KR100381727B1 - A Method for Identifying DNA Mutation Using Microwell and Kit Therefor - Google Patents

Abstract

The present invention relates to a method for identifying a DNA mutation using a microwell and a kit used in the method, which can inexpensively, easily and stably identify mutations such as substitution, insertion and deletion of bases in DNA. Identification method of DNA mutation using the microwell of the present invention comprises the steps of using a primer combined with biotin, and amplifying a portion of the DNA to identify the mutation by PCR method to obtain a biotin-bound base sequence; Constructing a probe consisting of a normal sequence of a DNA portion to be identified for mutation; Coupling the probe constructed in the previous step to an amine group in the microwell; Adding the biotin-bound nucleotide sequence obtained in the previous step to the probe-coupled microwell; Adding streptavidin-degrading enzyme to the microwells to bind the electrolytic enzyme to the biotin of the probe; And adding a substrate to the microwell to react with the electric streptavidin-degrading enzyme, and measuring color change or absorbance exhibited by the enzymatic reaction. According to the present invention, in any DNA mutation identification experiment, if a probe consisting of the nucleotide sequence of the DNA portion to be identified and a predetermined portion of the DNA can be amplified, it is cheaper, simpler and more stable than any conventional method. Mutations can be identified.

Description

A Method for Identifying DNA Mutation Using Microwell and Kit Therefor}

Long ago, methods for identifying which organisms or diseases by identification of genes have been widely used (Journal of Clinical Microbiology, 34 (1): 130-133 (1996)). In this case, various drugs are treated according to the type or disease of the organism, but when the disease is caused by the infection of the microorganism and new strains are generated through mutation, the existing drugs cannot be treated.

Conventionally, a method of searching for mutations such as substitution, insertion, and deletion of bases present in the DNA sequence includes dot blotting, restriction fragment-length polymorphism (RFLP), and single-strand conformational polymorphism (SSCP). Or nucleotide analysis of DNA sequences is used:

First, the dot blotting method consists of oligonucleotides of a certain size that are intended to be placed on a membrane to check DNA by using properties that do not bind to each other even if one nucleotide sequence is different over a certain temperature, which is Southern principle. It is a method of binding a nucleotide to an enzyme used for radioactivity, fluorescent material, and a color development reaction to check whether there is a mutation or not (see British Journal of Dermatology, 131 (1): 72-77 (1994)). . In addition, oligonucleotides may be bound to the membrane and DNA amplified to confirm their binding.

Second, the RFLP method uses restriction properties that restriction enzymes can only cleave a specific sequence. When a DNA sequence amplified by PCR is processed with a restriction enzyme, if a mutation exists in the restriction region of the restriction enzyme, this sequence is used. Will not be cut. Therefore, if a DNA sequence carrying a mutation in a normal DNA sequence and a restriction enzyme cleavage site is cut with the same restriction enzyme, the normal sequence is cleaved, but the nucleotide sequence having the mutation is not cleaved. After treating the restriction enzyme as described above, electrophoresis on the same gel results in a different number of electrophoretic bands of normal DNA and mutant DNA, which is a method of confirming by comparing DNA electrophoresis patterns in a gel state ( See Molecular Cell Biology, 1995: 279-281.

Third, the SSCP method takes advantage of the fact that the bases (A, T, C, G) constituting DNA are attracted to electricity. In other words, if the normal sequence and the sequence with mutations are denatured and made into single strands, then recombined, and then electrophoresed, it is possible to confirm even if one base is changed. This method is compared with that of Molecular Cell Biology (1995).

In addition, there is a method of identifying the presence of mutations by analyzing the DNA base sequence to be confirmed by gel or machine compared with the normal sequence (Molecular Cell Biology, 1995: 245-248). These methods use radioactivity, fluorescent materials, or enzymes. When using radioactivity, there are disadvantages in terms of stability and high cost in the treatment of wastes. Even without using radioactivity, membranes or acrylamide are used. ) Difficulty in handling due to electrophoresis on gel.

On the other hand, the presence or absence of mutations can be determined by applying a method of checking for the presence of a specific gene using a microwell, which is used to examine a protein using an antibody or to confirm the presence of a specific gene by binding DNA or oligonucleotides. . For example, by attaching a sample (sample) to the microwell, and making a probe by using a normal oligonucleotide to confirm the part to be confirmed, and attaching the sample amplified by PCR to the microwell, biotin ( Biotin) is a method of confirming the binding of enzymes by applying hybridization technology (Molecular Cell Probes, 6 (1): 79-85 (1992)). However, this method takes a lot of testing time because the microwell is attached to the patient and the sample is used. However, the presence or absence of mutation can be confirmed. In this case, the PCR amplified sample is used. Not only does the DNA have a secondary structure that can interfere with the probe, but the longer the DNA, the less likely it is to bind to the well (Analytical Biochemistry, 1991; 138-142). In addition, when using the sample to drop the double strands and then coupled to the well, there is a problem in that the recombination occurs easily during the experiment, so that the bond with the probe is reduced.

Summary of the Invention

Therefore, the present inventors have diligently researched to develop a technology that can identify DNA mutations cheaply and stably using microwells. As a result, the present inventors have made inexpensive mutations in DNA samples using enzymes that bind microwells and biotin. The present invention was completed by confirming that it can be judged simply and stably.

After all, the main object of the present invention is to provide a method for identifying a normal DNA mutation known in the base sequence using a microwell by PCR amplification.

Another object of the present invention to provide a kit for identifying a DNA mutation that can be easily carried out the method.

The present invention relates to methods and kits for identifying DNA mutations using microwells. More specifically, the present invention relates to a method for identifying a DNA mutation using a microwell and a kit used in the method, wherein the mutation, such as substitution, insertion, and deletion of a base in DNA, can be confirmed inexpensively, simply, and stably. It is about.

1 is a graph showing the results of confirming the mutation of the DNA according to the method of the present invention.

Identification method of DNA mutation using the microwell of the present invention comprises the steps of using a primer combined with biotin, and amplifying a portion of the DNA to identify the mutation by PCR method to obtain a biotin-bound base sequence; Constructing a probe consisting of a normal sequence of a DNA portion to be identified for mutation; Coupling the probe constructed in the previous step to an amine group in the microwell; Adding the PCR fragment obtained in the previous step to the probe-coupled microwell; Adding streptavidin-degrading enzyme to the microwell to bind the biotin of the probe; And adding a substrate that reacts with the degrading enzyme added to the microwell, and measuring color change or absorbance exhibited by the enzymatic reaction.

In addition, the kit for identifying DNA mutations of the present invention is used to remove microwells having an amine group attached to the inner surface, an EDC solution used when attaching a probe, a 1-methylimidazole solution having a pH of 7.0, and an unbound probe. 0.4M NaOH / 0.25% Tween-20 solution, dH ₂ O treated in microwells at blocking, solution consisting of 20XSSPE / 0.0167% Triton X-100, and Salmon sperm DNA (10 mg / mL), DNA 0.5x SSC, 0.1% Tween-20 solution, streptavidin-degrading enzyme that binds to biotin, biotin and strep, used for washing unbound DNA amplification sample after binding to amplification sample and probe attached to microwell 150 mM NaCl, 100 mM Tris-HCl (pH 7.5) solution used for binding of tavidin, 150 mM NaCl / 0.1% Tween-20, 100 mM Tris-HCl (pH 7.5) solution and streptavidin- for washing streptavidin It consists of a substrate that reacts with a degrading enzyme.

Hereinafter, the DNA mutation identification method of the present invention will be described in more detail by dividing step by step.

As used herein, the term “mutation” means a substitution in which one or more nucleotide sequences are changed, a deletion in which one or more nucleotide sequences are lost, or an insertion in which one or more nucleotide sequences are not present. .

Mutations caused by changes in one or more sequences in the form of mutations are mutations that occur commonly in microorganisms. For example, if a nucleotide sequence change at a certain position in Mycobacterium tuberculosis is known to be resistant to rifampicin (see Lancet 341: 647-650 (1993)), the presence or absence of a mutation, as well as any nucleotide sequence mutation It would be very useful for the treatment of the patient if it could be confirmed whether

In the method for identifying a mutation according to the present invention, as well as whether or not there is a mutation, it is easy to check how any nucleotide sequence is substituted, inserted or deleted, and thus prepares an amplified sample as described below. Preparing a sample, attaching the probe to the microwell, and then combining the probe with the sample to confirm the probe.

Step 1: Amplify the Sample

Using a primer bound to the biotin, and amplifying the portion of the DNA to be identified by the PCR method to obtain a biotin bound sequence: PCR is performed by binding the biotin to the primer, PCR When amplified by the method, there are complementary sequences in which the biotin is attached and the sequences in which the biotin is not attached.

Step 2: construct the probe

Construct a probe consisting of the normal nucleotide sequence of the DNA region to be identified for mutation: the constructed probe consists of 10 or more DNA base sequences, the 5 'end of the nucleotide sequence capable of binding to the amine groups of the microwells. Includes phosphate.

Step 3: Attach the Probe

The probe constructed in the previous step is combined with the amine group of the microwell: the constructed probe may naturally bond within the sequence, so that the probe is first constructed from 90 to 100 ° C. in order to single-chain the constructed probe. , Most preferably at 94 ° C., for 5 to 15 minutes, most preferably for 10 minutes, then immediately put on ice to cool. Cooled pH 7.0, 10 mM 1-methylimidazole solution and cooled pH 7.0, 10 mM 1-ethyl, which catalyze the reaction of phosphides of oligonucleotides with amine groups in microwells in a single chain probe 3- (3-dimethylaminopropyl) -carbodiimide (1-ethyl-3- (3-dimethylamino propyl) -carbodiimide, EDC) solution was added, and this was a microwell having an amine group (-NH ₂ ) inside After being placed in a tube, it is treated with a tape or the like so as not to evaporate, and left at 40 to 60 ° C., most preferably at 50 ° C. for 5 to 9 hours, most preferably 7 hours, and the probe adheres to the amine group of the microwell. . Probe-attached microwells are washed at room temperature with 0.4M NaOH / 0.25% Tween-20 and each well is washed again with distilled water.

Step 4: Add the Sample to the Microwell

The PCR fragment obtained in the previous step is added to the microwell to which the probe is bound: first, inside the well with a solution consisting of dH ₂ O, 20XSSPE / 0.0167% Triton X-100, and Salmon sperm DNA (10 mg / ml). 10 to 20 minutes, most preferably 15 minutes, at 40 to 60 ℃, most preferably 50 ℃ to prevent binding between the amine group and the biotin-bound sample of the well that does not bind to the probe. In order to make the double-chain DNA sample of the biotin-bound sample obtained in step 1 into a single chain so as to bind the probe, the biotin-coupled sample was 90 to 98 ° C, most preferably at 94 ° C for 5 to 15 minutes. Most preferably for 10 minutes and immediately put on ice. Subsequently, a single chain DNA sample was mixed by adding a solution consisting of dH ₂ O, 20XSSPE / 0.0167% Triton X-100, and Salmon sperm DNA (10 mg / ml), and then the mixed solution was added to the microwell. At 70 ° C., most preferably 60 ° C., for 5-15 hours, most preferably 10 hours. Samples are removed after the reaction and the microwells are washed with 0.5 × SSC, 0.1% Tween-20. If a mutated sample is added, the affinity of the sample for the microwell probe will be low, but if the sample is not mutated, the affinity of the sample for the microwell probe will be maintained at a high level. Thus, the biotin is bound to the microwells, and the difference between the mutated and non-mutated samples may be measured in terms of the density of the biotin bound to the microwells.

Step 5: Add Degrading Enzymes

Add the streptavidin-degrading enzyme to the microwells to bind the biotin of the probe: bind the enzyme to the biotin to measure the difference in density of the biotin bound to the microwells in order to measure the difference between the mutated and non-mutated samples. Let's do it. The degrading enzyme used may be an enzyme that can decompose the substrate and measure the color reaction or absorbance. For example, streptavidin-alkaline phosphatase can be used, which, if used with enzymes, first uses the microwells in 150 mM NaCl, 0.1% Tween-20, 100 mM Tris-HCl (pH 7.5) solution. And then streptavidin-alkalinephosphatase diluted with 150 mM NaCl, 100 mM Tris-HCl (pH 7.5) solution into a microwell, at about 35-45 ° C., most preferably at 40 ° C., 30-90 ° C. Minutes, most preferably 60 minutes. Then, the reaction solution was removed and 150 mM NaCl, 0.1% Tween-20, 100 mM Tris-HCl (pH 7.5) solution was added to the microwell, and then 50 to 70 ° C, most preferably at 60 ° C for 5 to 15 minutes. Preferably, it is left for 10 minutes and then washed.

Step 6: Confirm the Enzyme Reaction

Add a substrate reacting with streptavidin-degrading enzyme added to the microwell, and measure the color change or absorbance exhibited by the enzymatic reaction: the substrate exhibits a color change or absorbance when degraded by the enzyme-substrate reaction. Preferred peptide substrates are preferred. For example, when using streptavidin-alkaline phosphatase as a degrading enzyme, 100 μl of p-nitrophenyl phosphate (pNPP) is added to the microwell as a substrate to react thereto, and 60 to 120 minutes, most preferably 90 minutes. The reaction was stopped by addition of 1M NaOH and the absorbance was measured at 405 nm. Equipment for measuring the absorbance may use a conventional spectrophotometer, but in order to easily measure the enzyme-substrate reaction solution contained in the microwell, it is preferable to measure using an ELISA reader.

According to the present invention, there is also provided a kit comprising the following components for the method: a microwell having an amine group attached to its inner surface; PH 7.0, 10 mM EDC solution and pH 7.0, 10 mM 1-methylimidazole solution used to attach oligonucleotides; 0.4M NaOH / 0.25% Tween-20 solution used to remove unbound oligonucleotides; With dH ₂ O, 20XSSP / 0.0167% Triton X-100, and Salmon sperm DNA (10 mg / mL) used to bind to the DNA amplification sample to be processed or blocked at the time of blocking and oligonucleotides attached to the microwell. Composed solution; 0.5 × SSC, 0.1% Tween-20 solution, used to wash unbound oligonucleotides after binding to the DNA amplification sample and oligonucleotides attached to the microwells; Streptavidin-degrading enzyme that binds to biotin; 150 mM NaCl, 100 mM Tris-HCl (pH 7.5) solution used for binding biotin to streptavidin; 150 mM NaCl / 0.1% Tween-20, 100 mM Tris-HCl, pH 7.5 solution for washing streptavidin; And a substrate that reacts with streptavidin-degrading enzyme.

According to the present invention, the difference between the mutated sample and the non-mutated sample can be measured in terms of the density of biotin bound to the microwell. For this purpose, after binding the enzyme to biotin, the substrate reacts to the enzyme. By measuring the activity of the enzyme by adding, it is possible to indirectly confirm the type of mutation as well as whether the sample is mutated. Therefore, in any DNA mutation identification experiment, if a probe consisting of the normal nucleotide sequence of the DNA portion to be confirmed and a certain portion of the electric DNA can be amplified, it is cheaper, simpler and more stable than any conventional method. Mutations can be identified.

That is, the method of identifying DNA mutations using the microwell of the present invention has the following advantages as compared to the conventional methods used:

1. In the conventional method, the cost is excellent because the same effect can be obtained even if the amount is much smaller than the amount of oligonucleotide to be added to the microwell. Conventionally used methods put about 600ng / well in the same experiment (see Molecular and Celluar Probes, 12: 407-416 (1998)), but in the present invention, even if put 100ng / well, similar results compared to each other (Absorbance) is obtained.

2. In the present invention, microwells prepared by attaching oligonucleotides to which oligonucleotides have been attached can be prepared by attaching oligonucleotides to reduce costs in both oligonucleotides and microwells.

3. When confirming the mutation by attaching oligonucleotides to a conventional microwell having an amine group, the high absorbance value of the negative control is a problem, using the microwell used in the present invention, the absorbance value of the negative control Can drop significantly.

4. In the present invention, the secondary structure, low binding degree, and complementary binding between samples were solved by using a probe in the present invention.

5. Conventional methods, as described above, are very difficult to handle by using membranes or gels, and are difficult to identify a large amount of samples because they undergo a complex process of several steps, whereas the method of the present invention only Since all procedures are done in one well, the experiment can be performed simply.

6. Since the conventional method has variables such as sample and experimenter, it is impossible to experiment in a batch condition every time, while the kit prepared according to the present invention not only has the same condition but also can be stored for a long time, so Mutations can be detected for the sample. In particular, since there is no experimental error in humans when using a machine, it is possible to quantify experimental statistics or types of samples.

7. The method of the present invention can easily confirm the result by eye. That is, as a result of the experiment, even a simple color change can be identified with the naked eye, thereby giving the experimenter reliability.

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

Example 1 Identification of DNA Mutations Using Microwells

In the method of identifying DNA mutations using the microwell of the present invention, a probe having a normal nucleotide sequence of a DNA portion to be identified is coupled to a microwell, and the degree of binding between the probe and the DNA sample of the patient that complements the probe is measured. How to check for DNA mutations. In order to prove that the present invention is effective, a DNA sample of a patient having a normal base sequence is used for a direct sequenced DNA portion which is known to exist uniquely in the DNA of Mycobacterium tuberculosis BCG. The degree of binding was determined by binding to probes unrelated to the direct sequence as probes and negative controls of various types having base sequences with different mutations in one position of the direct sequence.

Example 1-1 : Use of a Probe Having a Normal Sequence

First, a primer b having a nucleotide sequence of 5'-GGTTTTGGGTCTGACGAC-3 '(SEQ ID NO: 1) having a biotin was prepared, and a primer b having a nucleotide sequence of 5'-CCGACAGGGGACGGAAAC-3' (SEQ ID NO: 2) was produced. , Korea). Then, 200 ng / μl of DNA solution containing the direct sequence of BCG, 0.5 μl of primer a (100 pmol / μl), 0.5 μl of primer b (100 pmol / μl), 0.4 μl of dNTP (25 mM), 10 × Taq buffer PCR was repeated for 30 minutes at 94 ° C for 1 minute, 55 ° C for 1 minute 30 seconds, and 72 ° C for 1 minute 30 seconds in a composition of 3 μl of MgCl ₂ (25 mM), 0.2 μl of Taq (5 unit / μl, Promega), and 40.4 μl of distilled water. Was performed.

Next, probe # 1 having a base sequence of 5'-TTGACCTCGCCAGGAGAGAAGATCA-3 '(SEQ ID NO: 3) having a normal DNA base sequence and probe # 2 having a base sequence of 5'-TCCGTACGCTCGAAACGCTTCCAAC-3' (SEQ ID NO: 4). (Bionia, Korea), each probe was dispensed to enter 10 pmol per well, and then heated to 94 ° C for 10 minutes. Then, after cooling for 10 minutes under ice-cooling, it was condensed with a centrifuge, and EDC and 1-methylimidazole were added to the condensed sample to pH 7.0 and 10 mM, respectively. Dispense 100 μl into microwells (Nunc, USA) on ice, leave for 7 hours at 50 ° C, and then add 138 μl dH ₂ O, 20XSSPE to each empty well to remove unbound PCR fragments. A solution consisting of /0.0167% Triton X-100, and 2 mu l of Salmon sperm DNA (10 mg / ml) was aliquoted and left at 50 ° C for 20 minutes.

The aliquots were removed and a mixture of 68 μl of dH ₂ O, 30 μl of 20 × SSPE / 0.0167% Triton X-100, 1 μl of Salmon sperm DNA (10 mg / ml) and 1 μl of DNA sample amplified by PCR was added to each well. After dispensing, the mixture was left at 60 DEG C for about 10 hours. The aliquot was removed, washed three times with 200 × of 0.5 × SSC, 0.1% Tween-20, and the same solution was added for 15 minutes and left at 60 ° C. Washed three times with the same solution, and then diluted 3000-fold with streptavidin-alkaline phosphatase with 100 mM Tris-HCl (pH 7.5) and 150 mM NaCl solution with 150 mM NaCl, 100 mM Tris-HCl (pH 7.5) solution. 100 μl was dispensed into the wells, and then left at 40 ° C. for 1 hour.

Next, the wells were washed three times with 150 mM NaCl / 0.1% Tween-20, 100 mM Tris-HCl (pH 7.5) solution, and then treated three times for 5 minutes at 60 ° C. with the same solution. 100 μl of pNPP (N7653, sigma, U.S.A.) was added to each well and reacted for 90 minutes, and the absorbance was measured at 405 nM (see FIG. 1).

Example 1-2 : Use of Probes with Substituted Sequences

For identification of substitution mutations, probe # 11 having a base sequence of 5'-TTGACCTCGCCAGAAGAGAAGATCA-3 '(SEQ ID NO: 5) by substituting G for the 14th base of probe # 1 in place of probe # 1 and probe # 2 with A Except for using the 14th base A of the probe # 2 with G to prepare a probe # 21 having a base sequence of 5'-TCCGTACGCTCGAGACGCTTCCAAC-3 '(SEQ ID NO: 6) (Bionia, Korea), DNA mutations were identified in the same manner as in Example 1-1 (see FIG. 1).

Example 1-3 Use of Probes with Inserted Sequences

To identify the insertion mutations, insert T into the 14th base of probe # 1 instead of probe # 1 and probe # 2 to obtain probe # 12 with a base sequence of 5'-TTGACCTCGCCAGTGAGAGAAGATCA-3 '(SEQ ID NO: 7). DNA mutations were confirmed in the same manner as in Example 1-1, except that they were prepared and used (Bionia, Korea) (see FIG. 1).

Example 1-4 : Use of Probes with Deleted Sequences

To identify the deletion mutation, remove G, the 14th base sequence of probe # 1, instead of probe # 1 and probe # 2, replacing probe # 13 with the base sequence of 5'-TTGACCTCGCCAGAGAGAAGATCA-3 '(SEQ ID NO: 8). DNA mutations were confirmed in the same manner as in Example 1-1, except that they were prepared and used (Bionia, Korea) (see FIG. 1).

Example 1-5 : Use of a Voice Control Probe

For use as a negative control of the present invention, probe #C comprising a nucleotide sequence of 5'-GGAGCTTTCCGGCTTCTATCAGGTA-3 '(SEQ ID NO: 9) containing a nucleotide sequence uniquely present in the DNA of H37Rv, a type of Mycobacterium tuberculosis DNA mutations were confirmed in the same manner as in Example 1-1, except that they were prepared and used (Bionia, Korea) (see FIG. 1).

FIG. 1 illustrates a DNA sample of a patient having a normal sequencing probe in a DNA sequence of BCG, which is known to exist solely in the DNA of tuberculosis bacterium. Several types of probes and negative controls with mutated nucleotide sequences, each of which was bound to a probe unrelated to the direct sequence, showing the degree of binding of each DNA. No. 1 is probe #C, a negative control; No. 2 is probe # 1 having normal sequence; No. 3 is probe # 11 which is a substitution mutant; No. 4 is probe # 13 which is a deletion mutant; No. 5 is probe # 12 which is an insert mutant; No. 6 is probe # 2 having normal sequence; And No. 7 shows the case where probe # 21 which is a substitution mutant was used, respectively.

As shown in FIG. 1, when the patient's DNA sample reacted with a probe having a normal DNA sequence, the absorbance was the highest, indicating that the patient's DNA sample was normal. On the other hand, if the DNA sample of the patient reacts with the probe with the mutated nucleotide sequence, it is determined that the DNA sample of the patient reacts with the probe with the normal DNA nucleotide sequence, although it varies depending on the type of mutation. Since the absorbance was significantly lower than the absorbance, the presence or absence of mutation of DNA was clearly confirmed.

By applying the present invention, when using a probe having a nucleotide sequence showing each mutation for each base sequence, it is possible to determine the position and type of the mutation, even if the mutation occurs in one or more bases, the position and type of mutation You can check it.

As described and demonstrated in detail above, the present invention has the effect of identifying substitutions, deletions and insertions as well as the presence or absence of one or more mutations, which is cheaper, simpler and more stable than any conventional method.

Claims (18)

(Iii) amplifying a portion of the DNA to be identified for mutation using a primer with a biotin conjugated by PCR to obtain a biotin-coupled base sequence; (Ii) constructing a probe consisting of the normal sequence of the DNA portion to be identified for mutation; (Iii) binding the probe constructed in step (ii) to the amine group of the microwell; (Iii) adding the biotin-bound base sequence obtained in step (i) to the microwell to which the probe is bound; (Iii) adding streptavidin-degrading enzyme to the microwell to bind the electrolytic enzyme and the biotin of the probe; And, (Iii) adding a substrate to the microwells for reaction with the electrical streptavidin-degrading enzyme and measuring the color change or absorbance exhibited by the enzymatic reaction, Method for Confirming DNA Mutation Using Microwells. The method of claim 1, Constructed probe is more than 10 sequences, and the 5 'end of the sequence It characterized in that it comprises a phosphate (phosphate) Method for Confirming DNA Mutation Using Microwells. The method of claim 1, (Iii) the step of preparing the probe in a single chain into the microwell, and reacting by adding a catalyst for reacting the phosphate of the single-chain probe with the amine group of the microwell, and then washing the microwell Method for Confirming DNA Mutation Using Microwells. The method of claim 3, wherein The catalyst was cooled to pH 7.0 with 10 mM 1-methylimidazole (1-methy- limidazole) solution and cooled to pH 7.0 10 mM 1-ethyl-3- (3-dimethyl Aminopropyl) -carbodiimide (1-ethyl-3- (3-dimethylamino- propyl) -carbodiimide, EDC) solution, characterized in that Method for Confirming DNA Mutation Using Microwells. The method of claim 3, wherein Washing was performed with 0.4M NaOH / 0.25% Tween-20 solution. Characterized Method for Confirming DNA Mutation Using Microwells. The method of claim 1, In step (iv), the sample obtained in step (i) Place in the well and combine with the probe, remove the sample and remove the microwell Characterized in that it comprises a process of washing Method for Confirming DNA Mutation Using Microwells. The method of claim 6, Sample obtained in step (i) is made into a single chain into a microwell Before, dH ₂ O, 20XSSPE / 0.0167% Triton X-100 and Salmon sperm A solution containing DNA (10 mg / ml) was added to a microwell for 20 minutes at 50 ° C. Characterized in that it further comprises the process of leaving the liver Method for Confirming DNA Mutation Using Microwells. The method of claim 6, When the sample obtained in step (i) is combined with the probe, dH ₂ O, 20XSSPE / 0.0167% Triton X-100 and Salmon sperm DNA (10 mg / Ml) is added Method for Confirming DNA Mutation Using Microwells. The method of claim 6, Washing was performed with 0.5x SSC, 0.1% Tween-20 solution. Characterized Method for Confirming DNA Mutation Using Microwells. The method of claim 1, Step (v) washes the microwells first and stops streptavidin-degrading enzyme. Place in microwells and combine with biotin, remove the reaction solution Characterized in that it comprises a second washing process Method for Confirming DNA Mutation Using Microwells. The method of claim 10, First wash 100 mM Tris-HCl (pH 7.5), 150 mM NaCl / 0.1% Characterized in that it is carried out with a Tween-20 solution Method for Confirming DNA Mutation Using Microwells. The method of claim 10, Streptavidin-degrading enzyme is 150 mM NaCl, 100 mM Tris-HCl (pH 7.5) Streptavidin-alkaline phosphatase diluted with solution alkaline phosphatase) Method for Confirming DNA Mutation Using Microwells. The method of claim 10, 2nd wash 150mM NaCl / 0.1% Tween-20, 100mM Tris-HCl (pH 7.5) The solution is placed in a microwell and left at 60 ° C for 10 minutes, Characterized by washing Method for Confirming DNA Mutation Using Microwells. The method of claim 1, Substrates that react with streptavidin-degrading enzymes can be degraded by enzyme-substrate reactions. Is a synthetic peptide substrate that exhibits color change or absorbance. Characterized by Method for Confirming DNA Mutation Using Microwells. The method of claim 14, If streptavidin-degrading enzyme is streptavidin-alkaline phosphatase, Substrate is characterized in that the p-nitrophenyl phosphate (pNPP) Method for Confirming DNA Mutation Using Microwells. The method of claim 1, Color change is characterized by measuring with the naked eye Method for Confirming DNA Mutation Using Microwells. The method of claim 1, Absorbance is measured using an ELISA reader. Characterized Method for Confirming DNA Mutation Using Microwells. (i) microwells with amine groups attached to the inner surface; (Ii) pH 7.0, 10 mM EDC solution and pH 7.0, 10 mM 1-methylimidazole solution; (Iii) 0.4M NaOH / 0.25% Tween-20 solution; (Iii) a solution comprising dH ₂ O, 20 × SSPE / 0.0167% Triton X-100 and Salmon sperm DNA (10 mg / ml); (Iii) 0.5 × SSC, 0.1% Tween-20 solution; (Iii) streptavidin-alkaline phosphatase; (Iii) 100 mM Tris-HCl, pH 7.5 solution containing 150 mM NaCl; (Iii) 100 mM Tris-HCl, pH 7.5 solution containing 150 mM NaCl / 0.1% Tween-20; And, (Iii) pNPP (p-nitrophenyl phosphate), DNA Mutation Identification Kit.