liquid-liquid extraction method such as a phenol-chloroform method or a solid-liquid extraction method using a carrier can be used. Also, a commercially available nucleic acid extraction method QIAamp
(Manufactured by QIAGEN), Smitest (manufactured by Sumitomo Metal)
Etc. can also be used.

Next, a hybridization reaction is carried out between the extracted nucleic acid component and the carrier for gene detection. The reaction solution is used in a buffer having an ionic strength of 0.01 to 5 and a pH of 5 to 10. It is possible to add dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant, and the like to this solution. The extracted nucleic acid component is added thereto, and denatured at 90 ° C. or higher. Insertion of the gene detection electrode immediately after denaturation,
Alternatively, it can be performed after quenching to 0 ° C. Further, it is also possible to carry out a hybridization reaction by dropping a solution on a substrate. During the reaction, the reaction rate can be increased by an operation such as stirring or shaking. The reaction temperature is in the range of 10 ° C. to 90 ° C., and the reaction time is 1 minute or more to about 1 night. After the hybridization reaction, the electrode is taken out and washed. For cleaning, ionic strength 0.01-5
And a buffer having a pH of 5 to 10 is used.

[0030] Among the nucleic acid chain-immobilized carriers obtained by the present invention, the nucleic acid-immobilized electrode using the electrode substrate is useful when performing detection using an electrochemically active DNA-binding substance. Detection using an electrochemically active DNA binding substance is performed by the following procedure. After washing the nucleic acid-immobilized array, a DNA-binding substance that selectively binds to the double-stranded portion formed on the surface of the electrode substrate is allowed to act, and an electrochemical measurement is performed. The DNA-binding substance used here is not particularly limited, but includes, for example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, tris intercalator, polyintercalator and the like. It can be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene, viologen and the like. The concentration of the DNA binding substance varies depending on its type, but is generally used in the range of 1 ng / ml to 1 mg / ml. At this time, ionic strength in the range of 0.001-5, pH5-10
Use a buffer in the range of After the electrodes are reacted with the DNA binding substance, they are washed and electrochemically measured. Electrochemical measurements are made with a three-electrode type, ie, reference electrode, counter electrode, working electrode, or a two-electrode type, ie, counter electrode, working electrode.

In this measurement, a potential higher than the potential at which the DNA binding substance electrochemically reacts is applied, and the reaction current value derived from the DNA binding substance is measured. At this time, the potential can be swept at a constant speed, applied as a pulse, or a constant potential can be applied. In the measurement, the current and the voltage are controlled using a device such as a potentiostat, a digital multimeter, and a function generator. Based on the obtained current value, the concentration of the target gene is calculated from the calibration curve. Gene detection device using gene detection electrode,
It is composed of a gene extraction section, a gene reaction section, a DNA binding substance reaction section, an electrochemical measurement section, a washing section, and the like.

Further, the nucleic acid chain-immobilized carrier obtained by the present invention can detect a gene by using fluorescence intensity as an index by pre-fluorescently labeling a sample or using a fluorescently-labeled nucleic acid chain as a second probe. Become. At this time, when used in combination with a fluorescence microscope or the like, it becomes possible to increase the degree of accumulation of nucleic acid chains.

As described above, by using the nucleic acid chain synthesis method according to the present invention, nucleic acid chains having different sequences can be synthesized continuously on the same carrier, and the operation of synthesizing nucleic acid chains can be simplified. In addition, nucleic acid chain synthesis can be performed at low cost. Furthermore, the use of the nucleic acid chain-immobilized electrode prepared by the method of the present invention enables electrochemical gene detection,
A simple and highly sensitive nucleic acid sequence test can be performed.

[0034]

The present invention will be described in more detail with reference to the following examples.

Example 1 Filter for Immobilizing Template Nucleic Acid Chain A 1.5 kb fragment obtained by amplifying rDNA of Escherichia coli was used for immobilization.
And synthetic DNA. For rDNA, two types of samples were prepared. One is heat-denatured 100 μg / mL (0.2mo
l / L sodium chloride, 5mmol / L phosphate buffer (pH7))
RDNA, and the other is alkali-denatured 100μg / mL
(0.2 mol / L sodium hydroxide) rDNA. As the synthetic DNA, a 20 μg / mL 90-mer oligonucleotide (10 mmol / L phosphate buffer (pH7)) was used. Spot 1 μL each on a nylon filter, allow to air dry, then run hybridization buffer (2xSSC-1mmo)
1 / L EDTA (pH 7)). Then, baking was performed in an oven at 80 ° C. for 1 hour.

Example 2: Electrode immobilized on a template nucleic acid chain For immobilization, a 1.5 kb fragment obtained by amplifying rDNA of Escherichia coli was used.
And synthetic DNA. For rDNA, two types of samples were prepared. One is heat-denatured 100 μg / mL (0.2mo
l / L sodium chloride, 5mmol / L phosphate buffer (pH7))
RDNA, and the other is alkali-denatured 100μg / mL
(0.2 mol / L sodium hydroxide) rDNA. A 20 μg / mL 90-mer thiolated oligonucleotide (10 mmol / L phosphate buffer (pH 7)) was used as the synthetic DNA. Each 0.2 μL was spotted on a 5 mm square gold electrode and allowed to air dry.

Example 3: Nucleic acid chain synthesis on a template nucleic acid chain-immobilized filter A nucleic acid chain was synthesized on the nylon filter of Example 1 on which the nucleic acid chain was immobilized. For the synthesis, a nucleic acid chain labeling kit (fluorescein isothiocyanate: FITC) manufactured by Pharmacia was used. 100 μL of a reagent comprising a nucleotide mix, a random primer, and a polymerase (klenow) was dropped on the filter, and reacted at 37 ° C. for 1 hour in a hybrid bag. After blocking and washing, alkaline phosphatase-labeled anti-FITC antibody and 1
Allowed to react for hours. After washing, when the luminescent substrate CDP-Star was dropped, luminescence was observed only from the spot where the DNA was spotted, indicating that the nucleic acid chain could be synthesized on the filter. However, no luminescence was observed from the spot of the synthetic DNA, indicating that it was not synthesized.

Example 4 Synthesis of Nucleic Acid Chain on Electrode with Immobilized Template Nucleic Acid Chain A nucleic acid chain was synthesized on the 5 mm square gold electrode of Example 2 in which the nucleic acid chain was immobilized. For the synthesis, a nucleic acid chain labeling kit (Fluorescein isothiocyanate: F manufactured by Pharmacia) was used.
ITC) was used. Reagent consisting of nucleotide mix, random primer and polymerase (klenow)
20 μL was dropped on the electrode and reacted at 37 ° C. for 1 hour without drying. After blocking and washing, the plate was reacted with an alkaline phosphatase-labeled anti-FITC antibody for 1 hour.
After washing, when the luminescent substrate CDP-Star was dropped, luminescence was observed only from the portion where the DNA was spotted. In addition, light emission was observed from the spot of the synthetic DNA on the gold electrode, and it was confirmed that synthesis was possible.

Example 5: Transfer from a template nucleic acid chain-immobilized filter A nucleic acid chain on a nylon filter synthesized by the method of Example 4 was transferred to another nylon filter. First, the nylon filter as a template was brought into contact with the nylon filter on the transfer side, and the electrophoresis tank was arranged so that an electric field was applied inside. The direction of the electric field is minus from the mold side to the transfer side →
Set to plus. After transferring for 1 minute at a potential gradient of about 20 V / cm, the filter on the transfer side was baked at 80 ° C. As a result of using the same luminescence detection system as in Example 4, it was confirmed that the nucleic acid chain synthesized on the filter was transferred to the transfer side. The template filter was recyclable at least three times.

Example 6 Transfer from Gold Electrode Immobilized on Template Nucleic Acid Chain The nucleic acid chain synthesized on the gold electrode by the method of Example 4 was transferred to a nylon filter. First, the gold electrode of the mold was brought into contact with the nylon filter on the transfer side, and the electrophoresis tank was arranged so that an electric field was applied inside. The direction of the electric field was set from minus to plus from the mold side to the transfer side. After transferring for 3 minutes with a potential gradient of about 10 V / cm, remove the filter on the transfer side.
Baking was performed at 80 ° C. As a result of using the same luminescence detection system as in Example 4, it was confirmed that the nucleic acid chain synthesized on the filter was transcribed on the transcription side. The mold electrode could be transferred at least three times.

Example 7 Detection of Nucleic Acid Chain on Duplicated Filter Gene was detected using a filter in which nucleic acid chain was duplicated. Using rDNA PCR product as target gene,
Labeling was performed at FITC. Heat-denatured target gene 10
0 μL was dropped on the filter and reacted at 37 ° C. for 1 hour in a hybrid bag. After blocking and washing,
The mixture was reacted with an alkaline phosphatase-labeled anti-FITC antibody for 1 hour. After washing, when the luminescent substrate CDP-Star was dropped, luminescence was observed only from the spot where DNA was spotted, indicating that the nucleic acid chain could be detected on the duplicated filter. Approximately the same level of detection was possible with a filter duplicated from the electrodes.

Example 8: Structure of an electrode for immobilizing a nucleic acid chain FIG. 1 is a plan view schematically showing one example of first and second electrode substrates used for immobilizing a nucleic acid chain in the present invention. It is. In the figure, reference numeral 1 denotes a 1 cm square glass substrate. On the substrate 1, 30 × 30 gold electrodes 2 of 100 μm square are patterned. Each electrode 2 is partitioned by a 50-micron insulating film 3, and a lead wire (not shown) is taken out from the back surface of each electrode.

Example 9: Synthesis of Nucleic Acid Chain Using Electrodes FIG. 2 is an explanatory diagram schematically showing an example of a manufacturing process of a nucleic acid chain immobilized array using an electrode substrate and a mechanism of nucleic acid synthesis. A first electrode substrate 4 on which a first nucleic acid chain 5 serving as a template for a probe nucleic acid chain is immobilized is prepared by applying a solution of a 30-mer oligonucleotide 5 having a different sequence to each of two electrodes of the electrode substrate of FIG. It was prepared by dripping with. Polybenzyl glutamate (PBLG) 21 having a molecular weight of 1,000 is bound to the terminal of the oligonucleotide 5 in advance, and a thiol group 22 is introduced to the terminal of the PBLG.

Next, the first electrode substrate 4 having the first nucleic acid chain immobilized thereon was treated with dNTPs, DNA synthase 23 and primer 9
And a complementary strand 6 (second nucleic acid strand) was synthesized. At the time of synthesis, first, the primer 9 is attached to the oligonucleotide, and further the DNA synthase 23 acts to synthesize the complementary strand 6. A thiol group 7 was introduced at the end of the synthesized second nucleic acid strand.

Next, the first electrode substrate 4 on which the first nucleic acid chain is immobilized and the second electrode substrate 8 are separated by a distance of 1 μm so that the gold electrode surface faces the gold electrode surface of the first electrode substrate. It was immersed in a 100 mmol / L phosphate buffer as it was. While heating to 95 ° C., a potential of 500 mV was applied to the second electrode substrate 8 with the first electrode substrate 4 as a counter electrode (24). As a result, an electric field was generated in the direction from the second substrate to the first substrate. By this operation, the second nucleic acid strand moves to the second substrate, and the thiol group 7 adheres to the gold electrode, and the 30 ×
The second nucleic acid strands having different sequences could be immobilized on the 30 pattern electrodes. The template electrode substrate 4 on which the first nucleic acid chain was immobilized could be reused at least 50 times repeatedly.

Example 10 Indirect Synthesis of Nucleic Acid Chain on Electrode FIG. 3 is an explanatory diagram schematically showing another example of producing a nucleic acid-immobilized carrier using an electrode substrate. In this embodiment, a first glass substrate 10 in which a first nucleic acid chain serving as a template is immobilized on a glass substrate is used as the first substrate. The first glass substrate 10 was prepared by dropping different cDNA 11 solutions in a 30 × 30 pattern using a micropipette. Next, the first glass substrate 10 on which the first nucleic acid strand was immobilized was immersed in a solution containing dNTPs, DNA synthase 25, and random primer 12 to synthesize a complementary strand (second nucleic acid strand) 13. A thiol group 14 was introduced at the end of the synthesized complementary chain. With the first glass substrate 10 interposed, a second electrode substrate 15 and another electrode 16 (counter electrode) similar to those described in the first embodiment were connected to glycine-N
Immerse in aOH buffer and add 5 between the counter electrode and the second electrode substrate.
A potential of 00 mV (relative to the counter electrode) was applied (26). By this operation, the second nucleic acid strand synthesized on the first glass substrate was transferred to and fixed on the second electrode substrate 15 having a pattern of 30 × 30. The first glass substrate 10 on which the first nucleic acid chain was immobilized could be reused at least 50 times repeatedly.

Embodiment 11: Duplication of Mold Substrate FIG. 4 is a view showing an example of a method for producing a duplicate substrate (second substrate) of a mold substrate (first substrate). The first mold
The glass substrate (first substrate) on which the nucleic acid chains are immobilized is different
The cDNA solution was prepared by dropping 30 × 30 paternins with a micropipette. Next, the glass substrate on which the first nucleic acid strand was immobilized was immersed in a solution containing dNTPs, DNA synthase 25, and random primers to synthesize a complementary strand (second nucleic acid strand). A thiol group was introduced into the terminal of the synthesized complementary chain via a polyethylene glycol having a molecular weight of 1,000. The second substrate is treated with N- (6-maleimidocaproyloxy) succinimide, Ns after preliminarily treated with γ-aminopropyltriethoxysilane.
The substrate was treated with uccinimidyl-6-maleimidohexanone (EMCS) to introduce a thiol-reactive functional group on the substrate surface. In the replication of the first substrate, first, a glass substrate of a mold serving as the first substrate and the replication substrate serving as the second substrate were interposed, and electrodes were provided outside both of them. The first substrate, the second substrate, and the two electrodes are immersed in a 10 mmol / L phosphate buffer (pH 7), and the temperature is raised to 80 ° C. while applying a potential of 500 mV (with respect to the counter electrode) between the two electrodes. Heated. By this operation, the synthesized nucleic acid chains on the first substrate were immobilized on the second substrate by SS bonding in a pattern of 30 × 30 each. The replica of the template thus prepared could be used as a template for a nucleic acid chain-immobilized substrate in the same manner as the template substrate, and could be reused at least 50 times repeatedly.

In the method of Example 9 and the method described above, the time required to produce one DNA array in which 10,000 probe nucleic acids were implanted was about 5 minutes. In contrast, (1) DNA on the array substrate
The method of sequentially extending the DNA strand of the probe for every unit nucleotide (US Pat. No. 5,889,165) requires 3 days,
(2) The method of immobilizing a DNA probe synthesized in advance on a substrate (US Pat. No. 5,807,522) required 60 minutes.

[0049]

By using the method for synthesizing a nucleic acid chain according to the present invention, it becomes possible to synthesize nucleic acid chains having different sequences continuously on the same carrier. Synthesis can be performed at low cost. Furthermore, by using the nucleic acid chain-immobilized electrode prepared by the method of the present invention, electrochemical gene detection becomes possible, and a simple and highly sensitive nucleic acid sequence test can be performed.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing an electrode substrate used for manufacturing a nucleic acid chain immobilized array in an example of the present invention.

FIG. 2 is a diagram illustrating an embodiment of a method for manufacturing a nucleic acid chain immobilized array using electrodes according to an embodiment of the present invention.

FIG. 3 is a view illustrating another embodiment of a method for producing a nucleic acid chain immobilized array using electrodes according to an embodiment of the present invention.

FIG. 4 is a view showing another embodiment of the method for producing a nucleic acid chain immobilized array using electrodes according to the present invention.

[Explanation of symbols]

1, 10: glass substrate, 2: gold electrode, 3: insulating layer, 4:
Template electrode, 4, 8, 15 ... electrode array, 5 ... oligonucleotide, 6, 13 ... synthetic DNA, 7, 14, 22 ... thiol group, 9 ... primer, 11 ... cDNA, 12 ... random primer, 16 ... counter electrode, 21 ... spacer, 23, 2
5 ... DNA synthase, 24 ... heat + potential, 26 ... alkali +
potential

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/552 G01N 33/566 33/566 37/00 102 37/00 102 C12N 15/00 F

Claims (12)

[Claims] 1. A method for producing a nucleic acid chain-immobilized carrier in which a second nucleic acid chain having a predetermined base sequence is immobilized on a second substrate, comprising: a base of the second nucleic acid chain on a first substrate; Providing a first nucleic acid chain-immobilized carrier on which a first nucleic acid chain having a base sequence complementary to a sequence is immobilized; and a first nucleic acid chain-immobilized carrier in a nucleic acid chain synthesis solution. Synthesizing a second nucleic acid strand having a sequence complementary thereto along the nucleic acid strand; arranging the second substrate so as to face the first nucleic acid strand side of the first substrate; By applying an electric field from the two substrates to the first substrate, the second nucleic acid strand synthesized along the first nucleic acid strand is caused to migrate to the surface of the second substrate and bind to the surface of the second substrate. And a method comprising the steps of: 2. The step of causing the synthesized second nucleic acid chain to migrate to the surface of the second substrate and to bind the second nucleic acid chain to the surface of the second substrate, wherein the double-stranded nucleic acid is denatured into a single strand in an electrolyte solution. The method of claim 1, wherein the method is performed under conditions. 3. A method for applying an electric field from the second substrate to the first substrate, comprising providing a pair of electrodes outside the second substrate and the first substrate, and applying a potential to the electrode pair. The method of claim 1, wherein the method comprises: 4. A substrate electrode having an electrode material surface is used as at least one of the first substrate and the second substrate in order to apply an electric field from the second substrate to the first substrate. The method of claim 1, wherein an electric potential is applied. 5. The electrode substrate, wherein an electrode film is coated on an insulating substrate, and the surface of the electrode film is divided into a plurality of electrode regions in an insulating layer region.
The method according to claim 4, wherein the first nucleic acid strand or the second nucleic acid strand having different sequences is fixed. 6. The method according to claim 3, wherein at least one of the first substrate and the second substrate is a substrate or a film made of synthetic resin or glass. 7. The method according to claim 7, wherein the first or second nucleic acid strand is RNA, DNA,
2. The method of claim 1, wherein the method is selected from the group consisting of PNA and analogs thereof. 8. The method according to claim 1, wherein the bonding between the second nucleic acid strand and the second substrate is performed via a bond selected from the group consisting of a covalent bond, an affinity bond, and an electrostatic bond. the method of. 9. An electrode material surface of the substrate electrode is made of gold, and a bond between the first nucleic acid chain and the second substrate is formed by a bond between sulfur bonded to the second nucleic acid chain and the gold surface. 5. The method according to claim 4, wherein the method is performed via affinity binding between the two. 10. The nucleic acid chain synthesis solution, comprising:
The method according to claim 1, comprising a nucleic acid synthase, a nucleotide monomer, and an electrolyte. 11. The method according to claim 2, wherein the condition under which the double-stranded nucleic acid changes to single-stranded is that the temperature of the electrolyte solution is 90 ° C. or higher. 12. The method according to claim 1, wherein the nucleic acid chain-immobilized carrier having the second nucleic acid chain immobilized on a second substrate is used for a genetic test, and the second nucleic acid chain is a probe.