KR100429967B1 - Method of analysing one or more gene by using a dna chip - Google Patents

Abstract

According to the present invention, a DNA capture chip in which DNA complementary to an analyte nucleic acid is arranged is immersed in a sample solution to hybridize the analyte nucleic acid to DNA on a DNA capture chip, transfer it to a detection DNA substrate, and sequence complementary to the nucleic acid to be analyzed. The present invention relates to a method of hybridizing fluorescently labeled probe DNA and analyzing the fluorescence of a detection DNA substrate to analyze information of nucleic acids in a sample. It is possible to manufacture a separate DNA detection chip that can be used to manufacture a DNA chip that puts the gene of an organism to be analyzed on a single substrate, and to analyze a number of gene sites with a single DNA chip to save time and cost required for DNA analysis. Can be reduced. In addition, since the genetic information is analyzed by combining the fluorescently labeled probe DNA prepared separately with the DNA detection chip, the gene analysis method of the present invention uses a reporter material on a nucleic acid immobilized on a sample DNA or a DNA capture chip for analysis of a mutant gene. No additional steps are required, and the signal levels detected are similar and interference can be reduced, allowing information on genes without complex image analysis.

Description

Gene analysis method using DNA chip {METHOD OF ANALYSING ONE OR MORE GENE BY USING A DNA CHIP}

The present invention relates to a genetic analysis method using a DNA chip and a DNA detection chip used therein. More specifically, using the complementary hybridization properties of DNA, one or more gene sites in a sample can be arranged in a specific position and competitive binding of fluorescently labeled probes thereof results in the determination of one or more genes present in the sample. It is about how to obtain genetic information at the same time.

As DNA sequence information is rapidly increasing due to the development of molecular biology technology, one of the important tasks of modern molecular biology is to develop fast and inexpensive technology to apply new genetic information to diagnosis. A recent development of DNA diagnostic tools and genetic analysis tools is a DNA chip in which DNA molecules are densely arranged on a two-dimensional surface. A DNA chip is a substrate in which a large number of DNAs are arranged and immobilized with high density. DNA chip is composed of cDNA chip with 100bp or more genes (full-length open leading frame or EST) and DNA synthesized by chemical reaction with less than 100bp. It can be divided into oligonucleotide chips arranged. DNA chips are arranged on the chip surface by synthesizing DNA molecules directly on the surface of the chip or by immobilizing pre-synthesized DNA molecules on the surface. The DNA chip may be classified into a pin microarray chip, an inkjet microarray chip, a photolithograph chip, and an electronic array DNA chip according to a specific manufacturing method thereof.

The ore plate DNA chip is an oligonucleotide arrayed on a substrate using an ore plate technique, which is widely used in a semiconductor process, and a specific method is described in detail in US Pat. No. 5,143854. Since the DNA chip manufacturing method requires a mask having each pattern for each DNA chip manufacturing process, the process is complicated in mass production and expensive equipment is required. Therefore, the manufacturing cost is high and the cleaning and mask arrangement process is required for each process. There is this.

Fin microsubstrate DNA chips are described in Schena M et al., "Quantitative monitoring of gene expression pattern with a complementary DNA microarray," Science, 270 (5253): 467-470, 1995. Specifically, it consists of three parts: a substrate (poly-L-lysine is treated on the glass surface for binding to a gene), probe DNA (DNA on 96-well amplified by PCR and fixed on the substrate), and pins are mounted. Robotic print head (moving DNA from the well to the glass substrate to fix). The inkjet microchip DNA chip is a method using a cartridge of the same principle as the cartridge used to eject the dye solution in the inkjet printer instead of the pins used in the pin microsubstrate chip. Genes are sprinkled onto a solid body by electric force, and there are thermal methods, thermal methods, solenoid methods, and piezoelectric methods. The advantage of this technology is that the gene can be sprinkled without touching the surface of the chip electrically, so that it can produce a large number of chips with the quantitative gene attached, but there is a drawback that occurs when the sample is injected and the injection device is operated. have. In addition, in the case of using piezoelectric printing, there is a method of producing an oligonucleotide on a surface by electrically releasing one of four bases, which is described in US Pat. No. 5,474,796. As a result, the DNA chips have a complicated manufacturing process, which is expensive in manufacturing cost and has a problem in mass production, and is expensive because many DNA chips need to be used when analyzing multiple genes.

The principle of gene analysis method using DNA chip is that cDNA or oligonucleotide of specific gene immobilized on DNA chip and sample DNA immobilized on DNA chip after adding fluorescent molecule or the like to reporter material. According to the intensity or type of fluorescence obtained by hybridization to determine the DNA DNA sequence and function of the sample by detecting the binding position of the sample DNA, or to analyze the expression level of specific DNA using the intensity of fluorescence. The DNA molecules immobilized on the surface of the DNA chip have a known sequence, and thus hybridize with unknown sample DNA, and thus, the DNA information of the unknown sample, such as information on the sequence, function, expression level, etc., from the position combined with the DNA sample. It can be seen. Specifically, in a genetic analysis method using a DNA chip prepared by the ore plate method, the nucleic acid to be analyzed is separated, amplified, labeled with a fluorescent reporter, hybridized with a nucleic acid (probe array) on a DNA chip, and hybridized with a probe on a substrate. The target DNA emits strong fluorescence so it is analyzed. Thus, since the sequence and position of the probe on the substrate are already known, the sequence of the target DNA can be known. An example of analyzing the expression level of a yeast gene using a plate micro substrate DNA chip.

Compared with the existing representative gene analysis methods such as Southern blot and Northern blot, mutation detection and DNA sequencing, the genetic analysis method using the DNA chip can search at least several hundred genes in a short time, The use of shapes allows for the attaching of very small amounts of genetic material at high densities, while at the same time searching for large numbers of genes. The detection of gene expression has the advantage that both cDNA chips and oligonucleotide chips are superior to conventional methods.

In addition, oligonucleotide chips can also be used for mutation detection, making a significant contribution to mutation detection, disease diagnosis or gene expression blueprints. Existing methods for analyzing mutated genes have various methods. Methods in Molecular Biology Vol. 9 "Procols in Human Molecular Genetics", ed. C. G. Mathew, Humana Press, NJ (1991). A commonly used mutation analysis method is DNA sequencing, and RFLP (restriction fragment length polymorphism) is a method of separating and analyzing fragments cut by gel electrophoresis using restriction enzymes that selectively cut specific sequences. If the restriction enzyme cuts the base position with, the size of the cut piece is changed, so the mutation of the gene can be known. In addition, a PCR technique using a primer suitable for the mutant portion, or ligase (ligase) may be used (two oligomers are added so that the mutant portion is ligated, and the ligase is combined to use a ligase that is not ligated). Various methods can be used, such as the detection by an oligomer probe by the Southern blot or the Northern blot method, and the use of a DNA chip. However, these conventional methods are interested in methods using DNA chips because only one mutation analysis can be performed at a time or complex gel electrophoresis analysis is required.

After all, cDNA chip is composed of genes consisting of at least 100 bp base, which is mainly used for research purposes such as finding information on protein expression and function of genes. There is a disadvantage in that it is difficult to detect the difference in genetic information due to mutations. In addition, the oligonucleotide chip is attached to the oligonucleotide of less than 100 bp to the substrate, unlike the cDNA chip has the advantage that can distinguish the difference between the single base, it is possible to search for mutations, but the gene bound to the oligonucleotide Because you don't know which gene is, you have to amplify only a specific part by PCR and limit the part of the gene that can be searched on a single chip. When analyzing using a DNA chip, a large number of gene analysis is performed at once. It is difficult and only performs its function by genetic analysis, and it has the disadvantage that the used DNA chip cannot be used for further use.

However, the genetic analysis method using the DNA chip requires a large number of DNA chips, which requires a lot of analysis time and requires a large number of expensive DNA chips in order to analyze the difference in the sequence between the various gene regions of the organism. There is a problem. Therefore, there is a need to provide a method for easily manufacturing a DNA chip of a plurality of gene sequences of an individual with a single DNA chip and analyzing these genes simultaneously to reduce the time and cost required for gene analysis.

In order to solve the problems of genetic analysis using the conventional DNA chip as described above, the present invention provides a) a DNA capture chip in which heterologous DNAs, including DNA complementary to the nucleic acid to be analyzed, and a nucleic acid to be analyzed. B) binding the nucleic acid to be analyzed to the DNA on the DNA capture chip, respectively, and removing the non-specifically hybridized nucleic acid molecule from the DNA capture chip to which the sample nucleic acid is hybridized, c. The present invention provides a method of manufacturing a DNA detection chip by transferring analyte nucleic acids bound to a DNA capture chip onto a new DNA substrate.

In another aspect, the present invention further comprises d) a DNA chip comprising a sequence specifically binding to a nucleic acid of interest and hybridizing the fluorescently labeled probe DNA with a nucleic acid of interest bound to a detection DNA chip to analyze fluorescence. It provides a method for analyzing genes using.

In addition, the present invention provides a method for analyzing a mutated gene using one or more probes that can distinguish abnormal and normal genes, point mutation genes in step d).

1 is a flowchart schematically illustrating a gene analysis process using a DNA chip according to an example of the present invention.

2 is a diagram illustrating a process of hybridizing a sample nucleic acid to a new substrate having a wall structure according to an example of the present invention and transferring the sample nucleic acid to a new substrate, and a method of manufacturing the DNA detection chip obtained thereby. FIG. 2A illustrates a structure of a DNA capture chip in which DNA having a specific base sequence is immobilized on a DNA capture chip substrate having a barrier rib structure. FIG. 2B illustrates a sample nucleic acid and a DNA capture chip, and FIG. 2C illustrates a sample nucleic acid. The structure of contacting the DNA capture chip hybridized to the DNA sequence with the substrate of the DNA detection chip for the movement of the sample nucleic acid is shown, Figure 2d is a detection of the DNA is fixed to the analysis target nucleic acid moved from the DNA capture chip formed according to the above process It represents a chip.

3 illustrates a method of manufacturing a DNA detection chip using a DNA capture chip having a through-hole plate structure according to an example of the present invention. 3A shows the structure of a DNA capture chip having a penetrating structure to which specific DNA is bound, FIG. 3B shows a sample nucleic acid and a DNA capture chip, and FIG. 3C shows a structure of a DNA capture chip to which a sample nucleic acid is bound and a sample. It shows the arrangement of DNA detection chip for the movement of nucleic acid.

FIG. 4 illustrates a method of moving a sample DNA fixed to a planar DNA capture chip having no barrier rib structure to a DNA detection chip using a through-hole array according to an example of the present invention.

5 shows a method of searching for a mutant gene using a DNA detection chip according to another example of the present invention.

6 illustrates a method of detecting a point mutation using a DNA detection chip according to another example of the present invention.

The present invention relates to a gene analysis method using a DNA chip and a detection DNA chip used therein. More specifically, by using the complementary hybridization characteristics of DNA, a plurality of gene regions in a sample can be arranged at specific positions, and the genetic information of one or more genes present in the sample can be simultaneously obtained through competitive binding with a probe. It is about a method.

The present invention relates to a method for producing a DNA detection chip arranged with the nucleic acid to be analyzed.

In one example of the invention, a method of manufacturing a DNA detection chip consists of the following steps:

a) preparing a DNA capture chip in which heterologous DNAs including DNA complementary to the nucleic acid to be analyzed and a sample solution containing the nucleic acid to be analyzed are prepared,

b) placing the DNA capture chip into the sample solution and hybridizing the nucleic acid of interest to the DNA on the DNA capture chip at an appropriate temperature and time to complementarily bind to the DNA on the DNA capture chip. Wash the hybridized DNA capture chip,

c) preparing a DNA detection chip by transferring the nucleic acids to be analyzed complementary to the corresponding DNA onto a new substrate,

By repeatedly performing the above steps, a plurality of DNA detection chips in which nucleic acids of the same sequence are arranged may be obtained. c) After washing the DNA capture chip and immersing it again in the sample solution, it is possible to obtain a DNA detection chip in which nucleic acids of the same sequence are arranged.

In addition, the present invention includes a sequence complementary to the DNA sequence of the DNA detection chip prepared by the above method, and the method for analyzing genes by searching for the presence of fluorescence and fluorescence generated by hybridization with a fluorescently labeled probe To provide. Multiple sample nucleic acids immobilized on the detection chip can be analyzed at one time. That is, a) preparing a DNA capture chip in which heterologous DNAs comprising DNAs complementarily bindable to the nucleic acid of interest and a sample solution containing the nucleic acid of interest, b) capturing DNA of the nucleic acid of interest Complementary binding to the DNA on the chip, respectively, removing the non-specific binding sample nucleic acid, c) moving the analysis target nucleic acids bound to the DNA capture chip on a new DNA substrate to prepare a DNA detection chip, and d) a method of analyzing a gene using a DNA chip comprising a sequence capable of specifically binding to the nucleic acid of interest and hybridizing the fluorescently labeled probe DNA with the nucleic acid of interest bound to the DNA detection chip to analyze fluorescence; To provide.

Specifically, the method for analyzing genes according to the present invention comprises the following steps:

a) preparing a DNA capture chip in which heterologous DNAs comprising DNAs complementarily bound to the nucleic acid of interest and a sample solution containing the nucleic acid of interest are prepared,

b) placing the DNA capture chip into the sample solution and hybridizing the nucleic acid of interest to the DNA on the DNA capture chip at an appropriate temperature and time to complementarily bind to the DNA on the DNA capture chip. Wash the hybridized DNA capture chip,

c) preparing a DNA detection chip by transferring the nucleic acids to be analyzed complementary to the corresponding DNA onto a new substrate,

d) Fluorescence analysis is performed by hybridizing a fluorescently labeled probe DNA with a sequence that can specifically bind to the nucleic acid of interest and the nucleic acid of interest bound to the detection DNA chip.

In another example of the present invention, a plurality of identical DNA detection chips are prepared to which an analyte nucleic acid is bound, and different DNA probe sets are hybridized to each DNA detection chip to obtain genetic information of genes having different genes or different mutation bases. You can also analyze

As used herein, the term "DNA detection DNA chip" refers to a DNA chip in which the DNA to be analyzed is transferred from a DNA capture chip onto a substrate. Therefore, when there are multiple kinds of DNA in the sample, each of them binds to the corresponding specific position on the capture chip and is then fixed in a mirror array at the corresponding position on the detection chip. The DNA arranged on the DNA capture chip is fixed by UV crosslinking in the case of the membrane or the covalent bond or the streptavidin-biotin bond through chemical reaction to the DNA chip. Sample nucleic acids that can be analyzed with the DNA detection chip of the present invention include DNA and RNA.

In the present invention, a substrate for preparing a DNA capture chip and a DNA detection chip, and a DNA capture chip may be purchased and used commercially (Insight / Synteni's UniGEM V (trade name), Affymetrix GeneChip (trade name), Methods of making the same are well known to those skilled in the art (Trends in Biotechnology vol 16 (1998) 301) The substrates of DNA detection chips and DNA capture chips may be the same or different, but both glass, And inorganic materials such as silicone, or polymer materials such as polystyrene, polypropylene, PET (polyethylene terephthalate), PMMA (polymethyl methacrylate), polyacrylate-based, or membranes such as nylon and nitrocellulose may be used. Also, a metal thin film such as gold or aluminum may be formed on the surface of a solid substrate such as glass or metal, or a polymer membrane such as nylon or nitrocellulose may be formed. It is also possible to use a film formed by coating a natural polymer such as dextran or a synthetic organic polymer such as polypropylene and polystyrene on the substrate. Preference is given to using elastic polymer materials, a technique for coating substrates and solid surfaces for immobilizing biomolecules such as DNA, RNA and PNA is also described in detail in WO 00/05316.

As used herein, the term "DNA capture DNA chip" refers to a DNA chip to which a nucleic acid that is complementary to a nucleic acid to be analyzed is immobilized. Preferably, each nucleic acid immobilized on a DNA capture chip comprises DNA, Peptide Nucleic Acid (PNA), etc., and the 5 'or 3' positions of these nucleic acids are covalent or streptavidin-biotin binding on the capture DNA chip. The back and the membrane is fixed by UV crosslinking, and when the nucleic acid is an oligomer, it is designed to selectively bind to only one nucleic acid in the sample solution containing the DNA to be analyzed.

When the sample nucleic acid hybridized to the nucleic acid immobilized on the DNA capture chip is transferred to the DNA detection chip, the DNA molecules are moved through the solution so that the solution at each position is not mixed so that the preferred DNA capture chip is a wall in the chip. A DNA chip having a structure (FIG. 2) or a DNA capture chip having a through hole plate structure (FIG. 4) can be used. In addition, in the case of using a general flat DNA chip as shown in FIG. 3, a hole plate structure punched at the same position as the position of the oligonucleic acid fixed on the capture DNA chip is sandwiched between the DNA capture chip and the DNA detection chip. Can also be used.

As used herein, the term "probe DNA" refers to a DNA for probe having a nucleotide sequence that can be complementarily bound to an assay nucleic acid. Conventionally, a gene analysis method using a DNA chip is labeled with a reporter molecule on a DNA or an analyte DNA immobilized on a DNA chip, and analyzes a fluorescence signal or radiation based on complementary binding of the probe DNA on the DNA chip to the analyte DNA. Although the information is obtained, the present invention has the advantage that it is not necessary to separately label the nucleic acid in the sample by moving the target nucleic acid on the detection DNA chip and preparing and binding a separate DNA probe. When analyzing several genes at the same time, a probe labeled with fluorescent molecules having different fluorescence characteristics is required for each gene, but the number of molecules that can be distinguished by fluorescence characteristics is limited to several dozen. According to the present invention, since a plurality of genes are arranged in a two-dimensional plane and the information of the genes can be distinguished according to the arrangement position, a fluorescent probe can be prepared and distinguished with a known sequence probe capable of binding to the gene with a limited number of fluorescent molecules. By combining the molecules and attaching the same probe to a plurality of genes at the same time to analyze the fluorescence wavelength at each position, it is possible to analyze multiple genes even with a limited fluorescence molecule. For example, when it is known that four mutations are possible for 100 genes, the oligonucleotide chip is analyzed using 100 chips according to each gene, but the method of the present invention can bind 100 genes. Make a DNA capture chip, bind genes, transfer them to a DNA detection chip, and bind four fluorescent molecules to probe DNA corresponding to each mutation for one gene. Even though there are four genes, the same fluorescent molecules can be used for each of the 100 genes, so the actual fluorescent molecules are 4 types.) When a total of 400 probes are hybridized with the DNA detection chip, the genes correspond to mutations for each gene. Since the fluorescence signal is analyzed, the variation of each gene from the sample is zero. You can see which sequences have the inverse, so you can get 100 genes in one analysis. Fluorescence molecule types of various wavelengths, their labeling methods, and methods for analyzing the results obtained from hybridized DNA molecules are well known to those skilled in the art. There are many kinds of fluorescent molecules that bind to DNA, such as fluorescein, FAM, JOE, TAMRA, ROX, Cy3, Cy5, and the method of binding these fluorescent molecules, for example, by synthesizing oligomers and using enzymes. There are various methods such as methods, and these are well known to those skilled in the art. Expression analysis from hybridized DNA molecules using fluorescence in DNA chips is described in Nature Genetics Supplement vol 21 p: 10, 1999.

The present invention also provides a method for analyzing a mutagenesis. In the analysis step d) provides a method for analyzing genes that distinguish between mutant and wild type genes using probes labeled with fluorescent molecules having different fluorescence wavelengths in DNA fragments that can hybridize to normal and abnormal genes, respectively.

In another example of the present invention, in step d), a probe in which fluorescent molecules having different fluorescence wavelengths are labeled on DNA fragments that can hybridize to an analysis site of each gene according to the base type of point mutation sites Thereby distinguishing the base type of the point mutation site.

In another example of the present invention, a plurality of identical DNA detection chips coupled with an assay nucleic acid may be prepared, and a different DNA probe set may be hybridized to each DNA detection chip to analyze genetic information of a mutation position.

Probes used can be manufactured with general oligonucleotide synthesizers, and many companies manufacture and supply oligonucleotides with desired sequences. Fluorescent molecules having various wavelengths are sold by oligonucleotide manufacturers and combined with oligonucleotides (a domestic company such as Bioneer, and a synthesizer manufactured by PE applied biosystem, etc.).

The present invention uses a chip, ie a detection chip, having a gene from a sample obtained by transferring a gene selectively positioned such as a DNA chip through denaturation to a new substrate by using hybridization and denaturation of nucleic acids. In addition, by using Southern (or Northern) blotting technique, oligonucleotide is used as a probe to find base sequence information of the region where the mutation occurs, and thus it is possible to read the sequence variation from the sample.

One of the major differences with conventional DNA chips is that RNA in the sample can also be transferred to a new substrate and probed in the same way as blotting. In the case of the conventional DNA chip, the DNA fragments are long in the case of the chip to be planted with the cDNA, and thus the sequence change due to the mutation cannot be analyzed. In the case of oligonucleotide chips, the number of genes that can be analyzed at a time is limited because the probe length is too short and the sample genes interact with the entire probe, so it is not possible to know which genes are bound. Therefore, methods such as PCR should be used to limit the genes to be analyzed from sample preparation.

And both have a problem in that the gene itself must be labeled with fluorescent molecules. Therefore, the method of the present invention allows the DNA capture chip to bind regardless of the nucleotide sequence like the cDNA chip so that the gene to be analyzed in the sample is bound to a specific position and transferred to a new substrate. Is arranged at a specific position on the substrate (the array information knows the genetic information of the DNA on the DNA capture chip, so the genetic information on the DNA detection chip can be predicted). When mixed, each probe binds to a gene position where the sequences can complementarily bind, and a large number of probes (all possible variants x gene type, including wild type) enter, but the fluorescent molecules that label the probe contain wild type. Only all kinds of possible variations are needed and only the wavelengths for the fluorescent molecules If it is seats. This is because each probe binds to a corresponding gene position in a competitive reaction, but only when the probe has the same nucleotide sequence.

In one example of the present invention, when searching for a mutant gene, DNA probes having sequences capable of hybridizing to wild type genes and mutant genes are labeled with fluorescent molecules having different fluorescence wavelengths.

In case of using a oligonucleotide chip to analyze a point mutation in which one base is different from a wild type, amplify only a specific gene region of a sample and hybridize the oligonucleotide chip by labeling a fluorescent molecule to obtain a fluorescent image. In the case of having similar sequences in other parts as well as related parts, hybridization occurs, resulting in a fluorescence signal, making it difficult to search for multiple gene sites at once. In the present invention, an oligonucleotide including a point mutation portion may be prepared for each gene, and four different fluorescence molecules may be labeled according to the type of the mutated base to make an oligonucleotide DNA probe having a length of about 25bp. . When the DNA probes for each gene are mixed and hybridized together, as shown in FIG. 6A, the DNA probe hybridizes oligonucleotides having the same sequence through a competition reaction, so that one fluorescence DNA probe is mainly bound to one position as shown in FIG. 6B. The DNA sequence of the point mutation position of the bound DNA probe can be known through the wavelength of the DNA chip fluorescence. More than four fluorescent molecules can be used to make DNA probes for mutations such as deletions or insertions.

Each oligonucleotide probe is designed to have a similar Tm, and because the probe is about 25bp long, it hybridizes to the same DNA sequence, but hybridizes at different sites of the gene with similar DNA sequences, resulting in fluorescence images. In order to reduce or eliminate such interference, when an oligonucleotide having a DNA sequence that binds by a few bp to a portion causing interference by mis-hybridization is added without a fluorescent molecule label, the interference oligonucleotide probe is hybridized. It can prevent the wrong signal from coming out.

When described with reference to the accompanying drawings a preferred embodiment of the present invention having the features as described above are as follows. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

Example 1 below shows an analysis method for the production method of the present invention using a DNA chip. DNA capture chip is a method of making the above-mentioned general DNA chip by fixing DNA having a sequence that selectively binds to a specific gene part in a sample solution in a two-dimensional array. The nucleic acid in the sample solution hybridizes to a fixed position of DNA capable of complementary binding to each other on the DNA capture chip, and transfers the hybridized nucleic acid to a new substrate to form a DNA detection chip. Probe DNA labeled with fluorescent molecules is added to the DNA detection chip and hybridized to obtain a fluorescent image of the DNA chip for analysis. Referring to FIG. 1, which shows a schematic analysis method, an analysis method of the present invention is described in detail as follows.

(A) Preparation of Sample Solution: Isolate and purify nucleic acids from samples such as blood and tissues. Nucleic acid to be used for analysis can be amplified by a method such as PCR in order to immediately obtain the amount required for analysis. In the case of RNA, RNA can be directly used or converted to DNA using reverse transcriptase and then amplified. Nucleic acid molecules having a nucleotide sequence complementary to that of the DNA immobilized on the DNA capture chip among the nucleic acids in the sample solution thus prepared are hybridized with DNA on the DNA capture chip and localized at a specific position on the DNA capture chip.

(B) Hybridization of an analyte nucleic acid to DNA immobilized on a DNA capture chip: The capture DNA chip may be prepared according to a conventional DNA chip manufacturing method, and a commercially available DNA chip may be purchased and used. DNA capture chips are washed to remove nonspecific hybridized nucleic acid molecules.

(C) Moving the target nucleic acid from the capture DNA chip to the detection DNA chip: A method for moving a sample nucleic acid bound to the DNA capture chip onto the DNA detection chip is characterized by complementary binding between the DNA immobilized on the DNA capture chip and the sample nucleic acid. To separate the sample nucleic acid to the substrate of the DNA detection chip. In order to transfer the sample nucleic acid immobilized on the DNA capture chip to the detection chip, any method capable of breaking the complementary binding between DNA can be used. For example, there is a method of raising the temperature or adding a chemical substance which has undergone hydrogen bonding such as modified buffer (5 X SSC, 0.4 M NaOH, 10 mM EDTA), formamide, or the like. If the DNA capture and DNA detection chips are metal or thin films, or are made by polymer membranes or coatings on the metal or thin film, the nucleic acid between the two chips may be transferred to the DNA Can apply voltage of about 2 V.

The DNA capture chip requires a means to prevent the mixing of the solution at each position since the nucleic acid molecules are moved through the solution when the hybridized nucleic acid is transferred to the DNA detection chip. The DNA capture chip has a wall structure (FIG. 2), or a through-hole plate in which a through-hole array is drilled at the same position as the DNA spot only in the process of movement between the DNA capture chip and the DNA detection chip. It may be a method (Fig. 4) or a DNA capture chip has a through-hole plate (Fig. 3) can also be used to chip the DNA molecules fixed on the same plane as a general DNA chip without a partition structure.

When the DNA capture chip is removed, the DNA in the DNA capture chip is fixed with strong binding force. Therefore, the DNA capture chip remains attached to the DNA capture chip and DNA molecules from the sample DNA solution are transferred to a new substrate to make a new DNA chip for detection. The DNA chip of the present invention is immobilized with a DNA molecule having a known DNA sequence, whereas the DNA detection chip of the present invention is two-dimensionally arranged according to a region to search for DNA information of a nucleic acid in a sample, and is mirrored with a DNA capture chip It becomes a DNA chip with a sequence.

(D) Genetic analysis using fluorescently labeled probes: Fluorescence analysis based on complementary binding between sample DNA immobilized on a DNA detection chip and fluorescently labeled probe DNA is known to those skilled in the art. The DNA chip obtained in the above process is hybridized with a fluorescently labeled DNA probe to search for mutation and genetic information.

In the case of general DNA chips, the difference in the DNA concentration in the sample solution is caused by the difference in the number of copies of genes or the amount amplified by PCR, resulting in a difference in signal in the fluorescence image. Since the DNA detection chip is made in proportion to the amount of DNA fragments, the intensity of the fluorescence signal at each position is almost constant, and only the difference in the fluorescence signal according to the wavelength is compared, thereby facilitating data processing.

(E) Repeating the steps (A) to (C) with one or more capture DNA chips to produce a plurality of DNA detection chips to search for nucleotide sequences of different positions for each gene site in (D). Hybridization of probes to analyze fluorescence images allows analysis of mutated parts of different positions of each gene.

According to a second embodiment of the present invention, there is provided an analysis method using a DNA capture chip having a wall structure. As illustrated in FIG. 2, in an example of the present invention, a method of manufacturing a DNA detection chip obtained by hybridizing a sample nucleic acid to a new substrate having a wall structure and transferring the sample nucleic acid to a new substrate is provided. to provide. A DNA capture chip in which DNA having a specific capture sequence is fixed to a DNA capture chip substrate having a barrier rib structure is prepared (FIG. 2A), and the DNA capture chip is immersed in a sample solution containing the nucleic acid to be analyzed (FIG. 2B). In order to move the nucleic acid of interest from the DNA capture chip hybridized to the DNA sequence complementary to the target nucleic acid, the DNA detection chip is contacted with the substrate of the DNA detection chip (FIG. To prepare (FIG. 2D).

According to a third embodiment of the present invention, a method of manufacturing a DNA detection chip using a DNA capture chip having a through-hole plate structure is shown. By fixing the DNA capable of complementarily binding to the nucleic acid to be analyzed on the inner surface of the through hole of the DNA capture chip, and hybridizing the nucleic acid to be analyzed, and then contacting the substrate for manufacturing the DNA detection chip to the lower portion of the DNA capture chip The hybridized sample nucleic acid is moved from the DNA capture chip to the DNA detection chip to prepare a DNA detection chip (FIG. 3). The contents of the present invention are not intended to simply manufacture a plurality of DNA chips. Instead, a DNA chip having genetic information of a sample is prepared, and a fluorescent probe is hybridized to the DNA chip and analyzed. The method of manufacturing a DNA chip from a substrate having a via hole is an example of one method of manufacturing such a DNA chip.

Attaching the DNA fragments to the inner wall of the through-holes can increase the surface area of the DNA fragments depending on the thickness of the substrate, thereby increasing the amount of sample nucleic acid transferred to the DNA detection chip. Since the top of the well of the through plate is open, the target nucleic acid is moved from the capture DNA chip to the detection DNA chip substrate in a state of blocking the top with a separate cover plate.

Through-holes are made by punching holes at regular intervals with a laser on a substrate made of inorganic materials such as glass and silicon, or polymer materials such as polystyrene, polypropylene, PET (polyethylene terephthalate), and PMMA (polymethyl methacrylate). In the case of this polymer material, it is produced by injection molding or the like. DNAs having different nucleotide sequences may be injected or synthesized by a micropipette or capillary tube to fix the DNA on the inner wall of each through hole.

If the DNA detection chip is a metal substrate or metal thin film, or is made of a polymer membrane or coating on the metal substrate or metal film, the DNA between the cover plate and the DNA detection chip may be used to assist in transferring the hydrogen-bonded nucleic acid to the DNA detection chip. You can also apply a voltage of about 2V.

According to the fourth embodiment of the present invention, the basic analysis method is similar to that of Example 1, but DNA detection of sample nucleic acid immobilized on a planar DNA capture chip having no barrier rib structure using only a through-hole array. A method of moving to a chip is provided (FIG. 4).

In a fifth embodiment of the present invention, the genetic analysis method of the present invention is particularly advantageous for searching for variant genes. 5 shows a method of searching for a mutant gene using a DNA detection chip. The mutant probe DNA having the sequence of the wild type probe DNA and the site where the mutation is formed in the specific region of the target nucleic acid is hybridized to the DNA detection chip to which the target nucleic acid is immobilized, and hybridized with the target nucleic acid immobilized on the DNA detection chip. How to check whether there is a mutation. In this case, DNA probes having sequences capable of hybridizing to wild type genes and mutant genes are labeled with fluorescent molecules having different fluorescence wavelengths, and hybridized by mixing these DNA probes corresponding to the respective genes. Through the DNA probe having the same sequence is hybridized with a nucleic acid molecule corresponding to each gene arranged on the DNA detection chip. In the case of wild type genes and mutant genes, they are hybridized with DNA probes labeled with different fluorescent molecules. Therefore, DNA detection chips can be imaged for two fluorescence wavelengths and the relative magnitudes of fluorescence obtained according to wavelengths for each gene can be compared. Is a wild type or a mutated gene. For each gene, if the fluorescence intensity of the DNA probe for the wild type is large at two wavelengths, the corresponding gene has the wild type genetic information. On the contrary, if the fluorescence wavelength of the DNA probe for the mutant gene is large, the corresponding gene is shown. Has mutated genetic information, and if the fluorescence intensity is similar, it has received wild type and mutant genes from parents because it has two genetic information received from parent and mother for one gene in the cells of diploid organisms. In this way, genes on the entire DNA detection chip can be analyzed to determine whether multiple genes are mutated at the same time. This method can be used when both wild-type and mutant genes are known, such as genetic search for disease as well as genetically engineered concentrate search, subtype search for viruses or bacteria. If multiple mutations are possible at one location, fluorescent probes with different fluorescence wavelengths (number of mutations to be analyzed + 1) can be used to construct DNA probes for wild-type and multiple mutation genes. Therefore, regardless of the number of genes to be analyzed in a single DNA detection chip, the number of fluorescent molecules required can be used (maximum value of the number of mutations to be analyzed + 1).

When multiple mutations are possible for each gene site, one or more identical DNA capture chips can be used to make multiple DNA detection chips and hybridize different sets of DNA probes to each DNA detection chip. In the case of using a single DNA capture chip, the process of (A) to (D) may be repeated by moving the analysis target nucleic acid bound to the capture DNA chip to a detection DNA chip and reusing the DNA capture chip.

In a sixth embodiment of the present invention, point mutations may be searched using the analysis method of the present invention. 6 illustrates a method of searching for point mutations using a DNA probe.

Individual differences in hereditary diseases or drug responses can also be caused by point mutations in which a single gene base changes. As described above, in the case of using a DNA detection chip to identify a point mutation different from a wild type, an oligonucleotide including a point mutation portion of each gene is prepared, and four different fluorescence wavelengths are generated depending on the type of the mutated base. Labeling fluorescent molecules with the oligonucleotide DNA probe of about 25 bp long. For example, the fluorescent molecules are FAM for cytosine, JOE for adenine, TAMRA for guanine, and ROX for thymine, and the maximum fluorescence emission wavelength of each of these compounds is 517 nm, 548 nm, 568 nm, and 591 nm. to be. When four DNA probes for each gene are mixed together and hybridized, the DNA probe is a strong reaction when a fluorescence image of the DNA probe chip is obtained according to the wavelength. Since the fluorescence wavelength that emits the fluorescence signal refers to the type of base (for example, the gene A has the largest fluorescence signal at 568 nm, and the gene B has the largest fluorescence signal at 517 nm, the point mutation is A Guanine and B is cytosine.) For each gene, you can determine the type of base at which the point mutation occurs. In this case, more than four fluorescent molecules may be used to prepare DNA probes for mutations such as deletion or insertion.

In the present invention, a DNA capture chip having a DNA complementary to the nucleic acid to be analyzed is immersed in a sample solution to hybridize the nucleic acid to be analyzed with DNA on the DNA capture chip, and then transferred to a detection DNA substrate, and has a sequence complementary to the DNA to be analyzed. The present invention relates to a method of hybridizing fluorescently labeled probe DNA and analyzing fluorescence to analyze information of nucleic acid in a sample. It is possible to manufacture a DNA chip that the gene of the organism to be analyzed on one substrate can be prepared, and by analyzing a plurality of gene sites with a single DNA chip can reduce the time and cost required for gene analysis. In addition, by combining the fluorescent label probe DNA prepared separately with the DNA detection chip to analyze the genetic information, the gene analysis method of the present invention can analyze the mutant gene, the reporter to the DNA fixed on the sample nucleic acid or DNA capture chip There is no need to add a substance, and the signal level detected is similar and the interference can be reduced, so that gene information can be obtained without the need for complicated image analysis.

Claims (14)

a) preparing a DNA capture chip in which heterologous DNAs including DNAs complementarily bindable to the nucleic acid of interest and a sample solution containing the nucleic acid of interest are prepared, b) complementarily binding the nucleic acid of interest to the DNA on the DNA capture chip, and removing the non-specifically hybridized nucleic acid molecule from the DNA capture chip to which the sample nucleic acid is hybridized; c) separating the analyte nucleic acid into a single strand form by breaking the bond between the analyte nucleic acid hybridized in step b) and the complementary DNA on the DNA capture chip by increasing the temperature or adding a base hydrogen bond decomposing material. Preparing a DNA detection chip by contacting a new substrate and binding the separated analyte nucleic acid onto a new substrate to move the analyte nucleic acids onto a new DNA substrate, d) a method of analyzing a gene using a DNA chip comprising a sequence capable of specifically binding to the nucleic acid of interest and hybridizing the fluorescently labeled probe DNA with the nucleic acid of interest bound to the DNA detection chip to analyze fluorescence; . The method of claim 1, wherein the substrate of the DNA capture chip and the DNA detection chip is an inorganic material that is glass or silicon, or a polymer material selected from acrylic compounds, polyethylene tetraphthalate, polycarbonate, polystyrene, and polypropylene. The method of claim 1, wherein a layer formed of any one of a metal thin film, a polymer membrane, and a polymer material / metal thin film is formed on the surface of the detection substrate. The method of claim 3, wherein the metal thin film is gold or aluminum, the polymer membrane is nylon or nitrocellulose, or the polymer material is dextran. The method of claim 1, wherein the nucleic acid to be analyzed is not mixed with each other by using a DNA capture chip having a wall structure or a through hole plate. The method according to claim 1, wherein the through hole plate is inserted between the DNA capture chip and the DNA detection chip so that a flat DNA capture chip can be used in step c). The method is characterized by moving the nucleic acid of interest. delete 8. The method of claim 7, wherein a voltage is applied between the DNA capture chip and the DNA detection chip to speed up the movement of the nucleic acid to be analyzed. The method of claim 1, wherein the method has a functional group capable of covalently binding to the DNA detection chip at the 5 'or 3' position of the nucleic acid to be analyzed. The method of claim 1, wherein the DNA detection chip has a structure in which a nylon or nitrocellulose membrane is mounted on a solid substrate, and the DNA of analysis is fixed to the membrane of the detection DNA chip by UV crosslinking. The method according to claim 1, wherein a probe is prepared for each gene by mixing probes labeled with fluorescent molecules having different fluorescence wavelengths in DNA fragments capable of binding to one gene region for the nucleic acid to be analyzed, and mixing them. A gene analysis method using a DNA chip that simultaneously hybridizes to a nucleic acid on a DNA detection chip to analyze a plurality of genes simultaneously. The method of claim 1, wherein in step d), the mutant and wild-type genes are distinguished by using probes labeled with fluorescent molecules having different fluorescence wavelengths in DNA fragments that can hybridize to normal and abnormal genes, respectively. Analysis method of gene. The method of claim 1, wherein in step d), a point mutation is performed using a probe in which fluorescent fragments having different fluorescence wavelengths are labeled on DNA fragments that can hybridize to an analysis site of each gene, depending on the type of base mutation point. Characterized by distinguishing the base type of the position. The method according to claim 1, 12, or 13, wherein a plurality of identical DNA detection chips are prepared to which an analyte nucleic acid is bound, and a different DNA probe set is hybridized to each DNA detection chip to analyze genetic information of a mutation position. .