KR20020083567A - A kit for manufacturing replica DNA chip, and a manufacturing method of replica DNA chip using the same - Google Patents

Abstract

PURPOSE: A kit for producing a replication DNA chip and a method for producing the replication DNA chip using the same kit are provided, thereby selectively selecting single nucleotide polymorphism, rapidly and correctly producing a desired replication DNA chip, and producing a desired sample-arrayed only replication DNA chip. CONSTITUTION: The kit for producing a replication DNA chip comprises (i) a lower layer part consisting of an original DNA fixed board, a first electrode on the board, alginate-strontium ion gel containing streptavidin on the electrode, and biotin-labeled DNA binding with the streptavidin; (ii) an upper layer part consisting of a replication DNA fixing board containing membrane or gold electrode and a second electrode on the board; (iii) a buffer filling a space between the lower layer part and the upper layer part; and (iv) one or two hole containing biological samples which are inserted into the buffer. The method for producing the replication DNA chip comprises the steps of: (i) applying negative charge into the first and the second electrodes to complementarily bind the biotin-labeled DNA and DNA in the biological sample; (ii) removing unbound biological sample in the lower layer part; and (iii) selecting DNA containing single base pair mismatch and applying positive charge into the first and the second electrodes to fix the selected DNA on the replication DNA fixing board.

Description

A kit for manufacturing a replica DNA chip and a method for manufacturing a replica DNA chip using the same {a kit for manufacturing replica DNA chip, and a manufacturing method of replica DNA chip using the same}

The present invention relates to a kit for producing a replica DNA chip and a method for manufacturing a replica DNA chip using the same. More specifically, streptavidin is entrapped in an alginate gel to which a certain concentration of strontium ions are bound. The present invention relates to a kit for producing a DNA chip to which a target DNA has been cloned by applying a specific voltage and a specific binding reaction of streptavidin-biotin from an original DNA chip.

Conventional DNA chips have been used to determine the abnormality of the sample DNA through a simple complementary binding reaction of DNA. That is, a method of measuring the DNA and oligomers having a specific base sequence highly integrated with a slide glass or a silicon substrate composed of electrodes and then reacting the sample DNA was measured. This DNA chip is an efficient way to analyze a sample by using a large amount of genetic information accumulated from the Human Genome Project (HGP), which can quickly analyze a large amount of genetic information with an automated technique in a short time. have.

Looking at the DNA chip manufacturing technology, there is a pin microarray method, an inkjet printing method, a photolithography method.

The pin microarray method is a method of arranging prepared DNA samples in a predetermined form on a slide using a pin (pin), such as a fountain pen nib. The pin microarray method is the simplest form of DNA chip development. The DNA is spotted and arranged on a slide using a dispenser controlled precisely by three axes of x, y and z. This method may not deliver a constant amount of DNA samples listed, and there may be a change in the shape of DNA spots due to pin wear.

The fabrication of DNA chips using inkjet printing differs in that the samples are arranged without direct contact with the slide surface using the inkjet printer head sprayer, rather than by direct physical contact with the DNA sample on the slide. . The advantage of this method is that the amount of sample delivered can be very precisely controlled and the shape of all spots is constant.However, it is difficult to clean the residual DNA sample in the cartridge and add new materials when changing the sample. Have

Photolithography combines photolithography using photomasks with DNA synthesis. The method involves covering the side chains of the bases, which are essential for the synthesis of DNA, with a blocking agent, a light-sensitive compound, and then removing the compounds with light when needed. If light is applied only to a desired area using a photomask and then a nucleotide is added to the compound, the synthesis reaction occurs only at the site where the blocking agent is lost by light. At this time, the nucleotide is also a light-sensitive blocking agent using a nucleotide covered with a side chain required for the reaction. Since DNA consists of only four bases of adenine, guanine, thymine, and cytosine, four layers of photomasks can be used to synthesize one layer of nucleotide sequences when the above process is repeated four times. Will be. Therefore, 100 photomasks are required to make oligonucleotides consisting of 25 nucleotide sequences. These methods cannot cope with small quantity batch production due to complicated process, time from order to production, and excessive cost of photomask manufacturing.

People differ in appearance and constitution because the three billion pairs of bases that make up the human genome are slightly different. Single base polymorphism, the most common form of DNA variation, appears in 1 form per 500 to 1000 bases.

Comparative studies of single nucleotide polymorphisms offer new breakthroughs in species evolution and phylogenetic studies. Specific changes in DNA that are conserved in the same series over a long period of time, when observed in more than 1% of the population, means a single nucleotide polymorphism, and when they appear only below 1%, they are simply called stools.

The present invention provides a kit for manufacturing a replica DNA chip in which a predetermined DNA is replicated from an original DNA chip using the electrical properties of the DNA, and a method of manufacturing a DNA chip in which the predetermined DNA is replicated using the same. .

1 is a schematic diagram of an original DNA chip in which agarose-strontium ion gel trapping streptavidin is formed.

2A is a schematic diagram of the original DNA chip to which the biotin-labeled DNA is bound,

FIG. 2B is a partially enlarged view of FIG. 2A;

3 is a schematic diagram of an original DNA chip in which various types of cDNAs are put in a hole,

4 is a schematic diagram of an original DNA chip in which only desired DNA is bound,

5 is a schematic diagram of a replica DNA chip selectively attached with the desired cDNA or oligomer,

Figure 6a is a 4 x 4 configuration of the original DNA chip from the top,

6B is a 4 × 4 perspective view of a replica DNA chip,

7 shows an example of biotin-labeled DNA, complementary bound DNA with a single base pair mismatch and intact complementary bound DNA,

8 is a view showing the binding state of each DNA shown in FIG.

9 is a view showing the separation of complementary bound DNA having a single base pair mismatch in the present invention,

10 is a view showing a state in which the DNA is completely complementary bound in the present invention.

Explanation of symbols for main parts of the drawings

1: glass substrate

2: gold or platinum electrode

3: wall constituting the hall

4: Alginette-strontium ion gel trapping streptavidin

5: hole

6: biotin-labeled DNA

7: replica DNA chip

8: original DNA chip

9: PBS buffer or 5X Standard Saline Citrate

10: electrode

11: glass substrate with membrane or gold electrode

12: potentiostat

13: wire

14: Biotin-labeled DNA bound to streptavidin

15: streptavidin

16: Arginine-Strontium Ion Gel

17: pools of different types of cDNAs or oligomers

18: Denaturation Solution

19: A hole in which an alginate-strontium ion gel is formed on an electrode

20: capture probe DNA

21: Complementary DNA without single base pair mismatch

22: DNA with a single base pair mismatch

The present invention relates to (i) a substrate to which an original DNA is attached, a first electrode formed on the substrate, an alginate-strontium ion gel trapping streptavidin formed on the electrode, and a biotin-labeled combination with the streptavidin. A lower layer made of DNA; (ii) an upper layer portion formed at a predetermined interval from the lower layer portion, the upper layer portion comprising a substrate for attaching a replica DNA including a membrane or a gold electrode and a second electrode formed on the substrate for attaching the replica DNA; (iii) a buffer filled between the lower and upper layers; And (iV) provides a kit for producing a duplicate DNA chip comprising one or more holes (hole) containing a biological sample introduced into the buffer.

In this case, (i) the lower layer of the alginette-strontium ion gel can adjust the binding degree of the gel by adjusting the amount of strontium addition, and (iv) the biological sample may include a DNA attached to the thiol group.

On the other hand, the present invention provides a method for producing a DNA chip in which a predetermined complementary binding DNA is replicated using the kit for producing a replica DNA chip.

That is, (i) applying a positive charge to the first electrode, a negative charge to the second electrode in order to complementarily bind the biotin-labeled DNA of the lower layer and the DNA in the biological sample in the replica DNA chip manufacturing kit; (ii) removing the biological sample not bound to the biotin-labeled DNA in the lower layer; And (iii) negative charge on the first electrode and second electrode to separate only the DNA having a single base pair mismatch from the DNA complementarily bound to the biotin-labeled DNA of the lower layer and attach the DNA to the replica DNA attachment substrate. Provided is a method of making a DNA chip in which complementary binding DNA with a single base pair mismatch is replicated, including the step of applying a positive charge.

In this case, the step (i) of applying positive charge to the first electrode and negative charge to the second electrode is preferably a step of applying a voltage of about 1.0V to about 2.0V with a potentiostat for about 10 minutes. In addition, the step (ii) of removing the unbound biological sample may be a step of washing with a PBS buffer, and the step (iii) of giving a negative charge to the first electrode and a positive charge to the second electrode may be about 1.0 to about It may be about 150 times to turn on and off the power at about 0.2 second intervals with a 2.0V voltage.

On the other hand, the present invention provides a method for producing a DNA chip is a DNA that is completely complementary binding using the kit for producing a duplicate DNA chip according to the present invention.

That is, (i) applying a positive charge to the first electrode and a negative charge to the second electrode to complementarily bind the biotin-labeled DNA of the lower layer and the DNA in the biological sample; (ii) removing the biological sample not bound to the biotin-labeled DNA in the lower layer; (iii) adding a strongly basic Denaturation solution to each hole; And (iv) applying negative charge to the first electrode and positive charge to the second electrode to separate DNA that is complementarily bound to the biotin-labeled DNA in the lower layer and attach the DNA to the replica DNA attachment substrate. It provides a method for producing a DNA chip in which DNA is duplicated.

In this case, the step (i) of applying positive charge to the first electrode and negative charge to the second electrode is preferably a step of applying a voltage of about 1.0V to about 2.0V with a potentiostat for about 10 minutes. In addition, step (ii) of removing the unbound biological sample may be a step of washing with PBS buffer, the strong basic separation solution of step (iii) is a solution consisting of 15X SSC, 2.0M NaOH and 50mM EDTA Can be used.

Hereinafter, the present invention will be described in detail.

The replica DNA chip according to the present invention is produced through a process of replicating a desired DNA using the electrical properties of DNA from the original DNA chip (8). That is, by using a predetermined ratio of strontium ions and an alginate gel, the streptavidin 15 is trapped in the gel and fixed on the first electrode 2 through their covalent bonds. Next, the binding of the desired DNA labeled with biotin with streptavidin by the specific reaction of streptavidin-biotin is formed. At this time, the step of capturing streptavidin using the predetermined ratio of strontium ions and alginate gel is strept through a specific binding reaction with calcium alginate, sodium alginate, agarose, and polyacrylamide. It can be replaced by the avidin capture step.

As the substrate to which the original DNA is attached, organic materials such as glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, silicon nitride, and the like are used, and a wall forming between the holes 5, a space where DNA is bound, is formed. (3) As the material, plastic, rubber, glass, metals, silicon substrates or the like can be used.

Meanwhile, the substrate 11 to which the replica DNA is attached is fixed to the second electrode 10, and the substrate 11 may be a glass substrate or a glass substrate including a membrane, gold, platinum electrode, or the like. At this time, when using a substrate containing a gold or platinum electrode, it is preferable to label the thiol (thiol) group at the end of the sample DNA.

In the present invention, hybridization and denaturation in the DNA chip 8 and the replica DNA chip 7 to which the original DNA is attached is performed in each hole 5 by PBS buffer or 5X SSC (standard). It may be performed after filling an aqueous solution such as saline citrate, in which case the hybridization applies a positive charge to the original DNA chip (8) and a negative charge to the replica DNA chip (7). 8) can be denatured by giving a negative charge and a positive charge to the replica DNA chip 7. Preferably, a voltage of 1.0 to 2.0 V is applied to the original DNA chip 8 and the replica DNA chip 7 with the potentiostat 12.

In this case, DNA hybridization and hybridization can be obtained quickly and accurately by using vias. Since DNA has a strong negative charge, it gives a positive charge to the original DNA substrate and a negative charge to the replica DNA substrate during hybridization to induce fast complementary binding, while negative charge is applied to the original DNA substrate during the DNA treatment. The positive charge is then transferred to the replica DNA substrate. Using this method, specific DNA arrays can be quickly replicated and repeated reproduction of the electron replica DNA chip in succession.

In the present invention, a strong base separation solution (Denaturation solution) is used to separate and replicate DNA that is completely complementary. The separation solution 18 is preferably made of 15X SSC, 2.0M NaOH, 50mM EDTA. After inserting the strong basic separation solution 18 into the hole 5, the original DNA chip 8 was negatively charged and the replicating DNA attachment substrate 7 was positively charged, applying a 1.0-2.0V voltage to the cDNA. Only comes to attach to the replica DNA attachment substrate (7), if the replication DNA attachment substrate (7) comprises a gold or platinum electrode is used as complementary DNA cDNA conjugated with a thiol group, when using a membrane There is no need for a specific functional group.

In this case, the nucleotide sequence to be replicated includes an oligomer having a short nucleotide sequence as well as cDNA.

On the other hand, the kit for producing a replica DNA chip of the present invention by applying a voltage only to each of the desired holes 19 can produce a replica DNA chip in which only the desired DNA is selectively arranged.

In addition, the present invention is applied to Single Nucleotide Polymorphism (SNP) by selectively selecting only the desired DNA from a DNA pool containing several kinds of DNA, and similar genes of living organisms including humans, animals, and plants are similar. Allows discrimination against single base-pair mismatches with one or two different sequences of functional DNA sequences.

That is, within a short time, the cDNA is complementarily combined including a single base pair mismatch, and then a single base pair mismatch is separated by applying a negative charge to the original DNA chip 8 and a positive charge to the replica DNA chip 7. Preferably, the power is supplied to 150 times with a voltage of 1.0 to 2.0V while the power is shortly applied and disconnected for about 0.2 seconds. At this time, the hole 5 is filled with a PBS buffer or 5X SSC (Standard Saline Sitrate) 9. As a result, only a single base pair mismatch portion is left in the replica DNA chip 7.

As such, the present invention can selectively distinguish a single base pair mismatch using an electronic cloning DNA chip technology, so that single nucleotide polymorphisms can be distinguished from each other. It is possible to make an electronic clone DNA chip accurately. Indeed, typical hybridization requires at least 12 hours or more, while electronic replication techniques can perform hybridization in minutes. By copying the original DNA, the same electron replica DNA chip can be produced continuously, and only the desired sample can be selectively arrayed by electrically controlling only the desired holes.

Other objects, features and advantages of the present invention will become apparent from the following detailed description of embodiments taken in conjunction with the accompanying drawings.

Referring to the accompanying drawings, preferred embodiments of the present invention having the features as described above are as follows.

Example

FIG. 1 boils 2% alginate and 0.075M strontium chloride and injects it into the hole 5 near 60 ° C. to fix it on the first electrode 2 formed on the glass substrate 1. . At this time, the first electrode 2 is fixed to the glass substrate 1, and fixed by injecting a calcium alginate and strontium mixed solution in which the streptavidin 15 is thinly layered at 1 mg / ml. . After the baking (baking) at 37 ℃ in a humidification chamber (humidity chamber) for 90 minutes. Or spin coating, spin coating the alginette and strontium ion mixture at 2000 RPM for 20 seconds using a spin coater, and then streptavidin (15) was mixed in a thin layer at 1 mg / ml. Spin coating calcium ginate and strontium mixture at 4000 rpm for 20 seconds.

Using this method, compared to ordinary agarose, covalent bonds between agarose and strontium ions form a tighter and narrower space to form agarose-strontium gel (4) that firmly captures streptavidin (15). Done. At this time, the degree of trapping depends on the concentration of strontium ions, and thus the degree of trapping can be controlled by adjusting the concentration. In other words, increasing the concentration of strontium ions leads to a stronger gel state, and decreasing the concentration of strontium ions leads to a loose gel state.

Alginate is a generic term for copolymers comprising mannuronic acid and guuronic acid with varying proportions and ordered arrangements. Gelation of alginates is achieved by the selective binding of divalent cations such as Ca ²⁺ to the guuronic acid blocks. When a divalent cation reacts with an alginate, it becomes an irreversible reaction if no other ions are present, and the rate of gelation occurs very quickly. When strontium ions interact with the alginate, the strontium ions bind with the guuronic acid to increase the hardness, thereby controlling the degree of curing and the degree of capture.

FIG. 2A shows that the biotin-labeled DNA 6 is streptized by placing a biotin-labeled DNA 6 in the PBS buffer 9 in a hole with a positive charge on the original DNA chip 8 and a negative charge on the replica DNA chip 7. Make a strong bond with Avidin (15). Since the DNA has a strong negative charge, it is attracted to the original DNA chip (8), collides with the alginet-strontium ion gel (4) that traps the streptavidin (15), and forms a streptavidin-biotin bond. The voltage at this time is applied to the potentiostat (12) voltage of 1.0 to 2.0V. The proper time for applying voltage is about 3 minutes.

2B is an enlarged view of the portion “A” of FIG. 2A, and shows a detailed view of the trapping of the streptavidin 15 in the alginate-strontium ion gel 16. The state of binding with biotin-labeled DNA 6 is shown. The streptavidin 15 and the biotin are strongly bound so that the biotin-labeled DNA 14 in combination with the streptavidin 15 is maintained without binding in the gel even when the electric field is changed.

3 is a DNA (17) of various types, including cDNA is separated by heating for 5 minutes at 95 ℃. Next, quench in an ice box, dissolve in PBS buffer (9) and pour into hole (5). Next, when positive charge is applied to the original DNA chip 8 and negative charge is applied to the replica DNA chip 7, a voltage of 1.0 to 2.0 V is applied to the potentiostat 12, and only cDNA having a complementary base sequence is biotin-labeled DNA. Combined with (6). At this time, the time to apply the voltage is about 10 minutes. Such hybridization allows reactions that take at least 12 hours under normal conditions to occur within minutes while being very sensitive to binding reaction conditions. After washing three times with PBS buffer to remove the unbound cDNA.

4 is a schematic diagram showing the results of hybridization of biotin-labeled DNA with cDNA having complementary sequences. Thereafter, washing with PBS buffer 9 and distilled water three times to wash unbound cDNA, resulting in only complementary cDNA.

FIG. 5 is a method of separating only bound cDNAs, and is separated using a denaturation solution 18 having a strong base. The composition of the separation solution was 15X SSC. It consists of 2.0M NaOH, 50mM EDTA. After inserting a strongly basic separation solution into the hole, the original DNA chip (8) is negatively charged and the replica DNA chip (7) is positively charged, applying a voltage of 1.0 to 2.0V. Come to (7) and attach. In this case, when the replica DNA chip 7 is gold or platinum, cDNA having a thiol group is used as complementary DNA, and when a membrane is used, no specific functional group is required. The time to apply the voltage is about 30 minutes at room temperature, and the temperature can be shortened by increasing the temperature, but the gel bond may loosen, so heat below 60 ℃.

After this process, only the desired cDNA remains in the replica DNA chip 7, and by repeating the above process, the same replica DNA chip 7 can be made as desired. The cloned replica DNA chip has a constant arrangement.

In addition, by selectively selecting only the desired DNA from several kinds of sample DNA pools 17, DNA sequencing that has similar functions among genes of living organisms including humans, animals and plants by applying to single nucleotide polymorphism Allows discrimination against single base-pair mismatches with either or two different sequences. That is, within a short time, the complementary DNA including a single base pair mismatch and the like is hybridized and then washed with PBS buffer 9 or the like. A negative charge is applied to the original DNA chip 8 and a positive charge is applied to the replica DNA chip 7 to separate a single base pair mismatch. Apply a voltage of 1.0 to 2.0V 150 times while turning off the power for about 0.2 seconds. At this time, the hole 5 is filled with the PBS buffer 9. Only a single base pair mismatch portion remains in the replica DNA chip 8.

At this time, as a method of separating only cDNA without a base pair mismatch, it is separated by using a denaturation solution 18 having a strong base. The composition of the separation solution 18 is composed of 15X SSC, 2.0M NaOH, 50mM EDTA. After inserting a strongly basic separation solution into the hole (5), the original DNA chip (8) is negatively charged and the replica DNA chip (7) is positively charged. Only the missing cDNA comes to the replica DNA chip and attaches. This is because cDNAs with single base pair mismatches are weakly bound and can be separated using only voltage under PBS buffer (9), but cDNAs without mismatches are strongly bound and can only be denatured using separation solution (18). Because.

FIG. 6A is a configuration diagram of the original DNA chip 8 composed of 16 holes 19 when viewed from above, and each hole is configured as described in FIG. 1. When the number of the holes 19 is a large number can be replicated by applying a voltage to the entire hole at once, or can be replicated in turn by applying a voltage to the desired hole. That is, only a desired sample can be selectively arranged on the DNA chip to be replicated by applying a voltage only to the desired hole 19. 6b is a 4x4 perspective view of a replica DNA chip according to the invention.

FIG. 7 shows a capture probe DNA (QC2) (20) having a single base-pair mismatch (RQC2 SBPM) 22 and biotin combined as an embodiment according to the present invention. ), The sequence of DNA (RQC2) 21 without a single base pair mismatch.

FIG. 8 is a view showing the combination of RQC2 SBPM 22, which is a DNA having a single base pair mismatch, and DNA 21 without a single base pair mismatch, to QC2, which is a capture probe DNA 20. FIG.

Here, DNA bases that perform similar functions among genes in humans, animals, plants, etc. are applied to single nucleotide polymorphism by selectively selecting only the desired DNA from various kinds of sample DNA pools 17. We present a method that allows the identification of single base pair mismatches that have different base sequences from one of the sequences.

9 is a diagram showing the separation of DNA having a single base pair mismatch.

Complementary DNA, including single base pair mismatches, is hybridized and washed with PBS buffer (9) and the like. A negative charge is applied to the original DNA chip 8 and a positive charge is applied to the replica DNA chip 7 to separate single base pair mismatches. Apply a voltage of 1.0 to 2.0V 150 times while turning off the power for about 0.2 seconds. At this time, the hole 5 is filled with a PBS buffer. This leaves only a single base pair mismatch portion in the replica DNA chip 7. This is because bonds with DNA with a single base pair mismatch are weakly bound and are separated first. Thus, only DNA with a single base pair mismatch can be found and separated.

FIG. 10 is a view showing the separation of DNA 21 that is completely complementarily bound without a single base pair mismatch. In this separation step, a strong base separation solution is used. The composition of the separation solution 18 is composed of 15X SSC, 2.0M NaOH, 50mM EDTA. After the strong base separation solution was placed in the hole 5, the original DNA substrate 8 was negatively charged, and the replica DNA chip 7 was positively charged, and a 1.0-2.0V voltage was applied. Only come to the replica and attach. That is, cDNA having a single base pair mismatch can be separated using only a voltage under PBS buffer (9) because the binding strength is weak, but cDNA without mismatch has strong binding strength and can only be separated using the separation solution (18). Because it can be denaturated.

According to the present invention as described above, first, single nucleotide polymorphism can be distinguished by selectively separating single base pair mismatches. Second, the electrical replication process can be used to quickly and accurately produce the desired replica DNA chip. Third, by continuously performing the DNA replication process of the DNA chip to which the original DNA is attached, the same replica DNA chip can be produced continuously. Fourth, since it is possible to replicate by applying voltage only to the desired hole, it is possible to manufacture a replica DNA chip in which only a desired sample is selectively arranged.

Those skilled in the art will appreciate that various changes and modifications can be made without departing from the technical spirit of the present invention.

Therefore, the technical scope of the present invention should not be limited to the above description but should be defined by the claims.

Claims (11)

(i) a substrate to which the original DNA is attached, a first electrode formed on the substrate, an alginate-strontium ion gel that captures streptavidin formed on the electrode, and biotin-labeled DNA bound to the streptavidin Lower layer; (ii) an upper layer portion formed at a predetermined interval from the lower layer portion, the upper layer portion comprising a substrate for attaching a replica DNA including a membrane or a gold electrode and a second electrode formed on the substrate for attaching the replica DNA; (iii) a buffer filled between the lower and upper layers; And (iV) Kit for producing a cloned DNA chip comprising one or two or more holes containing a biological sample introduced into the buffer. The method of claim 1, wherein the (i) the lower layer of the alginette-strontium ion gel is a duplicate DNA chip manufacturing kit, characterized in that the binding degree of the gel is adjusted by adjusting the amount of strontium added. The method of claim 1, wherein the (iv) biological sample comprises a DNA chip manufacturing kit, characterized in that containing a thiol group attached DNA. In the kit for producing a cloned DNA chip of claim 1, (i) applying a positive charge to the first electrode and a negative charge to the second electrode to complementarily bind the biotin-labeled DNA in the lower layer with the DNA in the biological sample; (ii) removing the biological sample not bound to the biotin-labeled DNA in the lower layer; And (iii) a negative charge on the first electrode and a positive charge on the second electrode to separate only the DNA having a single base pair mismatch from the DNA complementarily bound to the biotin-labeled DNA of the lower layer and attach it to the substrate for attaching the replicating DNA. Comprising the step of adding, Complementary binding DNA having a single base pair mismatch, DNA DNA method of production. 5. The single base pair of claim 4, wherein the step (i) of applying a positive charge to the first electrode and a negative charge to the second electrode comprises applying a voltage of about 1.0 V to about 2.0 V with a potentiostat for about 10 minutes. A method for producing a DNA chip in which complementary binding DNA having mismatches is replicated. The method of claim 4, wherein the removing of the unbound biological sample (ii) is a step of washing with PBS buffer. 5. The method of claim 4, wherein the step (iii) of applying a negative charge to the first electrode and a positive charge to the second electrode comprises turning the power on and off at intervals of about 0.2 seconds at a voltage of about 1.0 to about 2.0V. A method of manufacturing a DNA chip having a complementary binding DNA having a single base pair mismatch, wherein the step of performing about 150 times. In the kit for producing a cloned DNA chip of claim 1, (i) applying a positive charge to the first electrode and a negative charge to the second electrode to complementarily bind the biotin-labeled DNA in the lower layer with the DNA in the biological sample; (ii) removing the biological sample not bound to the biotin-labeled DNA in the lower layer; And (iii) adding a strongly basic Denaturation solution to each hole; (iv) applying negative charge to the first electrode and positive charge to the second electrode to separate DNA that is complementarily bound to the biotin-labeled DNA in the lower layer and attaching the DNA to the replica DNA attachment substrate. Method for producing a DNA chip cloned. 9. The method of claim 8, wherein the step (i) of applying a positive charge to the first electrode and a negative charge to the second electrode is a step of applying a voltage of about 1.0V to about 2.0V with a potentiostat for about 10 minutes. Method for producing a DNA chip in which the binding DNA is replicated. The method of claim 8, wherein the removing of the unbound biological sample (ii) is a step of washing with PBS buffer. The method of claim 8, wherein the strongly basic separation solution of step (iii) is a solution consisting of 15X SSC, 2.0M NaOH and 50mM EDTA.