KR20000012892A - Biomolecular microchip and preparation method thereof - Google Patents

Abstract

PURPOSE: A biomolecular microchip and a preparation method are provided, for detecting a gene more rapidly, reducing the time required, and being capable of application to broader field. CONSTITUTION: The biomolecular microchip is prepared by preparing a polyacrylamide solution; adding glycidylmethcrylate to the polyacrylamide solution on the solid surface such as a glass to prepare a gel by polymerization reaction; and forming a stable covalent bond by epoxy ring opening reaction of a biomolecule containing the glycidyl group and amine group on the gel or a compound containing amine group. Preferably the polyacrylamide solution contains a mixture of acrylamide and N,N'-methylenebisacrylamide of 90-99.9:0.1-10 (w/w), and a gelling-stimulating agent. The biomolecule is selected from DNA, RNA, a protein or an antibody, and is preferably DNA. A certain biomolecule is detected by the hybrid experiment attaching probes to DNA or RNA of the microchip.

Description

Biomolecule Microchip and Manufacturing Method Thereof

The present invention relates to a biomolecule microchip and a method of manufacturing the same. More specifically, the present invention provides a biomolecule microchip having covalent bonds of amine group-containing biomolecules or amine group-containing compounds to a polymerized gel by adding glycidyl methacrylate to a polyacrylamide solution, and having a stable bond. It relates to a manufacturing method. In general, hybridization of oligonucleotides in which an oligonucleotide or target DNA fragment is immobilized on a gel or solid surface is a technique that can be applied to various fields. Recently, many studies have been conducted to apply hybridization techniques of such oligonucleotide chips to DNA sequencing (Barinaga, M. (1991) Science 253: 1489; Cantor, CR, Mirzabekov, A. and Southern, E. (1992) Genomics 13, 1378-1383; Southern, EM, Maskos, U. and Elder, JK (1992) Genomocs 13, 1008-1017; Lipshutz, RJ, Morris, D., Chee, M., Hubbell, E., Kozal, MJ , Shai, N., Shen, N., Yang, R. and Fodor, SPA (1995) Biotechniques 19, 442-447). It is largely divided into a method of synthesizing oligonucleotide on the glass surface and a method of fixing the oligonucleotide synthesized on the solid surface or gel surface. For example, Southern and his colleagues used silicone rubber tubings to place oligonucleotides on a glass surface, using silicone rubber cement to attach them to a glass plate, and then layer them on the glass plate to be synthesized. Was developed to synthesize oligonucleotides by injecting the desired coupling solution into the specific sites and the remaining blocking sites by rotating the tubing in turn (Southern, EM, Maskos, U. and Elder, JK). (1992) Genomics 13, 1008-1017; Maskos, U. and Southern, EM (1992) Nucleic Acids Res. 20, 1675-1678; Maskos, U. and Southern, EM (1993) Nucleic Acids Res. 21, 2267- 2268; Williams, JC, Case-Green, SC, Mir, KU and Southern, EM (1994) Nucleic Acids Res. 22, 1365-l 367). A method of synthesizing oligonucleotides directly onto a glass surface by introducing photosensitive oligonucleotide synthesis mainly applied to DNA sequencing has been proposed. This method uses a light shielding mask on a surface where photosensitive protecting groups are induced on a 5 'hydroxyl group. It is a technique that forms free hydroxyl groups with light emitted and binds protected deoxynucleosides (Pease, AC, Solas, D., Sullivan, EJ, Cronin, MT, Holmes, CP and Fodor, SPA (1994) Proc. Natl. Acad. Sci. USA 91, 5022-5026; Sapolsky, RJ and Lipshutz, RJ (1996) Genomics 33, 445-456; Hoheisel, JD (1997) Trendsin Biotechnology 15, 465-469; Afftmetrix corp. In Russia, aldehyde groups are formed by activating oligonucleotides having 3-methyluridine at the 3'-end with sodium periodite (NaIO ₄ ) to bind amide groups with polyacrylamides activated by substituting hydrazide groups. Microchips (100 X 100 X 20 um) are listed (Yershov, G., Barsky, V., Belgovskiy, A., Kirillov, E., Kreindlin, E., Ivanov, I., Parinov, S., Guschin, D., Drobishev, A., Dubiley, S., and Mirzabekov, A. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918; Parinov, S., Barsky, V., Yershov, G., Kirillov, E., Timofeev, E., Belgovsky, A. and Mirzabekov, A. (1996) Nucleic Acids Res. 24, 2998-3004). In addition, a method of immobilizing oligonucleotides on an aminated polypropylene film (PP-NH ₂ ) by a phosphoramimidite-based synthesis method has been proposed (Matson, RS, Rampal, J., Pentoney, SL, Jr., Anderson, PD and Coassin, P. (1995) Analytical Biochemistry 224. 110-116). Another method is to bind and fix 3'-amino modified ringing nucleotides to a thin silicon dioxide (SiO ₂ ) film on the surface of a silicon chip in the detection of nucleic acid hybridization, with 3 'amino linkage and 3' A method is disclosed for binding 3'-amino modified oligonucleotides in an epoxy ring opening reaction between epoxysilane monolayers treated with glycidoxyoxytrimethoxysilane (Lamture, JB, Beattie). , KL, Burke, BE, Eggers, MD, Ehrlich, DJ, Fowler, R., Hollis, MA Kosicki, BB, Reich, RK, Smith, SR, Varma, RS and Hogan, ME (1994) Nucleic Acids Res. 22 , 2121-2125). In addition, a method of spotting oligonucleotides tailed with homopolymers (dTTP) on nylon membranes containing primary amines and inducing covalent bonds by activating thymine bases of oligonucleotides by UV irradiation Has been disclosed (Saiki, RK, Walsh, PS, Levenson, CH and Erlich, HA (1989) Proc, Natl. Acad. Sci. USA 86, 6230-6234), and improved amino-linker attached oligonucleotides and nylon A method of forming more stable bonds and increasing hybridization efficiency with amide bonds between carboxyl groups of a membrane is also disclosed (Zhang, Y., Coyne, MY, Will, SG, Levenson, CH and Kawasaki, ES (1991) Nucleic Acids Res) 19, 3929-3933). Hybridization by the addition of radiolabeled or non-radiolabeled target DNA to a DNA chip developed by the introduction of these methods has been successful in detecting RAS point mutations, cystic fibrosis deletions and multiple point mutations as well as DNA sequencing. (Cantor, CR, Mirzabekov, A. and Southern, E, (1992) Genomics 13, 1378-1383; Zhang, Y., Coyne, MY, Will, SG, Levenson, CH and Kawasaki, ES (1991) ) Nucleic Acids Res. 19, 3929-3933; Hacia, JJ, Brody, LC, Chee, MS, Fodor, SPA and Colliins, FS (1996) Nature Genetics 14, 441-447; Shoemaker, DD, Lashkari, DA, Morris , D., Mittmann, M. and Davis, RW (1996) Nature Genetics 14, 450-456; Sosnowski, RG, Tu, E., Butler, WF, 0'Connel, JP and Heller, MJ (1997) Proc. Natl. Acad. Sci. USA 94, 1119-1123), especially for genetic variation testing and polymorphism studies (Lipshutz, RJ, Morris, D., Ch). ee, M., Hubbell, E., Kozal, MJ, Shai, N., Shen, N., Yang, R. and Fodor, SPA (1995) Biotechniques 19, 442-447; Schena, M., Shalon, D , Davis, RW and Brown, PO (1995) Science 270, 467-470; Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, XC, Stern, D., Winkler, J., Lock, DJ, Morris, MS and Fodor, SPA (1996) Science 274, 610-614; De Rissi, J., Penland, L. and Brown, PO; Bittner, ML, Meltzer, PS, Ray, M., Chen, Y., Su, YA and Trent, JM (1996) Nature Genetics 14, 457-460). In addition, the sensitivity, unity and reproducibility of these methods are expected to be ideal tools for the analysis of mutations such as neoplasia and HLA typing, as well as for diagnosis of infections and genetic diseases (Saiki, RK, Walsh, PS, Levenson, CH and Erlich, HA (1989) Proc. Natl. Acad. Sci. USA 86, 6230-6234; Zhang, Y., Coyne, MY, Will, .SG, Levenson, CH and Kawasaki, ES (1991) Nucleic Acids Res. 19, 3929-3933.

Oligonucleotide placement by photolithographic synthesis or other synthetic methods directly on the glass surface is a very difficult high-level technique, and has security issues in the future. Such a problem is typically not suitable for quantitative analysis, since the product obtained on the solid surface is relatively low. In comparison, the DNA chip using the gel of the present invention can be arranged at the required concentration because the oligonucleotide synthesized by the conventional method can be directly placed on the surface thereof. It is known that polyacrylamide gels have a 50 mM DNA fixation capacity and can actually use DNA from 1.5 micromolar to 1.5 mmole in the placement and hybridization of oligonucleotide chips (Yershov, G., Barsky, V). ., Belgovskiy, A., Kirillov, E., Kreindlin, E., Ivanov, I., Parinov, S., Guschin, D., Drobishev, A., Dubiley, S., and Mirzabekov, A. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918; Parinov, S., Barsky, V., Yershov, G., Kirillov, E., Timofeev, E., Belgovsky.A. And Mirzabekov, A. ( 1996) Nucleic Acids Res. 24, 2998-3004). This corresponds to 0.5-50 fmol oligonucleotides per square 40 × 40 × 20 micrometers. This is more than 100 times higher than the two-dimensional fixation ability of the glass surface (Yershov, G., Barsky, V., Belgovskiy, A., Kirillov, E., Kreindlin, E., Ivanov, I., Parinov, S., Guschin, D., Drobishev, A., Dubiley, S., and Mirzabekov, A. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918). Therefore, the DNA chip using this gel has a relatively large field of application and is easy to manufacture.

Under this technology, the present inventor not only solved the above problem, but also, as a result of intensive efforts to manufacture biomolecule microchips using gels, glycidyl methacrylate was added to the polyacrylamide solution, and on the solid surface, After polymerizing glycidyl methacrylate with acrylamide to form a gel, it was confirmed that the amine group-containing DNA or the amine group-containing compound formed a stable covalent bond with the glycidyl group on the gel, The hybridization of DNA and complementary PCR products or oligonucleotides was confirmed to be specifically detected, and the sensitivity and specificity of the detection were examined to complete the biomolecule microchip of the present invention.

As a result, the main object of the present invention is to polymerize by adding glycidyl methacrylate to a polyacrylamide solution to prepare a gel on a solid surface, and to covalently bond a biomolecule or an amine group-containing compound such as an amine group-containing DNA. It is to provide a biomolecule microchip having a stable bond.

Another object of the present invention is to prepare a biomolecule microchip manufactured by the above method.

Still another object of the present invention is to provide a method for easily and quickly detecting DNA through hybridization of biomolecules such as DNA or RNA probes on DNA microchips.

1 is a diagram showing whether or not fluorescein amine is fixed to the gel of the present invention.

2 is a diagram showing the results of hybridization using a biotin-labeled single-stranded DNA probe after immobilizing a PCR product and oligonucleotide IPRO2 on an activated gel.

3 is a diagram showing the results of hybridizing oligonucleotide IPRO3 to an activated gel and then using a fluorescently labeled single-stranded DNA probe.

4 is a diagram showing the fixed rate of oligonucleotide FRA2-1 according to glycidyl methacrylate concentration.

5 is a photograph and quantitative analysis of the sensitivity and specificity of oligonucleotide hybridization by EtBr staining.

According to the present invention, a glycidyl methacrylate is added to a polyacrylamide solution on a solid surface to polymerize to form a gel, and covalently bonds an amine group-containing biomolecule such as an oligonucleotide or an amine group-containing compound to a stable bond. It is to provide a biomolecule microchip with and a method of manufacturing the same. The gel is formed by polymerizing a methacryl group of glycidyl methacrylate with an acrylamide acryl group in the polymerization reaction of polyacrylamide and glycidyl methacrylate unit. At this time, an appropriate amount of N, N, N ', N-tetramethylenediamine and 10% ammonium persulfate is added as a gelling accelerator in the solution of polyacrylamide for the gelation reaction. Thereafter, the biomolecule, for example, oligonucleotide attached to the amine group (NH ₂₎ as a linker to the 5 'end of the oligonucleotide, and are attached to the linker in an amine group (NH ₂₎ and the article rayisi dill article of methacrylate rayisi dilga epoxy (C ₃ H ₆ O ₂ ) Ring opening reaction results in stable oligonucleotide binding to the gel. This reaction can bond the macromolecules with amine groups to the gel very stably by the epoxy ring opening reaction.

In the gel preparation, the ratio of acrylamide and N, N'-methylenebisacrylamide in the polyacrylamide solution was 90 to 99.1: 0.1 to 10 (w / v), preferably 99: 1 (w / v). This facilitates the binding of oligonucleotides to the gel by relatively large pore size of the gel, and hybridization results in hybridization of the DNA immobilized on the gel with oligonucleotides or DNAs having homologous base sequences thereof. To facilitate. The polyacrylamide solution is prepared at a final concentration of 6 to 10%, preferably 8%. Glycidyl methacrylate was used at a concentration of 1 to 5% (w / v) relative to the total polyacrylamide solution. To detect hybridized products, the probes prepared and hybridized biotin or fluorescently labeled single-stranded DNA using PCR and the results were detected by color reaction or UV. In addition, the EtBr staining method, which hybridizes with unlabeled homologous oligonucleotides and is mainly stained on double helix, is used to perform experiments on its sensitivity and specificity to specifically target at least 5 pmol / μl and complementary one of three different oligonucleotides. Could be detected.

Accordingly, the biomolecular microchip of the present invention can be used to detect specific genes more quickly and simply than conventional PCR methods, as well as to be useful for DNA sequencing, genetic variation and polymorphism studies.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1: Amine Group Oligonucleotide Synthesis

An amine group oligonucleotide having an amine-linker attached to the 5 'end of an oligonucleotide to bind an oligonucleotide to a gel prepared by adding glycidyl methacrylate was prepared using a DNA synthesizer (Perceptive Biosystems Model 8909/8909; ( Note) synthesized on Bioneer). The base sequence of the amine group oligonucleotides used is as follows.

IPRO2: 5'-5CGGATAAATCCACTCTGGCTGC-3 '

IPRO3: 5'-5GTTATCACGGATCACAATGACGGCACTTAT-3 '

FRA2-1: 5'-5GAAGGTAGCGACTCGTATTAGTGAATACGA-3 '

FRA2-2: 5'-5GGCTACCATCAGGTACGTCTAATACTTCAT-3 '

In the above, 5 is an amine group (NH ₂ ).

Example 2 Preparation of Polyacrylamide Gel with Added Glycidyl Methacrylate

A polyacrylamide solution having a final concentration of 8% (w / v) was prepared by setting the ratio of acrylamide and N, N'-methylene bisacrylamide to 99: 1 (w / w). The amount of glycidyl methacrylate was 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5 for the total polyacrylamide solution, respectively. It was added to the concentration of% (w / v) and mixed in the mixer. Then, N, N, N ', N-tetramethylethylenediamine and 10% ammonium persulfate as gelling accelerators were added to the solution by 4 µl / ml and 10 µl / ml, respectively, for polymerization.

Example 3: Gel Immobilization on Glass Slide Surface

The surface of the microscope slide (76 × 26 mm) was treated with Binding Solution (5 μl 3- (trimethoxysilyl) propyl methacrylate; 5 μl acetic acid; 990 μl, ethyl alcohol) and dried. A polyacrylamide gel solution polymerized on the surface was added thereto, and a slide treated with a glass coat solution (Glass Coat Soluion, Sigma) was covered thereon and reacted for 2 hours or more. The covered slides were then separated to form activated polyacrylamide gels with addition polymerization of glycidyl methacrylate to a thickness of 20 μl or less.

Example 4 Fixation and Analysis of Fluoresceamine in Activated Gels

In the gel thus activated, 0.1M sodium borate pH9.5 was added to 0.5M fluoresceamine solution in order to know whether the glycidyl group and the amine group of glycidyl methacrylate reacted and fixed on the gel. The mixed solution of 1: 1 was spotted on the slide surface with the addition of glycidyl methacrylate and no addition to the gel with a diameter of 5 X 5 mm and reacted at room temperature for 30 minutes. After washing three times with water three times, and then using a UV transilluminator, as shown in Figure 1, the fluorescein amine is epoxy fluorescence was detected only in the gel with glycidyl methacrylate It can be seen that the ring-opening reaction is stably covalently fixed. In FIG. 1, lane 1 is a case in which fluoresceamine is fixed to a gel prepared without adding glycidyl methacrylate, and it can be seen that fluoresceamine is not fixed at all, and lane 2 is 5% (w / v). When fluoresceamine was immobilized on the gel prepared by adding glycidyl methacrylate, it was found that fluoresceamine was immobilized.

Example 5 Immobilization of Amine Group Oligonucleotides and PCR Products on an Activated Gel

295 or 129 base pair PCR products obtained from the synthesized amine oligonucleotide or inv gene of Yersinia genomic DNA were diluted 1: 1 in sodium borate (0.1M, pH9.3) and then activated. To a polyacrylamide gel with a maximum diameter of 3 × 3 mm. At this time, the PCR product was heat-treated at 100 ° C. for 5 minutes, immediately cooled on ice, and spotted by making single-stranded DNA. Immobilization of the amine oligonucleotide or PCR product was carried out for 30 minutes at room temperature and washed with water, and then reacted for 30 minutes by adding 1M ethanolamine, a blocking solution, to prevent the epoxy ring opening reaction by the amine group. Wash three times for 5 minutes with water. The slides were completely dried and stored at 4 ° C. for the next step. In addition, in order to measure the detection sensitivity of oligonucleotides by EtBr staining, serial dilution of the amine group oligonucleotide FRA2-1 to 0.5 fmol was fixed and placed on the gel as described above to confirm the specificity of the EtBr detection method. As a negative control group, two kinds of amine group oligonucleotides IPRO2 and FRA2-2 were immobilized on the gel.

Experimental Preparation Example: Probe Preparation by Polymerase Chain Reaction (PCR)

The oligonucleotide and PCR product were fixedly placed on the gel and hybridized. In this case, a product prepared by using a complementary oligonucleotide or PCR amplification was used. All PCR amplification reactions were performed with 20 μl PCR PreMix (1U, thermostable DNA polymerase; 250 uM, each dNTP; 50 mM, Tris-HCl, pH8.3; 40 mM, KCl; 1.5 mM, MgCl ₂ ; stabilizer; loading dye; ( Bioneer) was used. 2 pmol of IPRO2 sense primers and 20 pmol of KINV4 antisense primer (5'-CGTGAAATTAACCGTCACACT-3 ') to prepare 295 base pair biotin-labeled single-stranded DNA fragments of the Yersinia inv gene containing complementary base sequences And an asymmetric PCR reaction was performed using the PreMix PCR Kit to which 1 μM Biotin-11-dUTP (Boehringer Mannheim) was added. At this time, the amount of template Yersinia genomic DNA was 100ng. Similarly, asymmetric PCR was performed by adding 2 pmol of IPRO3 sense primer, 20 pmol of KINV4 antisense primer, and 3 nmole of Amersham Fluoro Green (Fluorescein-11-dUTP) to prepare a fluorescently labeled single-stranded DNA probe. 129 base pair fluorescently labeled single chain DNA fragments were prepared. At this time, 100 ng of template Yersinia DNA was used. All PCR conditions were 94 ° C., 30 seconds; 57 ° C., 60 seconds; The test was repeated 30 times at 72 ° C. for 1 minute 30 seconds, and pre-denaturation and last extension were performed at 94 ° C. for 5 minutes and 72 ° C. for 5 minutes.

Example 6 Hybridization Experiments with Biotin-labeled Single-Chain DNA Probes (295 Bases)

20 μl biotin-labeled single-chain PCR solution prepared using asymmetric PCR was heat treated at 95 ° C. for 5 minutes, and used as a probe (see FIG. 2). 0.5 mL of 295 base single-stranded DNA, 1 μL PCR solution and a final concentration of 0.5 nmol and 1 nmol of 22 base oligonucleotides (IPRO2) were immobilized on the gel, followed by 5 ml of hybridization buffer (5XSSC, 1% blocking). Reagent, 0.1% N-lauroyl sarcosine, 0.02% SDS) was added and prehybridized at 42 ° C. for at least 1 hour. The prepared biotin labeled single chain PCR solution was added and hybridized overnight at room temperature. After hybridization, the slides were washed twice with 2 × SSC, 0.1% SDS for 5 minutes, twice with 0.1 × SSC, 0.1% SDS for 5 minutes, 1M maleic acid, 0.15M NaCl for 10 minutes. In alkaline phosphate buffer solution (150 mM NaCl, 100 mM Tris-HCl, pH7.5) to which the streptavidin-AP conjugate was added, the slide was reacted and washed for about 1 hour, and then NBT and BCIP were added. Hybrid spots were detected by color reaction. As shown in FIG. 2, when the PCR product is spotted, the spot is identified, but the color sensitivity is weak and the spot shape bounces off like a tail, and when the oligonucleotide is spotted, the spot is almost spotted. Weak color sensitivity was observed to confirm the position. In Fig. 2 lanes 1 and 2 are 0.5 μl, lanes 3 and 4 are 1 μl of 295 base single chain DNA, and lanes 5 and 6 represent 1 nmol and 0,5 nmol of IPRO2, respectively. In addition, the color development method using streptavidin-AP was inconvenient to use in the biomolecule chip of the present invention because the experiment step was long and the number of washing was high.

Example 7 Hybridization Experiments Using Fluorescent Labeled Single Chain DNA Probes (129 Bases)

For hybridization experiments using fluorescently labeled single-stranded DNA probes, 1 nmol of IPRO3 oligonucleotides were immobilized on the slides, and the final 200 μl 1X hybridization buffer solution with 20 microliters of PCR solution prepared as a probe was added onto the slides. After the reaction at room temperature for 30 minutes, the mixture was washed three times with hybridization buffer for 5 minutes each. To examine the fluorescence, an fluorescence microscope (excitation = 490 nm, emission = 520 nm) or UV spectroscopy was used. The results are shown in FIG. In Figure 3 lane 1 is a gel prepared without the addition of glycidyl methacrylate before washing, lane 2 is also a gel prepared without the addition of glycidyl methacrylate before washing. In addition, lane 3 represents a gel prepared without the addition of glycidyl methacrylate after washing, and lane 4 represents a gel prepared by adding glycidyl methacrylate after washing. As expected, the DNA was immobilized on the gel with glycidyl methacrylate and it was demonstrated that it was fixed with stable binding even after washing. In addition, experiments using fluorescence-labeled single-stranded DNA probes showed stronger luminescence sensitivity and higher safety to detect hybridized spots than hybrid experiments with biotin-labeled single-stranded DNA probes. Could be shortened.

Example 8 Fixed Rate of Oligonucleotides According to Glycidyl Concentration

As an experiment to determine the fixation ratio of oligonucleotide according to the concentration of glycidyl methacrylate, FRA2-1 oligonucleotide was added to a gel as described above prepared at various glycidyl methacrylate concentrations of 1% to 5%. Immobilized and complementary oligonucleotide FRA2-1-2 (5'-TCGTATTCACTAATACGAGTCGCTACCTT C-3 ') was prepared in 1x hybridization buffer at a final concentration of 10 pmol and added onto slides. After reacting for 30 minutes at room temperature, the mixture was washed three times for 5 minutes with 1 × hybridization buffer. Thereafter, a final concentration of 1 mM EtBr solution in 0.1 × hybridization buffer was prepared, and the solution was added to a slide for 20 minutes, and washed with water several times. Hybrid oligonucleotides were detected using UV spectroscopy. As a result, as shown in FIG. 4, it was found that the oligonucleotide can be fixed at a glycidyl methacrylate concentration of 1%. Oligonucleotides that did not hybridize as a control were detected by staining with EtBr. As a result, EtBr could not be inserted into single-stranded DNA. In FIG. 4, lane 1 is fixed with oligonucleotide (FRA2-1) and then detected by EtBr without hybridization. Lane 2 is identically fixed and hybridized with complementary oligonucleotide (FRA2-1-2). It is then detected by staining with EtBr.

Example 9 Sensitivity and Specificity of Hybridization of Oligonucleotides by EtBr Staining

Experiments on the specificity and sensitivity of hybridization were performed using the method as described above. As shown in FIG. 5, the final concentrations of 500 pmol / μl to 0.5 fmol / μL oligonucleotides (FRA2-1) are listed on the slide (see A in FIG. 5), and at the same time 50Opmol / of non-complementary IPRO2 and FRA2-2, respectively. Μl and 50 pmol / μl were spotted (see FIG. 5B). FRA2-1-2 oligonucleotides complementary to FRA2-1 hybridized to the slide and stained with EtBr to detect up to 5 pmol / μl oligonucleotides and up to 50 pmol / µl specific hybridized DNA. Could. Although spots are seen in non-complementary oligonucleotides, quantitative comparison of light brightness shows that the value of OD is significantly lower. At 500 pmol / μl, the OD values of complementary oligonucleotides are 2,645 (FRA2-1), whereas the OD values of noncomplementary oligonucleotides are 610 (IPRO2) and 744 (FRA2-2), respectively. The OD value of FRA2-1 at 50 pmol / μl was 1,148 and IPRO2 and FRA2-2 were 478 and 320, respectively (images measured by Imager I were measured by the 1D Main program; Bioneer Co., Ltd.). In Figure 5, A is a sensitivity investigation, a-1: oligonucleotide FRA2-1 50Opmol / μl, a-2: 50 pmol/μl, a-3: 5 pmol/μl, b-1: 0.5pmol / μl, b- 2: 50 fmol / μl, b-3: 5 fmol / μl, c-1: 0.5 fmol / μl, c-2: non-complementary oligonucleotide FRA2-2 50 Opmol / μl, c-3: non-complementary oligonucleotide IPRO250Opmol / μl, B is a specificity test, a-1: FRA2-1 50Opmol / μl, a-2: FRA2-1 50 pmol/μl, b-1: IPRO2 50Opmol / μl, b-2: IPRO2 50 pmol/ Μl, c-1: FRA2-250Opmol / μl, c-2: FRA2-2 50 pmol/μl.

As described and demonstrated in detail above, the present invention is a biomolecule by forming a gel on a solid surface by polymerizing glycidyl methacrylate to polyacrylamide to fix the amine group-containing biomolecule on the gel, The amine group and the glycidyl group of glycidyl methacrylate in the epoxy ring opening reaction can form a stable bond. Therefore, the biomolecule microchip of the present invention can be used for hybridization experiments, so that the specific gene of interest can be detected more quickly, and the procedure and time can be shortened compared to the existing hybridization experiments. In addition, biomolecules such as nucleotides, proteins, and antibodies with amine groups can be attached onto the gel using this method, which enables applications in a wider range of fields than conventional DNA chips. DNA sequencing, genetic variation, and polymorphism It will be more useful for research.

Claims (11)

Preparing a polyacrylamide solution, Glycidyl methacrylate was added to the polyacrylamide solution on a solid surface and polymerized to prepare a gel. A method for producing a biomolecule microchip comprising forming a stable covalent bond by epoxy ring opening reaction of the gel glycidyl group with an amine group-containing biomolecule or an amine group-containing compound. The polyacrylamide solution according to claim 1, wherein the polyacrylamide solution contains acrylamide, N, N'-methylenebisacrylamide, and a gelling accelerator mixed at 90 to 99.9: 0.1 to 10 (w / w). Method for producing a biomolecule microchip. The method of claim 1, wherein the glycidyl methacrylate is added to the total polyacrylamide solution at a concentration of 1 to 5% (w / v). The method of claim 1, wherein the solid surface is a glass surface. The method of claim 1, wherein the biomolecule is at least one selected from the group consisting of DNA, RNA, protein, and antibody. 6. The method of claim 1 or 5, wherein the biomolecule is particularly DNA. Biomolecule microchip prepared according to any one of claims 1 to 6. 8. The biomolecule microchip of claim 7, wherein the biomolecule microchip is used in a method for detecting a specific biomolecule through hybridization experiments using DNA and RNA probes. 10. The biomolecule microchip of claim 8, wherein a fluorescent material-coupled probe is used as a biomolecule detection method. 10. The biomolecule microchip of claim 8, wherein a fluorescent substance selectively intercalating in the double helix is used as a biomolecule detection method. Use of biomolecule microchips prepared according to claim 6 in DNA sequencing, genetic variation and polymorphism studies.