KR20120107543A - Method for extracting nucleic acid using carbon nanotube charged electropositive - Google Patents

Abstract

Disclosed is a nucleic acid extraction method using positively charged carbon nanotubes that improve the recovery of nucleic acids using the positive-negative binding principle. To this end, the present invention comprises the steps of mixing a sample containing a nucleic acid and a positively charged carbon nanotubes, and reacting the same, and washing by adding a wash buffer to the mixture of the sample and the positively charged carbon nanotubes, and It provides a method for extracting a nucleic acid using a positively charged carbon nanotube comprising the step of recovering the nucleic acid by adding a DNA elution reagent to the washed mixture. According to the present invention, as compared with the conventional silica-based nucleic acid extraction method, not only the surface area to which the nucleic acid is bound is widened, but also the binding force with the nucleic acid is enhanced because the positive charge of the carbon nanotube proceeds by the negative charge and the electrostatic attraction of the nucleic acid. Enhancement has the effect of improving nucleic acid recovery.

Description

Nucleic acid extraction method using positively charged carbon nanotubes {METHOD FOR EXTRACTING NUCLEIC ACID USING CARBON NANOTUBE CHARGED ELECTROPOSITIVE}

The present invention relates to a nucleic acid extraction method using positively charged carbon nanotubes, and more particularly, to a nucleic acid using positively charged carbon nanotubes, which reduces extraction time and improves recovery of nucleic acids by using a positive-negative coupling principle. It relates to an extraction method.

Traditionally, ethanol precipitation methods (Lis and Schleif, Nucleic Acids Res., 2: 383-389 (1975)) have been used after using three buffers represented by dilution, disruption and neutralization of human blood-derived cells. However, because the method is inconvenient to handle and low in purity, US Pat. No. 5,707,812 has developed a nucleic acid extraction and purification kit comprising a column. (Horn et al., Human Gene Ther., 6: 565-573 (1995). Chandra et al., Anal. Biochem., 203: 169-172 (1992); Marquet et al., BioPharm .: 26-37 (1995) et al.)

The principle of nucleic acid extraction using such columns is the purification of nucleic acid molecules from proteins and other intracellular materials using the principle of structural interaction between water molecules and nucleic acids using glass fibers or silica membranes that specifically bind to nucleic acids. For this purpose, a centrifugation process was necessary.

Therefore, the DNA extraction kits conventionally used include (1) injecting blood into digestion buffer, (2) incorporating binding buffer into digested blood, (3) first centrifugation, and (4) centrifugation. Combining the sample, (5) second centrifugation step, (6) washing the centrifuged sample, (7) third centrifugation step, (8) eluting DNA from the sample, and ( 9) DNA was extracted through a fourth centrifugation step to extract DNA.

The above-mentioned method has been spotlighted as a high purity DNA extraction method, but it requires a lot of time and cost because it has to go through a culturing step and a centrifugation step, and there is a problem that a relatively large amount of blood is required for DNA extraction.

In addition, conventionally, organic solvents such as phenol and chloroform have been used to extract nucleic acids from prokaryotic and eukaryotic or complex biological samples. Nucleic acid extraction using the enzyme was digested enzymatically with proteolytic enzymes, cells were lysed with ionic surfactant, and nucleic acid was extracted with phenol or phenol / chloroform mixture. According to the extraction, the organic solvent phase and the water phase were separated, and the extracted nucleic acid could be separated by precipitating the nucleic acid distributed toward the water with alcohol. However, phenol or phenol / chloroform mixture has a dangerous effect on life or the environment and is considered a dangerous waste, so there is a problem that must be handled with care.

Accordingly, there is a need for development of compositions and methods for reducing the use of many reagents that are harmful to life or the environment, requiring no expensive equipment, and improving nucleic acid recovery.

Accordingly, an object of the present invention is to provide a method for extracting nucleic acids using positively charged carbon nanotubes which can shorten the extraction time of nucleic acids, increase the recovery of nucleic acids, and reduce the use of reagents.

In order to achieve the above object of the present invention, in one embodiment of the present invention, the step of mixing a sample containing a nucleic acid and a positively charged carbon nanotube, and reacting it, and the sample and the positively charged carbon nanotube It provides a method for extracting a nucleic acid using a positively charged carbon nanotube comprising the step of washing by adding a wash buffer to the mixture, and recovering the nucleic acid by adding a DNA elution reagent to the washed mixture.

In addition, in order to achieve the object of the present invention, in another embodiment of the present invention, the method comprising the steps of mixing and reacting a sample containing a nucleic acid and a CNT dispersion containing a positively charged carbon nanotubes, and the reaction of the sample and the CNT dispersion Centrifuging the mixture to separate the separated precipitates, adding washing buffers to the precipitates, drying the washed precipitates, and adding a DNA elution reagent to the washed precipitates. It provides a method for extracting a nucleic acid using a positively charged carbon nanotube comprising the step of recovering.

The nucleic acid extraction method according to the present invention increases the surface area of the carbon nanotubes to which the nucleic acid is bound compared to the conventional silica-based nucleic acid extraction method, thereby increasing the nucleic acid recovery rate.

In addition, since the nucleic acid extraction method of the present invention uses carbon nanotubes having positive charges instead of general carbon nanotubes or negatively charged carbon nanotubes, the positive charges of the carbon nanotubes are bound by the negative charge and the electrostatic attraction of the nucleic acid. As a result, the binding force with the nucleic acid is enhanced, and the nucleic acid recovery rate is increased compared with the case of using nonpolar elastic nanotubes or negatively charged carbon nanotubes.

In addition, the nucleic acid extraction method according to the present invention is not harmful to the human body because it does not use phenol, unlike the conventional organic solvent extraction method, and problems such as environmental pollution are minimized.

In addition, the nucleic acid extraction method according to the present invention has the effect of obtaining a nucleic acid economically and simply by using a small amount of a liquid sample or a solid sample such as blood, saliva.

1 is a flowchart illustrating a nucleic acid extraction method according to an embodiment of the present invention.
2 is a flowchart illustrating a nucleic acid extraction method according to another embodiment of the present invention.
Figure 3 is an electrophoretic picture of the nucleic acid purified by the nucleic acid extraction method according to the invention.

Hereinafter, a nucleic acid extraction method (hereinafter, referred to as a "nucleic acid extraction method") using positively charged carbon nanotubes according to preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, the nucleic acid is partially described, but the present invention can be applied to the extraction of RNA, which is a kind of nucleic acid.

1 is a flowchart illustrating a nucleic acid extraction method according to an embodiment of the present invention.

Referring to Figure 1, the nucleic acid extraction method according to an embodiment of the present invention is a reaction step (S100) to react by mixing the sample and carbon nanotubes (Carbon NanoTube: CNT), and the washing buffer in the mixture of the sample and CNT Washing step (S200) for adding and washing, and recovery step (S300) for recovering the nucleic acid by adding the DNA elution reagent to the washed mixture.

The CNTs may be single-wall nanotubes, multiwall nanotubes, or bundle nanotubes, but are not limited thereto. The CNTs have a long, thin tube-like structure with a diameter of several nm.

Referring to Figure 1, the reaction step (S100) according to the present invention is a step of reacting by adding a positively charged CNT to the sample containing the nucleic acid, by binding the nucleic acid of the sample to the surface of the CNT through the reaction Extract. Since the positively charged CNTs undergo the binding of the nucleic acid to the positively charged negative charge, the binding force between the nucleic acids is enhanced to increase the recovery rate of the nucleic acid from the sample compared to the negatively charged CNTs or the nonpolar CNTs. In this case, it is preferable to use CNT substituted with an amino group as a positively charged CNT.

In addition, the conventional non-polar CNT and nucleic acid binding principle is a π-π bond between the surface of the CNT and the base of the nucleic acid, a reaction time of several hours was required to bind the CNT and the nucleic acid. However, in the case of positively charged CNTs, the CNTs and the nucleic acid can be bound within about 5 minutes by introducing a positive charge on the surface of the CNTs.

In addition, CNT negatively charged have a variety of reagents, such as a for smooth engagement with the nucleic acid containing the NaCl, MgCl _2, KCl, CaCl ₂ salt solution, however, the use of CNT positively charged nucleic acids without reagents such as a salt solution And can be combined seamlessly.

In this reaction step (S100), 1 to 7 parts by weight of positively charged CNTs is added to 50 to 200 parts by weight of the sample to be extracted. At this time, the positively charged CNTs may be added in a powder form, but the powdered CNTs may be added in a dispersion state by mixing the powdered CNT with a solution so as to easily measure the exact amount used.

Here, the CNT dispersion is added 2 to 15 parts by weight based on 50 to 200 parts by weight of the sample. For example, 100-700 μl is added based on 5-20 mg of sample. As the solution, mainly water may be used, and a surfactant may be further added.

The sample containing the nucleic acid can be targeted to all cells such as animal cells, plant cells, microbial cells and the like. As a sample to be used for the nucleic acid extracting method, a biologically-derived sample containing cells is suitable, but the present invention is not limited thereto, and the nucleic acid extracting method can be applied to a sample not derived from a living body.

As the bio-derived sample, biological components of animals and plants may be appropriately used. Examples of animal-derived samples including humans include, for example, blood, tissue fluid, lymph fluid, cerebrospinal fluid, pus, mucus, runny nose, sputum, urine, feces, and fluids such as ascites, skin, lungs, liver, mucus, various And a cleaning liquid after washing tissues such as organs and bones, and nasal cavity, bronchus, skin, and various organs. In humans, dialysis drainage may also be used as a sample.

In addition, the nucleic acid targeted by the extraction method of this nucleic acid is not limited to the nucleic acid native to the cell contained in a sample, but can be applied also to nucleic acid, such as a nucleic acid of the virus infected by the cell, and the microorganisms in which the cell was taken. Therefore, biologically-derived samples containing phagocytic cells (white blood cells, etc.) of infectious patients are also suitable as samples of the hexin extraction method.

At this time, in order to use a biological-derived sample having a cell wall or cell membrane, a pretreatment step (not shown) for pretreating the sample with a cell decomposition reagent should be performed. In other words, in the case of using a bio-derived material, the sample uses a cell-degradation product that is a reaction between the cell-lysis reagent and the bio-derived material.

In one embodiment, in the pretreatment process, 150-250 μl of lysis buffer and 7-13 μl of proteinase K are mixed with 10 mg of beef tissue, which is about 1 at 50-70 ° C. Reaction over time produces a cell breakdown product for beef tissue.

In another embodiment, in the pretreatment process, 150 mg to 250 μl of DirGene ^® , a Lysis buffer from Sally Bio, is mixed with 10 mg of beef tissue, and reacted at room temperature for about 1 minute to prepare a cell degradation product for beef tissue.

Nucleic acid extraction method according to an embodiment of the present invention may further include a modification step before the reaction step (S100).

The reforming step is a step of modifying the nonpolar CNT into a positively charged CNT, and any method may be used as long as the CNT can carry a positive charge.

For example, the modification of the positively charged CNTs according to the present invention is the first step of adding a mixed acid formed of sulfuric acid (H ₂ SO ₄ ) and nitric acid (HNO ₃ ) to the non-polar CNT to generate a carboxyl-substituted CNT, And adding a diamine and a solvent to the CNT substituted with the carboxyl group to generate the CNT substituted with the amino group. At this time, sulfuric acid performs a catalytic role, nitric acid acts as an oxidizing agent.

More specifically, in the first step, 2 g of non-polar CNT, 50 ml of 97% sulfuric acid and 50 ml of 70% nitric acid are charged into a reflux apparatus, and the mixture of the reflux apparatus is stirred for 48 hours A step of adding distilled water as a solvent to the reflux apparatus to terminate the reaction, filtering the mixture with a filter, filtering the mixture until the pH of the mixture becomes neutral, and then drying the resultant, (CNT-COOH).

In the second step, 300 mg of CNT (CNT-COOH) substituted with the carboxyl group, 1 ml of hexamethylenediamine used as a diamine, and 150 ml of distilled water used as a solvent are put into a round flask After the addition, 1N HCL was dropped using a syringe to adjust the pH to 5, a step of stirring the mixture of CNT substituted with carboxyl group, hexamethylene diamine and distilled water for 6 hours, and The mixture is filtered and washed to neutralize the pH and then dried to produce CNT (CNT-NH ₂ ) substituted with an amino group.

Referring to FIG. 1, the washing step (S200) according to the present invention is a step of washing foreign substances such as cytolysis buffer by adding a washing buffer to the mixture generated through the reaction step (S100). In this case, ethanol or EDTA may be used as the washing buffer. Here, 70% ethanol is preferably used as ethanol.

In a particular embodiment, the washing step (S200) according to the present invention comprises the steps of adding a washing buffer to the mixture produced through the reaction step (S100), centrifuging the mixture to which the washing buffer is added, and the centrifugation It can be made through the step of removing the separated supernatant.

The centrifugation is carried out through a centrifuge for 8 to 12 minutes, preferably 10 minutes at 10,000 to 14,000 rpm, preferably 12,000 rpm. In addition, when the supernatant and the precipitate is separated through the centrifugation, the precipitate is separated using a sediment discharge device or the supernatant is removed to separate the precipitate.

Referring to Figure 1, the recovery step (S300) according to the present invention is a step of recovering the nucleic acid by adding a DNA elution reagent to the washed mixture through the washing step (S200). In this case, 0.1M Tris-HCl or PBS may be used as the DNA elution reagent.

In a particular embodiment, the recovery step (S300) according to the present invention comprises the steps of adding a DNA elution reagent to the washed mixture through the washing step (S200), and vortexing the mixture to which the DNA elution reagent is added (vortexing) And centrifuging the vortexed mixture, and recovering the separated supernatant through the centrifugation.

The centrifugation is carried out for 40 to 90 seconds, preferably 60 seconds at 10,000 to 14,000 rpm, preferably 12,000 rpm, and the vortexing is performed for 8 to 15 seconds.

2 is a flowchart illustrating a nucleic acid extraction method according to another embodiment of the present invention.

Referring to Figure 2, the nucleic acid extraction method according to another embodiment of the present invention is a reaction step (S100) to react by mixing the sample and CNT, the suspended matter separation step (S150) for removing the suspended matter from the sample, Washing step (S200) for washing by adding the washing buffer to the precipitate, drying step (S250) for removing the remaining washing buffer, and recovery step for recovering the nucleic acid by adding the DNA elution reagent to the washed precipitate (S300). It includes. At this time, the nucleic acid extraction method may include only any one of the suspended matter separation step (S150) and drying step (S250), it may be all included.

In this case, the washing step (S200) is divided into a step of adding a washing buffer to the precipitate separated through the suspended matter separation step (S150), centrifuging the precipitate to which the washing buffer is added, and the centrifugation through The supernatant may be removed.

2, the suspended matter separation step (S150) according to the present invention is a step for removing suspended matter such as protein, carbohydrates, fats and the like from the sample through a pretreatment step and a reaction step (S100). Centrifugation of the mixture of CNTs (or CNT dispersions) is carried out to separate the precipitates.

At this time, the centrifugation is carried out at 10,000 to 14,000 rpm, preferably 12,000 rpm for 8 to 12 minutes, preferably 10 minutes. In addition, when the supernatant and the precipitate is separated through the centrifugation, the precipitate is separated using a sediment discharge device or the supernatant is removed to separate the precipitate.

When the washing buffer is added without removing the floating matter through the floating separation step (S150), the protein may be reacted with the washing buffer to precipitate, and the precipitated protein is not removed even in the washing step. In other words, when the suspension separation step is performed, it is possible to react with the DNA elution reagent used in the recovery step to remove the elements that prevent the recovery of the nucleic acid.

On the other hand, the recovery step (S300) is a step of adding a DNA elution reagent to the precipitate dried through the drying step (S250), vortexing (vortexing) the precipitate to which the DNA elution reagent is added, and the vortexing Centrifuging the precipitate was carried out, and may be made of a step of recovering the separated supernatant through the centrifugation.

Referring to Figure 2, the drying step (S250) is a step for removing the washing buffer remaining in the sediment separated through the washing step (S200), all the washing buffer consisting of volatiles remaining in the precipitate is evaporated The precipitate is placed in a heat dryer to be removed and dried. For example, the precipitate is dried at 50 to 70 ° C. for 20 to 40 minutes inside the heat dryer.

The present invention will be described in more detail with reference to the following examples and comparative examples. However, the present invention is not limited to these examples.

Example 1

1. 2 g of CNTs, 50 ml of 97% sulfuric acid, and 50 ml of 70% nitric acid were added to a reflux apparatus to generate a mixture, and the mixture was reacted for 48 hours with constant stirring at 80 ° C.

2. After distilled water was added to the reflux device to terminate the reaction, the mixture was filtered through a filter, filtered through a filter until the pH of the mixture became neutral, and dried to produce a CNT substituted with a carboxyl group.

3. Put 300 mg of CNT substituted with the carboxyl group, 1 ml of hexamethylenediamine and 150 ml of distilled water into a round flask (250 ml), block the inlet with a rubber stopper, and drop 1N HCL into a round flask using a syringe. The mixture present in the flask was adjusted to pH 5 and stirred for 6 hours.

4. The mixture was filtered to adjust the pH to neutral, and then dried to produce CNT substituted with an amino group, and 200 mg of the CNT and 100 ml of distilled water were mixed to prepare a CNT dispersion containing positively charged CNTs.

5. Add 10 mg of beef tissue to the polypropylene tube (1.7 ml), add 200 μl of lysis buffer (containing 100 mM Tris-HCl, 50 mM NaCl, 150 mM EDTA, 1% SDS), and proteinase K 10. After the addition of μl and the reaction at 60 ℃ for 1 hour in a heater to prepare a first cell degradation product.

6. Inject 100 μl of the CNT dispersion and 200 μl of the first cell degradation product into a polypropylene tube (1.7 μl) to form a mixture, centrifuge the mixture at 12,000 rpm for 10 minutes, and separate by centrifugation. The supernatant was removed.

7. After adding 700 μl of 70% ethanol to the precipitate, centrifuging at 12,000 rpm for 10 minutes, removing the supernatant separated by centrifugation, separating the precipitate, and then putting the precipitate into a heating dryer. Dry at 60 ° C. for 30 minutes.

8. Inject the precipitate and the DNA elution reagent (0.1M Tris-HCl, pH 8.8) into a polypropylene tube (1.7 μl), perform vortexing for 10 seconds, and then centrifuge for 1 minute at 12,000 rpm, Purified nucleic acid was extracted by centrifugation to recover the separated supernatant.

Repeated extraction of purified nucleic acid under the above conditions was performed once more, and in order to distinguish two examples under the same experimental conditions performed as described above, each of [Example 1-1] and [Example 1-2]. Named.

[Example 2]

Extracting nucleic acid under the same experimental conditions as in [Example 1], but instead of the first cell degradation product of [Example 1],

20 μl of whole blood was added to a polypropylene tube (1.7 mL), 200 μl of Lysis buffer (containing 100 mM Tris-HCl, 50 mM NaCl, 150 mM EDTA, 1% SDS), and 10 μl of proteinase K were added thereto. After that, a second cell degradation product produced by reacting at 60 ° C. for 1 hour in a heater was used.

In order to distinguish the second embodiment under the same experimental conditions, the process of extracting the purified nucleic acid under the above conditions was repeated one more time, [Example 2-1] and [Example 2-2], respectively. Named.

[Example 3]

Extracting nucleic acid under the same experimental conditions as in [Example 1], but instead of the first cell degradation product of [Example 1],

10 mg of beef tissue was added to a polypropylene tube (1.7 ml), and 200 µl of DirGe ^® [Salal Bio, Korea] was added, followed by reaction at room temperature for 10 minutes.

Repeatedly extracting the purified nucleic acid under the above conditions one time, and in order to distinguish two examples under the same experimental conditions performed as described above [Example 3-1] and [Example 3-2], respectively. Named.

Example 4

Extracting nucleic acid under the same experimental conditions as in [Example 1], but instead of the first cell degradation product of [Example 1],

In the whole blood 20㎕ in polypropylene tubes (1.7㎖) and, DirGene ^® [saeneul Bio, Korea] was charged into a 200㎕, was used and the resulting by reacting for 10 minutes, a fourth cell decomposition products at room temperature.

Repeatedly extracting the purified nucleic acid under the above conditions one time, and in order to distinguish two examples under the same experimental conditions performed as described above [Example 4-1] and [Example 4-2], respectively. Named.

Comparative Example

1.10 mg beef tissue was added to a 1.5 ml tube, 200 μl of TE buffer and 20 μl of 10% SDS and 10 μl of proteinase K (20 mg / ml) were added to the tube, followed by 60 ° C. The reaction was carried out for 1 hour.

2. Once the beef tissue had loosened, the mixture was centrifuged at 12,000 rpm for 2 minutes through a centrifuge.

3. Transfer the separated supernatant to a new tube by centrifugation, and contain the genomic DNA binding buffer (6M Gu-HCl, 10mM Tris-HCl, 62.5mM MES, 7% Troton X-100) in the tube containing the supernatant. : pH 4.5) 200 μl was added and mixed.

4. After the binding column and the collection column were combined, the mixture injected with the genomic DNA binding buffer was transferred to the binding column.

5. After centrifugation of the mixture at 12,000 rpm for 2 minutes, the contents of the collection column were removed, and 70% wash buffer (containing 10 mM Tris-HCl, 80% Ethanol) at pH 7.5 in the combined column. Μl was injected.

6. The ethanol-added mixture was centrifuged at 12,000 rpm for 2 minutes through a centrifuge.

7. The contents contained in the collection column were removed, 500 μl of 70% ethanol was injected into the combined column, and then centrifuged at 12,000 rpm for 2 minutes.

8. The contents contained in the collection column were removed and centrifuged for 2 minutes at 12,000 rpm to completely remove the ethanol.

9. Purified nucleic acid was extracted by injecting 100 [mu] l of DNA elution reagent [10 mM Tris-HCl with pH 8.8, T & I, Korea].

[Experimental Example 1]

The concentration of the purified nucleic acid was measured through [Example 1-1] to [Example 4-2] using a UV spectrometer. The results are shown in Table 1 below.

[Table 1]

Here, T denotes beef tissue, B denotes blood, lysis denotes lysine buffer, and DLB denotes DirGene ^®, and the experiment was repeated under the same conditions to confirm the accuracy of the experiment. Looking at the Table 1, it can be confirmed that the addition of a positively charged CNT to beef tissue has excellent binding performance of the nucleic acid to the CNT. At this time, in the experiment of Example 1-2, the beef tissue contained more fat than the beef tissue in the experiment of Example 1-1, and it was determined that an error occurred in the repeated experiment result.

[Experimental Example 2]

Since the experiment was conducted with beef tissue and blood, the PCR reaction was confirmed using a cow's groping gene.

First, 12.5 μl of 2X PCR premix reagent [Senul Premix, Salal Bio, Korea] in a PCR tube (200 μl), 1 μl each of a pair of primers (MC1R Forward primer, MC1R Reverse primer) targeted to a bovine chromosome region, 2 μl of purified nucleic acid and 3.5 μl of distilled water were added and mixed through Example 1-1 to prepare a PCR reaction solution.

Subsequently, the tube containing the PCR reaction solution was amplified using a PCR instrument (GeneAmp PCR System 2700, Applied Biosystem, USA).

Then, 2 g of agarose [QA-Agarose ™, Q-bio gene, USA] was added to a Erlenmeyer flask (250 mL), and 0.5X TBE (tris boric acid EDTA) buffer solution [Tris-Base, Q-biogene, USA] 100 ml.

Subsequently, the mixture of the Erlenmeyer flask was dissolved in a microwave oven for 2 minutes, and then the mixture was placed in a gel container, and the mixture was solidified for 30 minutes to prepare a 2% agarose gel.

Next, fill the 0.5X TBE buffer solution with electrophoresis device (Mupid-α, Advance, Japan), and then 4µL of PCR reaction solution containing amplified nucleic acid and 6X Loading dye (0.25% bromophenol blue, 0.25% xylene cyanol). 0.8 μl of FF, 30% glycerol) [Bio Basic, CANADA] were mixed and loaded at 4.8 μl.

Then, electrophoresis was performed for 25 minutes at 100V.

In the following procedure, the gel was removed, stained with EtBr (ethidium bromide) [SIGMA ^® , USA] for 10 minutes, and the EtBr that did not bind to the nucleic acid was washed with distilled water for 10 minutes.

Subsequently, the agarose gel was placed on a UV transilluminator [CTOC KOREA, Korea] to confirm the presence of nucleic acid. The results are shown in Fig.

Subsequently, the experiment was conducted in the same manner using the purified nucleic acids obtained through the remaining examples, that is, [Example 1-2] to [Example 4-2], and the results are shown in FIG. 3.

Comparative Example

The presence or absence of nucleic acid was confirmed under the same experimental conditions as in [Experimental Example 2], but the nucleic acid purified through [Comparative Example] was used instead of the nucleic acid purified through [Example 1-1]. The results are shown in Fig.

FIG. 3 is an electrophoretic photograph after gene amplification of a nucleic acid purified by a nucleic acid extraction method according to the present invention through a PCR reaction. If ten lanes are shown, lanes 1 and lane 2 are sequentially selected from the left lane for convenience of description. , Lane 3, .... lane 10.

In FIG. 3, the first lane is the result obtained using the nucleic acid extracted through [Comparative Example], and the second lane is a 100bp marker. In addition, the third lane is the result obtained using [Example 1-1], the fourth lane is [Example 1-2], the fifth lane is [Example 2-1], and the sixth lane is [Example Example 2-2], the seventh lane is [Example 3-1], the eighth lane is [Example 3-2], the ninth lane is [Example 4-1], and the tenth lane is [Example 4]. -2] is the result obtained using the nucleic acid extracted through. In this case, as described above, the third and fourth lanes, the fifth and sixth lanes, the seventh and eighth lanes, the ninth and tenth lanes are repeated under the same conditions to confirm the accuracy of the experiment. This is the result of the experiment.

As shown in Figure 3, using the nucleic acid extraction method according to the present invention, it was confirmed that the nucleic acid recovery can be excellent as well as the nucleic acid recovery method compared to the silica-based nucleic acid extraction method (comparative example). .

While the above has been described with reference to a preferred embodiment of the present invention, those skilled in the art will be able to variously modify and change the present invention without departing from the spirit and scope of the invention as set forth in the claims below. It will be appreciated.

Claims (10)

(Iii) mixing a sample containing a nucleic acid with a positively charged carbon nanotube and reacting the same;
(Ii) washing by adding a wash buffer to the mixture of the sample and the positively charged carbon nanotubes; And
(Iii) extracting a nucleic acid using a positively charged carbon nanotube comprising the step of recovering the nucleic acid by adding a DNA elution reagent to the washed mixture. The method of claim 1, wherein prior to said step (iii)
A method of extracting a nucleic acid using a positively charged carbon nanotube, further comprising the step of modifying the non-polar carbon nanotubes with positively charged carbon nanotubes. The method of claim 2, wherein the positively charged carbon nanotubes are
Adding a mixed acid formed of sulfuric acid and nitric acid to the non-polar carbon nanotube to produce a carbon nanotube substituted with a carboxyl group, and
A method for extracting nucleic acids using carbon nanotubes having positively charged carbon nanotubes, wherein the carbon nanotubes are substituted by adding diamines and solvents to the carboxyl group-substituted carbon nanotubes to produce carbon nanotubes substituted with amino groups. The method of claim 1, wherein steps (iii) and (ii)
Centrifuging the mixture of the sample and the positively charged carbon nanotubes to separate the separated precipitate characterized in that it further comprises the step of extracting a nucleic acid using a positively charged carbon nanotubes. The method of claim 1, wherein steps (ii) and (iii)
The method of extracting a nucleic acid using a positively charged carbon nanotubes, characterized in that it further comprises the step of drying the washed mixture. The method of claim 1, wherein step (ii)
Adding a wash buffer to the mixture,
Centrifuging the mixture to which the wash buffer is added, and
Extracting a nucleic acid using a positively charged carbon nanotubes, characterized in that consisting of the step of removing the separated supernatant through the centrifugation. The method of claim 1, wherein step (iii)
Adding a DNA elution reagent to the washed mixture;
Vortexing the mixture to which the DNA elution reagent is added,
Centrifuging the mixture subjected to the vortexing, and
A method for extracting nucleic acids using positively charged carbon nanotubes, characterized in that the step of recovering the separated supernatant through the centrifugation. The method of claim 1, wherein the sample
A nucleic acid extraction method using a positively charged carbon nanotubes, characterized in that the biological wall in which the cell wall or cell membrane is decomposed by a cell decomposition reagent. The method of claim 8, wherein the cell lysis reagent is
A method for extracting nucleic acids using positively charged carbon nanotubes, characterized in that DirGene ^® Lysis buffer. (Iii) mixing the sample containing the nucleic acid and the CNT dispersion containing the positively charged carbon nanotubes and reacting the same;
(Ii) centrifuging the mixture of sample and CNT dispersion to separate the separated precipitate;
(Iii) washing by adding washing buffer to the precipitate;
(Iii) drying the washed precipitate; And
(Iii) recovering the nucleic acid by adding a DNA elution reagent to the washed precipitate, the nucleic acid extraction method using a positively charged carbon nanotube comprising a.

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