KR20000074881A - Method and kit for identifying DNA mutation using the microwell - Google Patents

Abstract

PURPOSE: A method and a kit for identifying DNA mutation using microwell that can safely confirm replacement, defect and insertion as well as existence of base sequence of the mutation with low cost are provided. CONSTITUTION: The identifying method is characterized by the followings : (i) preparing a sample of base sequence with biotin by amplifying a desired part of DNA with PCR(Polymerase Chain Reaction) ; (ii) making oligoneucleotide as a probe composed of more than 10 complimentary base sequences with phosphate on the end of 5¬1 of the base sequence ; (iii) bonding the probe to amine group of microwell ; (iv) adding the sample to the microwell and binding with the probe ; (v) adding enzyme to the microwell and joining with the biotin ; and (vi) then identifying mutation by determining color change or absorptivity by the enzyme. The kit comprises microwell, a solution of EDC(Ethylene Dichloride) and 1-methylimidazole to bond the oligoneucleotide, a solution of 0.4M NaOH/0.25%tWeen 20, a solution composed of dH2O 138microliters, 20XSSP/0.0167% TritonX-100 and Slamon sperm DNA 2microliters for blocking, a solution composed of dH2O 68microliters, 20XSSP/0.0167% TritonX-100 30microliters and Slamon sperm DNA 1microliters for binding the sample with the oligoneucleotide, 0.5*SSC and 0.1%Tween solution for washing unbonded oligoneucleotide, streptabidin, a solution of 100mM Tris-HCI(pH7.5) and 150mM NaCl, a solution of 100mM Tris-HCI(pH7.5) and 150mM NaCl/0.1%Tween 20, and substrate reacting with the enzymes.

Description

Method and kit for identifying DNA mutation using the microwell

The present invention relates to a method and kit for identifying DNA mutations using microwells. More particularly, the present invention relates to a plastic microchip capable of inexpensively, simply and stably identifying mutations such as substitution, insertion and deletion of one base in DNA. The present invention relates to a method for identifying a DNA mutation using a well and a kit used in the method.

In general, a method of determining which organism or disease is present by the presence of a gene has been used for a long time (Journal of Clinical Microbiology 1996 Jan; 34 (1): 130-133). In this case, depending on the type or disease of the organism to treat it using different drugs, in which case new varieties are generated that can not be treated with existing drugs. Many of these variants are made through mutations.

Conventional methods for detecting mutations such as substitution, insertion, and deletion of bases present in the base sequence of DNA include dot blotting, RFLP (Restriction Fragment-Length Polymorphism, and SSCP (Single-Strand Conformational Polymorphism). Alternatively, nucleotide analysis is used to analyze the nucleotide sequence of DNA.

First of all, the dot blotting method (British Journal of Dermatology 1994 Jul; 131 (1): 72-77) is characterized by the fact that one base sequence does not bind to each other even if it is above a certain temperature, which is the Southern principle. By attaching DNA to the membrane and binding the oligonucleotide consisting of nucleotide sequences of a certain size to check for radioactivity, fluorescent materials, and enzymes used for color reactions to check whether there is a mutation with or without the signal. . In addition, oligonucleotides are bound to the membrane and DNA is amplified to confirm their binding. Second, RFLP (Molecular Cell Biology 1995: 279-281) method utilizes the property that restriction enzymes can only cut a specific sequence. When a DNA sequence sequenced by PCR is treated with a restriction enzyme, the sequence will not be cleaved if a mutation exists at the cleavage site of the restriction enzyme. Therefore, if a DNA sequence carrying a mutation in a normal DNA sequence and a restriction enzyme cleavage site is cleaved with the same restriction enzyme, the normal sequence is cleaved, but the nucleotide sequence having the mutation is not cleaved. After treatment with the restriction enzyme as described above, electrophoresis on the same gel will give a different number of electrophoretic bands of normal DNA and DNA with mutations. Thus, it is a method of confirming by comparing the DNA electrophoresis form in a gel state. Third, SSCP method (Molecular Cell Biology 1995: 289) takes advantage of the fact that the bases (A, T, C, G) constituting DNA are attracted to electricity. Methodically denatured DNA with normal and mutant is made into single strands and then recombined and electrophoresed, even if one base is changed. Thus, the method of confirming the pattern of DNA in the electrophoresis gel state compared with the normal one. In addition, there is a method of identifying the presence or absence of mutations by comparing DNA sequences to be confirmed by gel or machine (Nolecular 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, membrane or acrylamide is used. Electrophoresis on the gel is difficult to manipulate.

Microwells, on the other hand, are used to test proteins using antibodies and to confirm the presence of specific genes by binding DNA or oligonucleotides to be tested. In this way, the presence or absence of a mutation can be confirmed by applying a method of checking whether a specific gene exists using a microwell. For example, there is a method of attaching a sample (sample) to a microwell and confirming the part to be confirmed by making a probe with normal oligonucleotide (Molecular Cell Probes 1992 Feb; 6 (1): 79-85). . This method is a method of attaching a sample amplified by PCR to a microwell and confirming the binding of the biotin-attached probe using the hybrid method using an enzyme. However, this method takes a lot of testing time because the sample is taken from the patient and attached to the microwell, and the presence or absence of a mutation can be confirmed. In this case, the PCR amplified sample is used. In addition to having a secondary structure of DNA, it may interfere with the binding of the probe, the longer the DNA is less bound to the well (Analytical Biochemistry, 1991; 138-142). In addition, when the sample is used, the double strands are dropped and then bound to the wells, and thus recombination occurs easily during the experiment, thereby decreasing the binding degree with the probe.

Therefore, an object of the present invention is to identify DNA mutations using plastic microwells that can solve the problems of existing methods for identifying DNA mutations by PCR amplification of normal DNA mutations having known sequence sequences. To provide.

Another object of the present invention is to provide a kit which can easily carry out the method.

Confirmation method of the present invention for achieving the above object comprises the steps of providing a nucleotide sequence of the organism as a biotin attached sample (sample) through PCR; Preparing a probe consisting of a base sequence complementary to the sample and having a phosphate attached to a 5 'end of the base sequence; Coupling the probe to an amine group in a microwell; Adding the sample to the microwell to bind the probe; Adding an enzyme to the microwell to bind biotin; And identifying the mutation by measuring color change or absorbance by the enzyme.

The kit of the present invention for achieving the above another object is to remove the microwells attached to the inner surface of the amine group, the EDC solution used when attaching the oligonucleotide, the 1-methylimidazole solution having a pH of 7.0, unbound oligonucleotides 0.4 M NaOH / 0.25% Tween 20 solution, 138 μl dH ₂ O, 20 × SSPE / 0.0167% TritonX-100, and ₂ μl Salmon sperm DNA (10 mg / ml) treated in microwells at blocking The solution consists of 68 μl of dH ₂ O, 30 μl of 20XSSPE / 0.0167% TritonX-100, and 1 μl of Salmon sperm DNA (10 mg / ml) used to bind the solution, DNA amplification sample and oligonucleotides attached to the microwells. 0.5 × SSC, 0.1% Tween 20 solution, streptavidin-enzyme that binds to biotin, combined with amplified sample, DNA amplification sample and oligonucleotides attached to microwells, and then used to wash unbound oligonucleotides. 100 mM Tris-HCl (pH 7.5), 150 mM NaCl solution, 100 mM Tris-HCl (pH 7.5) for washing streptavidin, 150 mM NaCl / 0.1% Tween 20 solution, used for binding iotin and streptavidin, and It consists of a substrate that reacts with enzymes.

1 is a graph showing a result of confirming the mutation (constant sequence presence and absence, substitution, deletion, insertion) of the tuberculosis bacterium (BCG) according to an embodiment of the present invention.

Hereinafter, the method of the present invention will be described in more detail.

As mentioned above, the forms of mutations include substitutions in which one or more nucleotide sequences are altered, deletions in which one or more existing nucleotide sequences are lost, and insertions in which one or more nucleotide sequences are not present. Mutations that occur due to a change in one or more sequences in the form of a mutation are mutations that commonly occur in microorganisms. For example, it was found that a change in one nucleotide sequence at a certain position in Mycobacterium tuberculosis is resistant to rifampicin (Lancet 1993; 341: 647-650). Therefore, if the presence or absence of the mutation as well as which nucleotide sequence can identify the mutation, it will be very useful in the treatment of patients.

As described above, the present invention is a method capable of easily confirming not only the presence or absence of mutations, but also how the nucleotide sequences are substituted or inserted and deleted.

The method of identifying a mutation according to the present invention is to prepare a sample (sample), prepare a probe (probe), attach the probe to the microwell, and then combine the sample and the probe, and then confirm the process Rough

Preparation of Samples

Amplify the portion of the DNA to identify the mutation by PCR method. At this time, it is prepared by binding the biotin (biotin) to one of the primers. When amplified by the PCR method, a base sequence without biotin and a base sequence without biotin are complementarily present in the amplified portion. In the present invention, a base sequence with biotin is used.

Making of the probe

Prepare oligonucleotides to be used as probes with DNA sequencing (or normal DNA sequencing, if mutation is confirmed) complementary to the nucleotide sequence of the DNA to be identified. The oligonucleotide prepared is composed of 10 or more base sequences and binds phosphate to the 5 'end of the base sequence.

According to the present invention, in order to confirm the presence or absence of substitution, deletion, or deletion of one or more bases in a DNA base sequence, an oligonucleotide having a base sequence complementary to a normal base sequence is prepared and used as a probe. These probes bind normal DNA but not mutated DNA. When you want to know the nucleotide sequence of the substituted mutant, all four parts except the base to be identified have the base sequence complementary to the normal nucleotide sequence, and four oligos each having the base of the site to be identified are A, C, G, and T. A nucleotide is made and used as a probe. The base binds complementarily and thus binds to three bases that are complementary but not complementary. As a positive control, oligonucleotides prepared by the nucleotide sequence of the part to be amplified are used, and a negative control uses oligonucleotides arranged with a DNA base sequence not present in a sample without the oligonucleotide or the DNA to be identified.

Attachment of the probe

The oligonucleotides prepared as described above are boiled at about 94 ° C. for about 10 minutes to form a single strand, and then immediately put on ice and cooled. 1-methylimidazole and cooled 1-ethyl-3- (3) at pH 7.0, which catalyze the reaction of the phosphate of oligonucleotides with the amine groups of the microwells on the cooled sample. -Dimethylaminopropyl) -carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) is added. The sample thus prepared is placed in a microwell having an amine group (-NH) on the inner surface used for PCR. If it is treated with a tape or the like so as not to evaporate and treated at about 50 ° C. for about 5 hours or more, the probe is attached.

wash

Probe-attached microwells are washed with 200 μl of 4M NaOH / 0.25% Tween 20 at room temperature, and each well with 200 μl of distilled water.

Combination of Probes and Samples

To prevent the inside of the wells that do not bind oligonucleotides from binding to the sample, the inside of the wells with a solution of 138 μl of dH ₂ O, 20XSSPE / 0.0167% TritonX-100, and 2 μl of Salmon sperm DNA (10 mg / ml) Treat at about 50 ° C. for about 20 minutes. Since the DNA amplification sample prepared above is double stranded and cannot be combined with a single stranded probe in the well, it should be made single stranded. To do this, boil at about 94 ° C for about 10 minutes and immediately place on ice. The single stranded DNA amplification sample is added to each well as a solution having the following composition, and incubated at about 60 ° C. or more for about 5 hours or more,

68 μl of dH ₂ O, 30 μl of 20 × SSPE / 0.0167% TritonX-100, 1 μl of Salmon sperm DNA (10 mg / ml), and 1 μl of DNA amplification sample by PCR.

Removal of Unbound DNA Amplification Solution

Discard the sample after incubation. Wash each well with 200 μl 0.5 × SSC, 0.1% Tween 20 solution. Into each well, add 200 µl of 0.5x SSC, 0.1% Tween 20 solution, and wash with 200 µl of 0.5x SSC, 0.1% Tween 20.

In the present invention, when removing the portion that does not bind with the solution when the probe and the sample are combined, the solution is changed differently to remove DNA that is not bound more clearly.

Combination of Biotin and Enzyme

After washing with 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20, the enzyme (streptavidin-Alkalinephosphatase) was added with 100 mM Tris-HCl (pH7.5), 150 mM NaCl. Dilute to a volume ratio of about 1: 3000 and place 100 μl in each well, and then incubate at about 40 ° C. for about 1 hour to bind biotin and streptavidin attached to the enzyme. This is because the sample contains biotin. Meanwhile, after incubating the enzyme and biotin, each well was washed with 200 μl of 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20 solution, and 100 mM Tris-HCl (pH 7.5), 150 mM NaCl / 0.1% Incubate at 200 ℃ or higher with 200µl of Tween 20 solution. next. Wash with 200 μl of 100 mM Tris-HCl, pH 7.5, 150 mM NaCl / 0.1% Tween 20 solution.

Confirm

In order to confirm the binding of each well probe and DNA, 100 μl of pNPP (p -Nitrophenyl Phosphate) solution (sigma N7653) was added to each well and incubated at room temperature for about 1 hour. 1 M NaOH was added to stop the reaction, and the absorbance was measured at 405 nm with an ELISA reader.

The method of the present invention can be manipulated more easily than the methods used so far to find mutations present in DNA. This is possible by providing a microwell with a prefabricated probe. In other words, well-known mutations can provide microwells industrially by constructing a probe with their complementary sequences in advance. These microwells can be used to identify mutations even in the absence of specialized knowledge. Therefore, the present invention can be mass-produced by providing a microwell having a probe attached with a base sequence complementary to a mutation of a specific organism and a reagent used in the method of the present invention. For example, microwells with amine groups attached to the inner surface, EDC solution used to attach oligonucleotides, 1-methy ₇ (1-methylimidazole) solution at pH 7.0, 0.4M used to remove unbound oligonucleotides Solution consisting of NaOH / 0.25% Tween 20 solution, 138 μl dH ₂ O, 20XSSP / 0.0167% TritonX-100, and ₂ μl Salmon sperm DNA (10 mg / ml) treated in microwells at blocking, DNA amplification A solution consisting of 68 μl of dH ₂ O, 30 μl of 20XSSPE / 0.0167% TritonX-100, and 1 μl of Salmon sperm DNA (10 mg / ml) used to bind the sample and oligonucleotides attached to the microwells. 0.5x SSC, 0.1% Tween 20 solution, streptavidin-enzyme that binds biotin, biotin and streptavidin to bind to amplification samples and oligonucleotides attached to microwells 100 mM Tris-HCl (pH7.5), 150 mM NaCl solution, 100 mM Tris-HCl (pH 7.5), 150 mM NaCl / 0.1% Tween 20 solution for washing streptavidin, and enzymes used in the binding of Kits consisting of substrates can be provided industrially.

According to the present invention, if only DNA oligonucleotides can be produced as a nucleotide sequence to be confirmed in any DNA mutation identification experiment, and a certain portion of DNA can be amplified, the experimenter who is not familiar with the experiment using the kit components in order by the method of the present invention. They can be used to find the mutation of DNA simpler than any conventional method.

As described above, the present invention is safer than any existing methods and can be easily tested at low cost. In addition, the presence or absence of mutations, as well as nucleotide sequence changes in the mutations can be easily identified. That is, the method of the present invention is inexpensive in terms of cost compared with the conventional method using the microwell. In the conventional method, the cost is excellent because the same effect can be achieved even if the amount of the oligonucleotide to be put in the well is much lower. The conventional methods are used to put about 600ng / well in the same experiment, but in the present invention 100 ng / well (Molecular and Celluar Probes, 1998 (12); 407-416). Comparing them, you get similar results (absorbance). In addition, the price of the microwells treated to attach the oligonucleotides used in the prior art is more expensive than the microwells used in the present invention. That is, oligonucleotides can be added to reduce the cost of both oligonucleotides and microwells in manufacturing the product. In addition, when the mutation was identified by attaching oligonucleotides to an existing microwell having an amine group, the high absorbance value of the negative control became a problem. Using the microwell used in the present invention, it is possible to significantly reduce the absorbance value of the negative control, thereby solving these problems. In addition, the secondary structure, low binding degree, and binding between the sample complementary to the DNA length, which is a problem in the mutation detection method used by attaching a sample to a conventional microwell, can be solved by using oligonucleotides in the present invention.

In addition, the existing methods are difficult to handle by using a membrane or a gel as described above, as well as difficult to identify a large amount of samples because it undergoes a complicated process of several steps, while the method of the present invention is only one Since all procedures are done in the well, anyone can easily perform the experiment without any special technique, which is more convenient than any method.

In addition, while existing methods have variables such as sample and experimenter, it is impossible to perform experiments in a batch condition every time, while the kit prepared according to the present invention not only has the same conditions but also can be stored for a long time. Mutations can be detected for. In other words, when the microwells are first created for the identification of 100 mutations, they are identical to the microwells used for the 100th year after the first one. In particular, the use of a machine does not have the experimental error of humans, so it can make statistical statistics of experimental statistics or types of samples. This advantage is a feature of the invention that cannot be obtained by any existing method.

The method of the present invention can easily confirm the result even by eye. In other words, the result of the experiment can be identified by a simple color change, so it is possible to provide reliability to the experimenter first of all because it is possible without a machine to check in the simple verification process. In addition, there is a feature that can easily know the base mutation of the substitution mutation of one or more bases present in the base sequence.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

Example 1

Sample Preparation

PCR probes were prepared using sites of direct sequence (name of specific site) that were found to be unique in the DNA of tuberculosis bacterium BCG. Biotin-attached primer a (5'-GGTTTTGGGTCTGACGAC-3 ') and primer b (5'-CCGAGAGGGGACGGAAAC-3') were prepared (Bionia, Korea). Oligonucleotide No20 with a nucleotide sequence found to be unique among the sites amplified by these primers; 5'-TTTTTTTTTTTTGACCTCGCCAGGAGAGAAGATCA-3 ', and No35; 5'-TTTTTTTTTTTCCGTACGCTCGAAACGCTTCCAAC-3 'was produced (Bionia, Korea). Oligonucleotide No3 having a nucleotide sequence uniquely present in the DNA of H37Rv, a bacterium of another type among tuberculosis bacteria; 5'TTTTTTTTTTATAGAGGGTCGCCGGTTCTGGATCA-3 ', and No41; 5'-TTTTTTTTTTGGAGCTTTCCGGCTTCTATCAGGTA-3 'was produced (Bionia, Korea).

For identification of substitution mutations, each of the 13th sequence was replaced with a different base with the only base sequence present in the DNA of the BCG, and the mutations of 5'-TTTTTTTTTTTTGACCTCGCCAGAAGAGAAGATCA-3 ', which are mutations of oligonucleotide No20, 5'-TTTTTTTTTTTCCGTACGCTCGAGACGCTTCCAAC-3 'was produced (Bionia, Korea). In order to confirm the insertion and deletion mutations, the following oligonucleotides were prepared by manipulating the positions of the 13th bases of each base sequence (for confirming insertion mutations of No20; 5'-TTTTTTTTTTTTGACCTCGCCAGTGAGAGAGAAGATCA-3 ', for confirming deletion mutations in No20; 5'-TTTTTTTTTTTTGACCTCGCCAGAGAGAAGATCA-3 '). All oligonucleotides produced were prepared such that 10 T's were present on the 5 'base.

DNA amplification

DNA amplification by PCR was performed under the following conditions. 200ng / μl of DNA solution of BCG, 0.5 / μl of primer a (100pmol / μl), 0.5μl of primer b (100pmol / μl), 0.4μl of dNTP (25mM), 10 × Tag buffer, 3μl of MgCl (25mM) , 5 μg of Taq (5 unit / μl, promega) and 40.4 μl of distilled water were treated at 94 ° C. for 5 minutes, and then amplified repeatedly at 94 ° C. for 1 minute, 55 ° C. for 1 minute 30 seconds, and 72 ° C. for 1 minute 30 seconds. It was.

Attaching Oligonucleotides to Microwells

Each oligonucleotide prepared by the base sequence was dispensed into 10 pmol of each well, and then boiled at 94 ° C for 10 minutes. Immediately driven into ice, cooled for 10 minutes, then condensed with a centrifuge. EDC and 1-methylimidazole were added to the measured sample to be 10 mM. Thus prepared samples were dispensed 100 μl into a microwell (Nunc) on ice. After preserving at 50 ° C. for 5 hours or more, washing was performed three times by treating 200 μl of 0.4M NaOH / 0.25% Tween 20 to remove unbound oligonucleotides. After standing at 50 ° C. for 15 minutes, 200 μl of 0.4M NaOH / 0.25% Tween 20 was washed three times. 200 µl of distilled water was washed three times. Treatment was performed at 50 ° C. three times for 5 minutes. 200 µl of distilled water was washed three times.

Each empty well was dispensed with a solution consisting of 138 μl of dH ₂ O, 20 × SSPE / 0.0167% TritonX-100, and ₂ μl of Salmon sperm DNA (10 mg / ml) and left at 50 ° C. for 20 minutes. After discarding the solution, dispense 68 µl of dH ₂ O, 30 µl of 20XSSPE / 0.0167% TritonX-100, 1 µl of Salmon sperm DNA (10 mg / ml), and 1 µl of DNA amplification sample by PCR. It was left for hours. The sample was discarded and washed three times with 200 μl of 0.5 × SSC and 0.1% Tween 20. The same solution was added for 15 minutes and placed at 60 ° C. After washing 3 times with the same solution, 100 μl of streptavidin enzyme solution diluted 100 times with 100 mM Tris-HCl (pH 7.5) and 150 mM NaCl solution was dispensed into each well. This was processed at 40 degreeC for 1 hour. The wells were washed three times with 100 mM Tris-HCl (pH 7.5), 150 mM NaCl solution / 0.1% Tween 20, and then treated three times for 5 minutes at 60 ° C. with the same solution. This was washed 3 times with the same solution. 100 μl of pNPP (sigma N7653) was added to each empty well and reacted for 90 minutes, and then absorbance was measured at 405 nm. The results are shown in FIG. 1. In FIG. 1, NoDNA is a control without oligonucleotide, A is a No3 negative control oligonucleotide, B is a No20 positive control oligonucleotide, and C is a substituted mutant oligonucleotide of No20. D is a deletion mutant oligonucleotide of No20 and E is an insertion mutant oligonucleotide of No20. F is a positive control oligonucleotide of No35, G is a substitution mutant oligonucleotide of No35, H is a negative control oligonucleotide of No41.

As can be seen from Figure 1, it can be seen that the method of the present invention can confirm the substitution, insertion, deletion, etc. of one base. In addition, it can be confirmed that even if more than one base can be confirmed through this.

As described above, the present invention is safer than any of the existing methods and has the effect of identifying substitutions, deletions and insertions as well as the presence or absence of one or more mutations at a low cost.

Claims (3)

Providing a nucleotide sequence of the organism as a sample (sample) to which biotin is attached by PCR; Preparing a probe consisting of a base sequence complementary to the sample and having a phosphate attached to a 5 'end of the base sequence; Coupling the probe to an amine group in a microwell; Adding the sample to the microwell to bind the probe; Adding an enzyme to the microwell to bind biotin; And The method of identifying a DNA mutation using a microwell, comprising the step of identifying a mutation by measuring the color change or absorbance of the enzyme. The method of claim 1, wherein the complementary nucleotide sequence comprises a nucleotide sequence complementary to the mutant nucleotide sequence of the sample. A microwell having an amine group attached to an inner surface thereof; EDC solution used when attaching oligonucleotide and 1-methylimidazole solution having pH of 7.0; 0.4M NaOH / 0.25% Tween 20 solution used to remove unbound oligonucleotides; A solution consisting of 138 μl of dH ₂ O, 20 × SSPE / 0.0167% TritonX-100 and ₂ μl of Salmon sperm DNA (10 mg / ml) treated in the microwells at blocking; A solution consisting of 68 μl of dH ₂ O, 30 μl of 20XSSPE / 0.0167% TritonX-100, and 1 μl of Salmon sperm DNA (10 mg / ml) used to bind the DNA amplification sample and the oligonucleotide attached to the microwell; 0.5 × SSC, 0.1% Tween 20 solution used to wash DNA unbound and oligonucleotides after binding to amplification samples and oligonucleotides attached to the microwells; Streptavidin-enzyme that binds to biotin; 100 mM Tris-HCl (pH 7.5), 150 mM NaCl solution used for binding biotin to streptavidin; 100 mM Tris-HCl, pH 7.5, 150 mM NaCl / 0.1% Tween 20 solution for washing streptavidin; And a kit for identifying a DNA mutation using a microwell, comprising a substrate reacting with enzymes.