KR20050006417A - Method for detecting a target nucleic acid by using labelled control nucleic acid - Google Patents

Abstract

PURPOSE: A method for detecting a target nucleic acid using unlabeled target nucleic acid is provided, thereby detecting or quantifying the target nucleic acid without labeling the target nucleic acid. CONSTITUTION: The method for detecting a target nucleic acid using a labeled control nucleic acid comprises the steps of: (a) providing a nucleic array fixing a polynucleotide probe; (b) hybridizing the target nucleic acid which is not labeled with the polynucleotide probe in the presence of the labeled control nucleic acid; and (c) measuring the hybridization signal of the labeled control nucleic acid from the hybridized product, and calculating the hybridization degree of the unlabeled target nucleic acid.

Description

Method for detecting a target nucleic acid by using labeled control nucleic acid}

The present invention relates to a method for detecting a target nucleic acid through hybridization using a microarray, which can be detected or quantified without labeling the target nucleic acid.

After the human genome sequence is known, microarrays have been widely used for analysis of nucleic acids such as nucleotide variation analysis of genes, qualitative and quantitative analysis of mRNA, and sequencing. This conventional analysis method was performed by hybridizing a target nucleic acid, usually labeled with a marker such as fluorescence, with a complementary probe nucleic acid immobilized with a microarray, and then stimulating it with a laser to measure the fluorescence signal generated.

One example of a conventional method for detecting or quantifying target nucleic acids using such labeled target nucleic acids is shown in FIG. 1. In Figure 1, (A) is a plan view of a nucleic acid microarray, 2 represents each spot, WMP represents a wild type matched probe, and MMP represents a mutant type complementary probe. matched probe). (B) is a side view of the microarray, 4 represents a polynucleotide probe, and 6 represents a substrate. (C), (D), (C '), and (D') are diagrams showing the hybridization state and the detection result after hybridization. According to the conventional method, each probe is hybridized with the labeled wild type target nucleic acid 8 or the labeled mutant target nucleic acid 10, and the hybridization result is measured. At this time, the presence or absence of a target nucleic acid can be detected through the presence or absence of a signal from each corresponding spot, and the amount can be measured from the intensity of the signal.

However, since markers such as the fluorescent substance are usually dependent on PCR or enzymatic reactions and biological reactions, the adhesion of the markers may not be uniform, thereby making errors in the analysis results using the labeled target nucleic acid. Can cause. In addition, the efficiency of attaching the fluorescent material to the target nucleic acid has the disadvantage of bringing errors due to low fluorescence signal in the analysis and the price increases. Therefore, if the nucleic acid can be analyzed without labeling the target nucleic acid, the cost of preparing the target nucleic acid can be reduced, and the error of the analysis result due to the non-uniform labeling can be reduced, thereby increasing the accuracy of the analysis.

Recently, a technique for analyzing nucleic acid without attaching a fluorescent label to a target nucleic acid has been announced. Gerber et al. Have applied nanomechanical cantilever to femtomol level DNA analysis. ( Proceedings of the National Academy of Sciences, 99, 9783-9788, 2002). In addition, Farkas et al. Have been used for analysis of mutations of hemochromatosis using electrical signals ( Journal of Molecular Diagnostics, 3, 74-84, 2001). Walt et al. Have been applied to diagnostic studies of cystic fibrosis using molecular beacons on fiber optic arrays ( Nature Biotechnology , 18, 91-94, 2000).

However, despite such efforts, there has been a demand for a simple and convenient method for detecting or quantifying a target nucleic acid without labeling the target nucleic acid.

Accordingly, it is an object of the present invention to provide a method capable of detecting or quantifying target nucleic acids without labeling the target nucleic acids.

1 is a diagram schematically showing a method for detecting a target nucleic acid using a conventionally labeled target nucleic acid.

2 is a diagram schematically illustrating a method for detecting a target nucleic acid according to an embodiment of the present invention.

3 is a diagram schematically showing a method for detecting a target nucleic acid according to another embodiment of the present invention.

The present invention provides a method for detecting a target nucleic acid, comprising the following steps:

(a) providing a nucleic acid array to which a polynucleotide probe is immobilized;

(b) hybridizing said polynucleotide probe with an unlabeled target nucleic acid under hybridization conditions in the presence of a labeled control nucleic acid; And

(c) measuring the hybridization signal of the labeled control nucleic acid from the hybridization reaction obtained and calculating the degree of hybridization of the unlabeled target nucleic acid.

Herein, the target nucleic acid and the control nucleic acid include sequences complementary to the polynucleotide probe.

The method may also be a method of detecting a target nucleic acid comprising the following steps.

(d) control hybridization obtained by hybridizing the hybridization signal to hybridization conditions of the polynucleotide probe with the labeled control nucleic acid in the absence of the target nucleic acid in step (b) and measuring the hybridization signal of the control nucleic acid from the hybridization reaction product. Comparing with the signal.

Here, when the hybridization signal is reduced than the control hybridization signal, it is determined that the target nucleic acid is present, and when the hybridization signal has no change compared to the control hybridization signal, it is determined that the target nucleic acid is not present.

The method may also be a method of detecting a target nucleic acid comprising the following steps.

(d) hybridize with the polynucleotide probe and wash with varying concentration ratios of the unlabeled target nucleic acid and the labeled control nucleic acid, and then measure the hybridization signal of the labeled control nucleic acid, thereby measuring the concentration ratio and the hybridization signal. Obtaining a correlation of; And

(e) determining the concentration of the target nucleic acid by comparing the correlation with the signal of the target nucleic acid obtained in step (c).

The present invention also provides a method for detecting a target nucleic acid comprising the following steps.

(a) providing a nucleic acid array to which a polynucleotide probe is immobilized;

(b) hybridizing and washing the polynucleotide probe and the labeled control nucleic acid under hybridization conditions;

(c) measuring a hybridization signal of the labeled control nucleic acid;

(d) adding an unlabeled target nucleic acid to the hybridization reaction to hybridize under hybridization conditions and wash; And

(e) measuring the hybridization signal of the labeled control nucleic acid from the hybridization reaction obtained, and determining the degree of change of the hybridization signal in comparison with the hybridization signal of the control nucleic acid obtained in step (c).

Here, the target nucleic acid and the control nucleic acid have a sequence complementary to the polynucleotide probe.

In the present invention, in the step (e), if the hybridization signal is reduced may determine that the target nucleic acid is present, and if the hybridization signal does not change may further comprise determining that the target nucleic acid is not present. .

The present invention may also be a method for detecting a target nucleic acid, further comprising the following steps.

(f) performing the steps (b) to (e) by varying the concentration ratio of the target nucleic acid to the control nucleic acid to obtain a correlation between the concentration ratio and the change of the hybridization signal; And

(g) comparing the change value of the hybridization signal obtained in (e) with the correlation to determine the concentration of the target nucleic acid.

In the present invention, the nucleic acid array is well known in the art (eg, US Pat. Nos. 5,445,934 and 5,744,305). Nucleic acid array generally refers to the arrangement of different polynucleotides in a certain region or more in a certain region on a solid substrate. The polynucleotide preferably has a polynucleotide in a known region on the substrate at a density of at least 50 polynucleotides / cm ² , more preferably at least 300 polynucleotides / cm ² and even more preferably at least 400 polynucleotides / cm ² . It is fixed. The nucleic acid array may be prepared by extending the nucleotides one by one through in situ synthesis using photolithography technology of the polynucleotide to be immobilized on the substrate. In addition, the nucleic acid array can be prepared by immobilizing a nucleic acid already synthesized or native on an activated substrate. The array used in the present invention can be used without any limitation to the manufacturing method.

In the present invention, a "polynucleotide probe" is a polynucleotide immobilized on a nucleic acid array substrate and has a complementary sequence specific to a target nucleic acid, so that the target nucleic acid can be detected or quantified from a sample in which several nucleic acids are mixed. Refers to a polynucleotide. In addition, in the present invention, the term "target nucleic acid" refers to a polynucleotide containing a nucleic acid sequence to be detected or quantified as a nucleic acid complementary to the polynucleotide probe and capable of specific hybridization. In addition, "control nucleic acid" refers to a polynucleotide comprising a sequence complementary to the polynucleotide probe, and refers to hybridization with the polynucleotide probe competitively with the target nucleic acid. Therefore, the degree of hybridization of the control nucleic acid with the polynucleotide probe varies depending on the relative amounts of the control nucleic acid and the target nucleic acid. Thus, the signal obtained as a result of hybridization with the amount of the target nucleic acid in the presence of a constant control nucleic acid from a signal obtained from the control nucleic acid labeled with the polynucleotide probe for the various relative amounts of the target nucleic acid and the control nucleic acid and labeled as a result of the hybridization. Correlation with As a result, while using an unlabeled target nucleic acid, the presence or absence thereof can be measured.

In the present invention, the label is not particularly limited and includes fluorescence, radioactivity and electrical signals. The label is preferably a fluorescent label. In the present invention, an unlabeled target nucleic acid sample to be detected or quantified is used. Therefore, in the prior art, the labeling process is not normally performed. On the other hand, in the hybridization reaction of the present invention, instead of using an unlabeled target nucleic acid, a control nucleic acid comprising a sequence complementary to the polynucleotide probe is used.

The method of the present invention will be described exemplarily through the drawings (see FIGS. 2 and 3). First, hybridizing the wild-type target nucleic acid 8 to the probe (WMP) complementary to the wild-type target nucleic acid in the presence of the fluorescently labeled control nucleic acid 12 results in the competitive hybridization of the wild-type target nucleic acid with the fluorescently labeled control nucleic acid. . Thus, the hybridization signal is reduced by competition (see A, B, C and D in FIG. 2). On the other hand, hybridizing the mutant target nucleic acid 10 to the probe (WMP) complementary to the wild type target nucleic acid in the presence of the fluorescently labeled control nucleic acid 12 hybridizes with only the fluorescently labeled control nucleic acid. Thus, the hybridization signal does not decrease relatively compared to when there is competition (see A, B, C 'and D' in Fig. 2). In Figure 2, 2, 4, 6, 8, 10, and 12 represent spots, polynucleotide probes, substrates, wild type target nucleic acids, mutant target nucleic acids and fluorescently labeled control probes, respectively.

In addition, the present invention hybridizes and washes wild-type target nucleic acids or mutant target nucleic acids that are not further labeled for positive spots (eg, mutant target nucleic acids) where the hybridization signal is not reduced by the above method, By measuring the hybridization signal, the presence or absence of a target nucleic acid can be quantified. Alternatively, the presence or absence of quantification of the target nucleic acid can be achieved by hybridizing and washing the wild type target nucleic acid or mutant target nucleic acid which is not further labeled with the labeled control nucleic acid hybridized, and measuring the hybridization signal. First, for a spot to which a probe complementary to a wild-type target nucleic acid is immobilized, if the wild-type control nucleic acid is hybridized, hybridization with the wild-type target nucleic acid hybridizes to the hybridized nucleic acid because it hybridizes with the fluorescent nucleic acid labeled control nucleic acid. The signal is reduced (see A, B, C and D in FIG. 3). On the other hand, hybridization with a mutant target nucleic acid leaves the hybridization signal intact, as no competitive hybridization with the fluorescently labeled control nucleic acid occurs (see A, B, C 'and D' in FIG. 3). Therefore, after confirming the target nucleic acid through the first hybridization reaction and the detection of the hybridization signal, the presence or absence of the target nucleic acid can be further confirmed through whether the hybridization signal is reduced by performing the hybridization reaction secondly.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example

In this embodiment, the PCR (Polymerase Chain Reaction) method is carried out using a full-length DNA of a wild type hepatocyte nuclear factor-1 (HNF-1α) gene isolated from the human genome as a target nucleic acid and the HNF-1α gene as a template. The exon 2 DNA of the HNF-1α gene obtained using was used as a target nucleic acid.

Example 1: Preparation of DNA Array by Spotting

The nucleic acid array used in this example was prepared by spotting a mixture of radial polyethylene glycol, crosslinking agent and polynucleotide probe having an epoxy group on a substrate.

(1) Synthesis of Radial Polyethylene Glycol Derivative Having Epoxy Group.

Epichlorohydrin (7.5 mL) and tetrabutylammonium bromide (0.32 g) were added to the aqueous NaOH solution (50 wt%, 2 mL), followed by stirring, followed by pentaerythritol ethoxylate (1 g). Was added slowly and stirred at room temperature for 18 hours. The reaction was confirmed to be terminated by TLC (thin layer chromatography). When the reaction was not terminated, the mixture was further stirred at 60 ° C. for 1 hour. Then, water (30 mL) was added and diluted, followed by extraction three times with methylene chloride (40 mL). The organic solution layer was washed again with saturated NaHCO ₃ (40 mL) three times and separated, anhydrous MgSO ₄ was added, dried and the solvent was removed at low pressure. The residue of epichlorohydrin was then removed by drying in vacuo for two days. The radial polyethylene glycol derivative having the epoxy group thus synthesized was confirmed by titration of an epoxy group using H NMR and 0.1 N HBr / acetic acid (glacial).

(2) Synthesis of Diamine Crosslinking Agent

Penta (ethylene glycol) di-tosylate (5 g, 9.2 mmol) was dissolved in DMF (40 mL) solution, NaN ₃ (4.2 g, 64.1 mmol) and pyridine (0.5 mL) were added sequentially, and then at 140 ° C. Stir for 18 hours. The solvent was removed at low pressure, stirred with addition of water (200 mL), followed by extraction with methylene chloride (100 mL) and the organic solvent layer washed three times with brine (100 mL). Anhydrous MgSO ₄ was added to dryness, the solvent was removed at low pressure, and column chromatography (flash column chromatography, EA: nHex = 1: 2) yielded a di-azide intermediate. The intermediate was dissolved in methanol (30 mL), 10% Pd-C (0.1 equivalent) was added, and then the reduction reaction was performed for 18 hours using hydrogen gas. The catalyst was removed using a Celite pad and the pad was washed with ethanol. The filtrate and the ethanol wash were mixed and the solvent was removed at low pressure to obtain a diamine crosslinker.

(3) Preparation of Gel Matrix Solution Using Diamine Crosslinking Agent

The radial polyethylene glycol derivative (100 mg) having the epoxy group was added to water (4 mL), stirred, and the diamine crosslinking agent (5.8 mg) was added thereto, stirred at room temperature for 18 hours, and stored at 4 ° C. in solution.

(4) Preparation of nucleic acid array by spotting gel matrix-DNA conjugate solution.

Each polynucleotide probe (SEQ ID NOS: 1-10) was added to 100 mM NaHCO ₃ (pH = 9.0) solution and stirred and left at 37 ° C. for 1 hour. The polynucleotide probe was added to the gel matrix solution prepared in the above (3) so that the equivalent ratio of the radial polyethylene glycol having the epoxy group, the crosslinking agent, and the polynucleotide probe was 4: 1: 4, and stirred to 37 ° C. The matrix-DNA conjugate was prepared by standing for 14 hours at, which was then used as a spotting solution.

The spotting solution was spotted onto a glass surface surfaced with amine groups and then left in a wet chamber at 37 ° C. for 4 hours. The reaction was then carried out and stored in a drier so that the glass surface amine groups in unspotted positions were negatively charged to prevent the target nucleic acid from adhering to the glass surface, i.

Example 2 Isolation of HNF-1α Gene from Human DNA.

Isolation of genomic DNA was performed using a nucleic acid extraction kit (QAIGEN, USA). To 10 ml of human blood, 10 ml of buffer solution (1.28M sucrose, 20mM MgCl ₂ , 4% Triton X-100, 40mM Tris-HCl, pH 7.5) and 30 ml of distilled water were allowed to react. The mixture was placed in a centrifuge and centrifuged at 4,000 rpm for 15 minutes. The supernatant was removed, and blood cells were separated by resuspending and centrifuging twice with the buffer solution. 5 ml buffer (800 mM guanidine-HCl, 30 mM EDTA, 5% Tween-20, 0.5% Triton X-100, 30 mM Tris-Cl, pH 8.0) was added to the isolated blood cells, vortexed and resuspended. The proteinase K solution (10mg / ml) was added and reacted for 1 hour in a 50 ° C water bath to completely destroy the blood cells. The cell lysate was placed in a Genomic-tip 100 / G filter, centrifuged at 4,000 rpm, buffered (1.25M NaCl, 15% isopropanol, 50mM Tris-Cl, pH 8.5), and centrifuged again to genomic DNA. Recovered. Isopropanol was added to the recovered DNA solution, mixed well, centrifuged at 4,000 rpm for 15 minutes, and the supernatant was removed. The precipitate was washed twice with 70% ethanol and centrifuged again at 4,000 rpm to give a precipitate. The precipitate thus obtained was well dried and then completely dissolved by addition of 10 mM Tris-HCl (pH 8.0) solution to selectively obtain pure genomic DNA.

Purified DNA was measured at a concentration using a spectrophotometer, and the concentration was constant at 50ng / μl to efficiently perform a polymerase chain reaction (hereinafter, referred to as PCR) in the following examples. Adjusted.

Example 3: Exon 2 Amplification of HNF-1α Gene by PCR

In order to amplify the exon 2 fragment of the HNF-1α gene from genomic DNA of a normal human isolated in Example 2, polymerase chain reaction (50 µl of total reaction solution) was performed as follows.

50 ng human genomic DNA, 2.5 U thermostable DNA polymerase, 200 μM dNTP (dATP, dTTP, dGTP, dCTP), 50 mM Tris-HCl (pH8.3), 40 mM KCl, 1.5 mM MgCl2, 25 pmol sense primer (5'- Using CGAAGATGGTCAAGTCCTACCT-3 ': SEQ ID NO: 11) and 25 pmol of antisense primer (5'-GCCACCTCTCGCTGCTTGC-3': SEQ ID NO: 12), denaturation (94 ° C, 30 seconds), annealing (55 ° C, 30 seconds), extension (72 ° C., 45 sec) was repeated 35 times with one cycle. Pre-denaturation and last extension were performed at 94 ° C. for 5 minutes and 72 ° C. for 5 minutes.

The PCR product (175 bp) thus amplified is used for the hybridization test of the DNA chip of Example 4 below.

Example 4 Detection of Target Nucleic Acids Using Fluorescently Labeled Control Nucleic Acids

In this embodiment, the probe nucleic acid (SEQ ID NOs: 1 to 10) and the target nucleic acid (the PCR amplification product) are hybridized in the presence of a fluorescently labeled control nucleic acid, and the fluorescent signal is measured to determine the presence or absence of the target nucleic acid. It was.

The fluorescence-labeled control nucleic acid had a sequence complementary to the probe nucleic acid and 20 nM nucleic acid labeled Cy3 at the 5 'end. Hybridization also results in a microarray immobilized with 20 nM of target nucleic acid and probe nucleic acid dissolved in 0.1% 6SSPET (Saline Sodium phosphate EDTA buffer containing 0.1% Triton X-100) solvent. The reaction was carried out at 16 ° C. for 16 hours. Next, the microarray was washed with 0.05% 6SSPET and 0.05% 3SSPET at room temperature for 5 minutes, dried at room temperature for 5 minutes, and scanned (GenePix 4000B model; Exxon). The spot size of the probe nucleic acid of the microarray used in the present invention was 170 ± 5 μm, and the spacing between the spots was adjusted to 375 μm.

The spot-to-spot variation of each spot group was 8% CV of the same probe group, and the chip-to-chip variation was 10.6% CV.

The fluorescent signal emitted by the fluorescently labeled control nucleic acid hybridized to the DNA microarray was detected at a resolution of 10 μm using a ScanArray Scanner. As a result of comparing the fluorescence intensity of the normal sample and the patient sample, the fluorescence intensity of the patient sample was increased by about 2 times compared to the normal sample.

Example 5: Detection of Target Nucleic Acids Using Fluorescently Labeled Control Nucleic Acids

In this example, each of the probe nucleic acids (SEQ ID NOs: 1 to 10) and the control nucleic acid labeled with fluorescence were hybridized under hybridization conditions, washed, and the fluorescence signal was measured. Next, the hybridized material was hybridized with the target nucleic acid (the PCR amplification product) under hybridization conditions, washed, and then the fluorescent signal was measured to determine the presence or the amount of the target nucleic acid.

The fluorescence-labeled control nucleic acid had a sequence complementary to the probe nucleic acid and used 20 nM nucleic acid labeled Cy3 at the 5 'end. In addition, hybridization was performed using a microarray immobilized with 20 nM of control nucleic acid and probe nucleic acid dissolved in 0.1% 6SSPET (Saline Sodium phosphate EDTA buffer containing 0.1% Triton X-100) solvent. The reaction was carried out at 16 ° C. for 16 hours. Next, the microarray was washed with 0.05% 6SSPET and 0.05% 3SSPET at room temperature for 5 minutes, dried at room temperature for 5 minutes, and scanned (GenePix 4000B model; Exxon). Hybridization reaction was performed by adding 29 nM of target nucleic acid under the same conditions.

The fluorescent signal emitted by the fluorescently labeled control nucleic acid hybridized to the DNA microarray was detected at a resolution of 10 μm using a ScanArray Scanner. As a result of comparing the fluorescence intensity of the normal sample and the patient sample, the fluorescence intensity of the patient sample was increased by about 2 times compared to the normal sample.

According to the method for detecting the target nucleic acid of the present invention, it is possible to detect the presence or the amount thereof without labeling the target nucleic acid.

<110> SAMSUMG ELECTRONICS CO., LTD. <120> Method for detecting a target nucleic acid by using labelled          control nucleic acid <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid probe <400> 1 atggtcaagt ctacct 16 <210> 2 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid probe <400> 2 cgctgtgtga tgt 13 <210> 3 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid probe <400> 3 cctcccactg tgg 13 <210> 4 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Nucleic aicd probe <400> 4 cacctcctgc tg 12 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid probe <400> 5 ggagatggtc gatac 15 <210> 6 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid probe <400> 6 aggtggaact ggtt 14 <210> 7 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid probe <400> 7 accagtccta cct 13 <210> 8 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid probe <400> 8 gcagaatcgg gc 12 <210> 9 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid probe <400> 9 gaagtgggcc g 11 <210> 10 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid probe <400> 10 cgggccaccc tg 12 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 cgaagatggt caagtcctac ct 22 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 12 gccacctctc gctgcttgc 19

Claims (6)

(a) providing a nucleic acid array to which a polynucleotide probe is immobilized; (b) hybridizing said polynucleotide probe with an unlabeled target nucleic acid under hybridization conditions in the presence of a labeled control nucleic acid; And (c) measuring the hybridization signal of the labeled control nucleic acid from the hybridization reaction obtained, and calculating the degree of hybridization of the unlabeled target nucleic acid, Wherein said target nucleic acid and control nucleic acid comprise a sequence complementary to said polynucleotide probe. The method of claim 1, (d) control hybridization obtained by hybridizing the hybridization signal to hybridization conditions of the polynucleotide probe with the labeled control nucleic acid in the absence of the target nucleic acid in step (b) and measuring the hybridization signal of the control nucleic acid from the hybridization reaction product. And comparing with a signal, wherein if the hybridization signal is reduced than the control hybridization signal, it is determined that a target nucleic acid is present, and if the hybridization signal has no change compared to the control hybridization signal, A method for detecting a target nucleic acid, characterized in that it is determined to be absent. The method of claim 1, (d) hybridize with the polynucleotide probe and wash with varying concentration ratios of the unlabeled target nucleic acid and the labeled control nucleic acid, and then measure the hybridization signal of the labeled control nucleic acid, thereby measuring the concentration ratio and the hybridization signal. Obtaining a correlation of; And (e) comparing the signal with the correlation of the target nucleic acid obtained in step (c) and determining the concentration of the target nucleic acid. (a) providing a nucleic acid array to which a polynucleotide probe is immobilized; (b) hybridizing and washing the polynucleotide probe and the labeled control nucleic acid under hybridization conditions; (c) measuring a hybridization signal of the labeled control nucleic acid; (d) adding an unlabeled target nucleic acid to the hybridization reaction to hybridize under hybridization conditions and wash; And (e) measuring a hybridization signal of the labeled control nucleic acid from the hybridization reaction obtained, and comparing the hybridization signal of the control nucleic acid obtained in step (c) to determine the degree of change of the hybridization signal, Wherein said target nucleic acid and control nucleic acid have a sequence complementary to said polynucleotide probe. The method of claim 4, further comprising determining that the target nucleic acid exists when the hybridization signal decreases, and determining that the target nucleic acid does not exist when the hybridization signal does not change. How to. The method of claim 4, wherein (f) performing the steps (b) to (e) by varying the concentration ratio of the target nucleic acid to the control nucleic acid to obtain a correlation between the concentration ratio and the change of the hybridization signal; and (g) comparing the change value of the hybridization signal obtained in (e) with the correlation to determine the concentration of the target nucleic acid.