KR101380909B1 - Absorbents for purification of nucleic acid and a purification method using the absorbents - Google Patents

Abstract

The present invention, in an adsorbent for purifying nucleic acids from a biological specimen, relates to an adsorbent for nucleic acid purification comprising more than one from a group consisting of zeolites, activated carbon, bentonite, white clay, and red clay, a purification column comprising the same, a purification kit, and a purification method using the adsorbent. An adsorbent of the present invention does not go through an additional complicated and longtime centrifugal process but can promptly isolate non-nucleic acid substances from the biological specimen thereby easily obtaining high yield of nucleic acids.

Description

Absorbents for purification of nucleic acid and a purification method using the absorbents}

The present invention provides an adsorbent for purifying nucleic acids from biological samples, wherein the adsorbent for purifying nucleic acids comprising any one or more from the group consisting of zeolite, activated carbon, bentonite, clay and loess, a purification column comprising the same, and a kit for purification thereof. It relates to a purification method using an adsorbent.

The technology of purely separating nucleic acids from biological samples is an essential technology in all of biotechnology such as biochemistry, molecular biology, and medicine that deal with genes.

Nucleic acids present in cells are divided into genomic DNA, plasmid DNA, and RNA, and different methods are used depending on the type of nucleic acid to be purified. However, the basic principle of purifying nucleic acid in a biological sample is to dissolve the cell and to remove the nucleic acid. Cell components are removed by various methods and ethanol is added to the aqueous nucleic acid solution to precipitate and recover the nucleic acid.

Methods for lysing cells include large physical, chemical and enzymatic methods. Representative examples of the physical method include a method of mixing a glass egg and a cell and then vigorously mixing the cell and using an osmotic pressure. Chemical methods include surfactants that explode cell membranes, and enzymatic methods include enzymes that selectively degrade cell walls. In addition, a combination of two or more methods may be used, such as lysing cells by heating cells treated with surfactants and enzymes. This method has been widely used to date since the development in the 1970s (Sambook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

With the development of carriers that specifically adsorb nucleic acids only from a variety of materials contained in cell lysates, ethanol precipitation has been replaced by a method that can isolate nucleic acids in a short time with high efficiency using nucleic acid adsorbents. Most products for nucleic acid purification have been used to produce a nucleic acid adsorbent such as silica gel or glass fiber in a columnar form.

The most widely used product for nucleic acid purification is a kit for purifying plasmid DNA from microorganisms, and all of these kits purely separated plasmid DNA using an alkali lysis method (Birnboim HC & Doly J., 1979, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Res 7 (6), 1513-1523). The alkali dissolution method generally consists of nine steps. First, centrifugation of the microbial culture to recover the microbial cells, suspending in the buffer solution containing the enzyme inhibitor, lysing the cells by adding an alkaline solution consisting of 0.2N NaOH, 1% SDS (Sodium Dodesyl Sulfate) At this stage, genomic DNA with large molecular weight is irreversibly denatured. Neutralizing the cell lysate by adding an acidic buffer solution such as 3 M potassium acetate and pH 5.5, separating the aqueous layer and the cell precipitate by centrifugation, and supernatant containing the plasmid DNA. Selectively binding the plasmid DNA to the column, passing the washing solution containing ethanol again to remove defective impurities in the column, further centrifuging to completely remove the washing solution, and finally distilled water. B) eluting the plasmid DNA using an appropriate buffer solution.

To date, technology for developing a plasmid DNA purification kit has been proposed to improve the nucleic acid adsorbent (US Pat. Korean Patent 10-0622606) and the like have been developed, but all did not overcome the limitations of the existing method using the alkali dissolution method.

In the alkaline lysis method, in order to use the purified plasmid DNA for the enzymatic reaction, it was necessary to completely remove the detergent component and excess salt added during the cell lysis. Therefore, a high purity plasmid DNA through the alkali lysis method requires a number of solutions and has the disadvantage of requiring more than 30 minutes for nucleic acid purification because of the cumbersome steps of centrifugation.

Boiling method of boiling cell suspension instead of alkali lysis to dissolve cells and degenerate genomic DNA is more convenient than alkali lysis method, but purity of purified plasmid DNA is lower than alkali lysis method and requires boiling water. Due to the high viscosity of the cell lysate, it was not commercialized because centrifugation was required for more than 10 minutes to remove the cell lysate. Thus, most labs rarely use it (Sambook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Therefore, there is a need for the development or purification of a new adsorbent that solves the problems of the complex and long time addition of the solution of the alkali dissolution method or the boiling method and the centrifugation process and can purify the nucleic acid in a high yield in a short time. ought.

United States Patent Registration Publication No. 5075430 United States Patent Registration Publication No. 5155018 U.S. Patent Registration Publication No. 5658548 United States Patent Application Publication No. 588041 Korean Patent Registration Publication No. 0622606

Sambook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 Birnboim H. C. & Doly J., 1979, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Res 7 (6), 1513-1523).

It is an object of the present invention to provide an adsorbent for selectively adsorbing only non-nucleic acid materials for purifying only nucleic acids from various biological samples in high yield in a short time. In addition, an object of the present invention is to provide a purification method capable of purifying only nucleic acid in a high yield in a short time using the adsorbent.

The above object of the present invention can be achieved by providing an adsorbent for purification of nucleic acid from one or more selected from the group consisting of zeolite, activated carbon, bentonite, clay and loess in the adsorbent for purifying nucleic acids from biological samples.

The present invention relates to an adsorbent for purifying nucleic acids from biological samples, wherein the adsorbent for purifying nucleic acids comprises at least one selected from the group consisting of zeolite, activated carbon, bentonite, clay and loess.

Specifically, the adsorbent is to selectively adsorb the non-nucleic acid material for separating the nucleic acid, the particle size of the adsorbent is preferably 50 to 250 mesh, the nucleic acid is selected from the group consisting of DNA, RNA, plasmid DNA, mRNA and rRNA It may be one or more, preferably plasmid DNA. The adsorbent is preferably composed of zeolite and activated carbon in a ratio of 1: 1 to 2: 1.

The present invention also relates to a nucleic acid purification column comprising the adsorbent. An adsorbent may be used in the column of 1 to 10 mg, and the adsorbent may be attached to a filter. The filter may be a synthetic resin filter or a membrane filter having pores of 0.1-10 μm.

The present invention also relates to a nucleic acid purification kit comprising the adsorbent.

The present invention provides a method for purifying a nucleic acid from a biological sample, the method comprising: a) recovering cells by centrifuging a biological sample and suspending it in a buffer solution; b) lysing the cell suspension and passing the column containing the sorbent for purification of nucleic acid to purify the nucleic acid; And c) recovering the purified nucleic acid. Preferably, in step b), lysozyme may be added to lyse the cells, and microwave irradiation may include the step of lysing the sample. Specifically, the microwave can be irradiated for 30 to 60 seconds and the lysozyme is preferably added 0.1mg to 10mg per column.

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

The present inventors have developed an effective nucleic acid purification method for shortening the processing time of the long time due to the various solutions and the centrifugation step in the conventional alkali dissolution method. The adsorbents of the present invention can purify nucleic acids with high yields in a significantly shorter time compared to conventional processes.

The biological sample of the present invention can be used for any biological sample such as microorganisms consisting of Escherichia coli, bacteria, yeast and bacillus, animal tissues, plant tissues, blood, secretions, and samples in which the sample is transformed with a recombinant gene.

The adsorbent of the present invention is used to separate non-nucleated substances, which are insoluble aggregates consisting of protein denatured aggregates and cell debris particles, from nucleic acid materials after lysing cells from biological samples. It is. In the case of using the adsorbent of the present invention, the adsorbent adsorbs the non-nucleic acid material to lower the viscosity of the cell lysate, thereby increasing the concentration of the nucleic acid, thereby obtaining a high yield of purified nucleic acid by only one centrifugation.

The present inventors have experimented with various materials capable of adsorbing organic materials and found that activated carbon, zeolite, bentonite, white clay and loess selectively adsorb only non-nucleic acid materials efficiently.

Therefore, the present invention can purify the nucleic acid using an adsorbent including at least one component of activated carbon, zeolite, bentonite, clay and loess. The average particle diameter of the adsorbent is preferably 50-250 mesh. If the average particle diameter is less than 50 mesh, the adsorption capacity may drop, and if the average particle diameter exceeds 250 mesh, it may be difficult to process the adsorbent.

Nucleic acid refers to DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) includes one or more selected from the group consisting of genomic DNA, RNA, plasmid DNA, mRNA and rRNA, preferably may be plasmid DNA.

As a result of comparing various components and concentrations of the adsorbents, activated carbon and zeolite were simultaneously added in a ratio of 1: 1 to 1: 2, which showed the highest yield.

The adsorbent of the present invention can be used by directly adding to the cell lysate or mounted on a column. In the case of mounting to a column, in order to dispense a small amount of sample under a constant state, the adsorbent is dispersed and then dispensed and dried. Alternatively, the adsorbent may be attached to the filter and mounted. The filter is preferably a synthetic resin filter or a membrane filter having pores of 0.1-10 탆. If the pore is less than 0.1㎛ nucleic acid does not pass through the yield may be low, if it exceeds 10㎛ non-nucleic acid can also pass through the nucleic acid purification effect is reduced.

In addition, the adsorbent is preferably 1 mg to 10 mg per column. If less than 1mg does not show sufficient adsorption effect, it takes a long time to purify the nucleic acid, and if it exceeds 10mg, the recovery of nucleic acid was rather reduced due to too many adsorbents.

In addition, the present invention can be commercialized as a nucleic acid purification kit containing the adsorbent. In the case of a kit using a conventional alkali dissolution method, it is cumbersome and takes a long time by dissolving and centrifugation in several steps, whereas in the case of the kit of the present invention, nucleic acids can be purified in high yield in a short time, thereby providing an effective purification kit. can do.

A method of purifying nucleic acid from a biological sample, comprising: a) centrifuging a biological sample to recover cells and suspending them in a buffer; b) lysing the cell suspension and passing the column containing the sorbent for purification of nucleic acid to purify the nucleic acid; And c) recovering the purified nucleic acid. Preferably, in step b), lysozyme may be added to lyse the cells, and microwave irradiation may include the step of lysing the sample. Specifically, the microwave can be irradiated for 30 to 60 seconds and the lysozyme is preferably added 0.1mg to 10mg per column.

Common boiling methods use surfactants to lyse cells. Since surfactants are included in the plasmid DNA solution, they act as inhibitors of enzymatic reactions such as restriction enzyme treatment or polymerase chain reaction. need. In addition, since the viscosity of the cell lysate is high, centrifugation of 10 minutes or more is required in order to separate from the supernatant. Therefore, a method for rapidly separating the cell lysate and the aqueous solution is required.

In the present invention, a method of lysing cells using only lysozyme and using a microwave to irradiate microwaves instead of using boiling water was used. The microwave frequency of the microwave was about 2.45GHz, and the results of the experiment showed that heating for 30 to 60 seconds showed that the nucleic acid was purified with high yield.

As a result of comparing the amount of lysozyme added, the nucleic acid was purified in the desired yield when 0.1 mg or more was added. If the lysozyme is less than 0.1mg, it is difficult to obtain a sufficient amount of nucleic acid because the cell wall is hardly dissolved, and if it exceeds 10mg, it is preferable to contain less than 10mg because it contains more than sufficient amount for cell wall lysis.

Figure 1 (a) schematically shows a conventional alkali purification method. Typically, purification by this method took more than about 30 minutes of 9 steps in total. On the other hand, according to the purification method of the present invention of Figure 1 (b), Example 1 took 4 minutes in a total of four steps. This is about 7 times shorter time and shows the effect that a high yield of nucleic acid can be obtained in a simple process in a short time compared to the conventional method. As can be seen in Patent Experimental Examples 7 and 8, it was confirmed that the plasmid DNA purified by the present invention can be obtained in a better yield compared to the DNA purified by the conventional method, and in particular, although the experiment time was shortened. It was confirmed that there was no effect on the purity and sequence of the purified plasmid DNA.

The adsorbent of the present invention was able to quickly separate non-nucleic acid material without going through a separate complicated long time centrifugation process, and showed an effect of easily obtaining a high yield of nucleic acid. In particular, the purification method of the present invention can lyse cells using only lysozyme, so that the nucleic acid can be easily purified without using a separate surfactant and without a washing step such as ethanol precipitation. have.

Figure 1 (a) is a schematic diagram of a conventional nucleic acid extraction method, (b) is a schematic diagram of a nucleic acid extraction method according to the present invention.
2 is an electrophoretic diagram of DNA recovered using different adsorbents for each concentration of activated carbon.
Lane 1: Comparative Example 1
Lane 2: Example 1 (0.5 mg of activated carbon)
Lane 3: Example 2 (1 mg of activated carbon)
Lane 4: Example 3 (1.5 mg of activated carbon)
Lane 5: Example 4 (2 mg of activated carbon)
Lane 6: Comparative Example 2
Lane 7: size indicator
3 is an electrophoretic diagram of DNA recovered using different adsorbents for each concentration of zeolite.
Lane 1: Comparative Example 1
Lane 2: example 5 (zeolite 2 mg)
Lane 3: Example 6 (zeolite 4 mg)
Lane 4: Example 7 (zeolite 6 mg)
Lane 5: Example 8 (zeolite 8 mg)
Lane 6: comparative example 2
Lane 7: size indicator
Figure 4 is an electrophoresis of the recovered DNA when using different concentrations of activated carbon and zeolite at the same time.
Lane 1: Comparative Example 1
Lane 2: Example 9 (0.5 mg of activated carbon + 2 mg of zeolite)
Lane 3: Example 10 (activated carbon 1 mg + zeolite 4 mg)
Lane 4: Example 11 (Activated Carbon 1.5 mg + Zeolite 6 mg)
Lane 5: Example 12 (activated carbon 2 mg + zeolite 8 mg)
Lane 6: Comparative Example 2
Lane 7: size indicator
Figure 5 is the electrophoresis of the recovered DNA when using a different ratio of zeolite and activated carbon.
Lane 1: Example 13 (1 mg of activated carbon + 2 mg of zeolite)
Lane 2: Example 14 (2 mg of activated carbon + 4 mg of zeolite)
Lane 3: Example 15 (4 mg of activated carbon + 4 mg of zeolite)
Lane 4: Example 16 (1 mg of activated carbon + 4 mg of zeolite)
Lane 5: Example 17 (2 mg of activated carbon + 8 mg of zeolite)
Lane 6: comparative example 2
Lane 7: size indicator
6 is an electrophoresis diagram of the DNA recovered when the microwave heating time is different.
Lane 1: Comparative Example 3
Lane 2: Example 18 (Microwave 10 Second Heating)
Lane 3: Example 19 (microwave 20 seconds heating)
Lane 4: Example 20 (microwave oven 30 seconds heating)
Lane 5: Example 21 (Microwave 40 sec Heating)
Lane 6: Example 22 (Microwave 50 sec Heating)
Lane 7: Example 23 (microwave oven 60 seconds heating)
Lane 8: Example 24 (microwave oven 70 seconds heating)
Lane 9: Example 25 (80 seconds of microwave heating)
Lane 10: comparative example 2
Lane 11: size indicator
Figure 7 is the electrophoresis of the recovered DNA when the amount of lysozyme used. Lane 1: comparative example 4
Lane 2: Example 26 (lysozyme 0.01 mg)
Lane 3: Example 27 (lysozyme 0.1 mg)
Lane 4: Example 28 (lysozyme 1 mg)
Lane 5: size indicator
8 is an electrophoretic diagram of purified DNA and DNA treated with the restriction enzyme thereof.
Lane 1: Example 1
Lane 2: Example 1 (treated with restriction enzyme EcoRI)
Lane 3: Comparative Example 1
Lane 4: Comparative Example 1 (treated with restriction enzyme EcoRI)
Lane 5: size indicator
9 is a saline sequence analysis.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are illustrative only and are not intended to be limiting.

Example 1

Nucleic Acid (Plasmid DNA) Purification Protocol

E. coli DH5a transformed with a 6 kbp plasmid derived from pBluscript and pBluscript was seeded in 1 L LB culture medium (LB broth, 10 g tryptone, 5 g yeast extract, 10 g sodium chloride) containing 100 μg / ml ampicillin. Shake culture was performed for 18 hours at a temperature of 37 ℃. 1.5 ml of each of the E. coli shake cultures was dispensed into 1.5 ml tubes and centrifuged to precipitate and stored at −20 ° C. in a cell mass, if necessary, to be taken out. All reagents were purchased from Sigma-Aldrich and used.

100 μl of TE (Tris-HCl 10 mM, 1 mM EDTA, pH8.0) buffer solution was added to the cultured cell masses dispensed by the above method, and the cells were completely suspended by stirring for 10 seconds by vortexing. 0.5 mg of activated charcoal was suspended in distilled water, loaded on the column, and dried in a dryer. Transfer the cell suspension to a column containing 0.5 mg of activated carbon adsorbent, add 1 mg of lysozyme, stir for 10 seconds, heat for 40 seconds in microwave to irradiate microwave, and centrifuge at 13,000 rpm for 2 minutes at room temperature. Plasmid DNA was eluted.

Example 2 -17

The cell suspension of Example 1 was passed in the same manner as in Example 1 except for passing through 0.5 mg of activated carbon through a column containing an adsorbent of the following components.

Example No. absorbent Example 2 Activated carbon 1mg Example 3 Activated carbon 1.5mg Example 4 Activated carbon 2mg Example 5 Zeolite 2mg Example 6 Zeolite 4mg Example 7 Zeolite 6mg Example 8 Zeolite 8mg Example 9 Activated Carbon 0.5mg + Zeolite 2mg Example 10 Activated Carbon 1mg + Zeolite 4mg Example 11 Activated Carbon 1.5mg + Zeolite 6mg Example 12 Activated Carbon 2mg + Zeolite 8mg Example 13 Activated Carbon 1mg + Zeolite 2mg Example 14 Activated Carbon 2mg + Zeolite 4mg Example 15 Activated Carbon 4mg + Zeolite 4mg Example 16 (Example 10) Activated Carbon 1mg + Zeolite 4mg Example 17 (Example 12) Activated Carbon 2mg + Zeolite 8mg

Examples 16 and 17 were carried out under the same conditions as in Examples 10 and 12, respectively.

Examples 18-25

The cell suspension of Example 1 was passed through a column containing 2 mg of activated carbon and 4 mg of zeolite adsorbent instead of 0.5 mg of activated carbon, and the irradiation time of microwaves was changed in the same manner as in Example 1 except that It was.

Example No. Microwave irradiation time Example 18 10 seconds Example 19 20 seconds Example 20 30 seconds Example 21 40 seconds Example 22 50 seconds Example 23 60 seconds Example 24 70 seconds Example 25 80 seconds

Examples 26-28

The cell suspension of Example 1 was passed through a column containing 2 mg of activated carbon and 4 mg of zeolite adsorbent instead of 0.5 mg of activated carbon, and the amount of lysozyme used was changed in the same manner as in Example 1. .

Example No. Usage of Lysozyme Example 26 0.01mg Example 27 0.1mg Example 28 1mg

Comparative Example 1

The same procedure as in Example 1 was carried out except that no adsorbent was used in the cell suspension of Example 1. Instead of using the sample once purified, it was analyzed after each experiment.

Comparative Example 2

Plasmid DNA was purified by alkaline lysis according to the method provided by the manufacturer using the Hybrid-Q Plasmid mini kit purchased from GeneAll Co., Ltd. in the same cell mass as in Example 1.

Comparative Example 3

The cell suspension of Example 1 was passed through a column containing 2 mg of activated carbon and 4 mg of zeolite adsorbent instead of 0.5 mg of activated carbon, and was subjected to the same method as Example 1 except that no microwave was irradiated.

Comparative Example 4

The cell suspension of Example 1 was passed through a column containing 2 mg of activated carbon and 4 mg of zeolite adsorbent instead of 0.5 mg of activated carbon, and was carried out in the same manner as in Example 1 except that lysozyme was not used.

Experimental Example 1

In order to observe the effect on the concentration of nucleic acid purification of the adsorbent consisting of activated carbon, 2 μl of the eluted plasmid DNA solution of Examples 1 to 4 was taken and subjected to electrophoresis analysis in 0.5x TAE buffer using 0.9% agarose gel. It was isolated and stained with ethidium bromide and confirmed by ultraviolet irradiation. And for the comparative experiments, the eluted plasmid DNA solutions of Comparative Examples 1 and 2 were analyzed in the same manner. The electrophoretic analysis results are shown in FIG. 2, and the examples or comparative examples according to the lane numbers are shown in Table 4 below.

Electrophoresis Lane Number Examples or Comparative Examples One Comparative Example 1 2 Example 1 (0.5 mg of activated carbon) 3 Example 2 (1 mg of activated carbon) 4 Example 3 (1.5 mg of activated carbon) 5 Example 4 (2 mg of activated carbon) 6 Comparative Example 2 7 Size indicator

As can be seen in FIG. 2, when 1-2 mg of activated carbon was added, it was confirmed that the product was purified in a similar yield compared to a commercialized product.

Experimental Example 2

In order to observe the effect on the concentration of nucleic acid purification of the adsorbent consisting of zeolite, 2μl of the eluted plasmid DNA solution of Examples 5 to 8 was taken and subjected to electrophoresis analysis in 0.5x TAE buffer using 0.9% agarose gel. It was isolated and stained with ethidium bromide and confirmed by ultraviolet irradiation. And for the comparative experiments, the eluted plasmid DNA solutions of Comparative Examples 1 and 2 were analyzed in the same manner. The electrophoretic analysis results are shown in FIG. 3 and the examples or comparative examples according to the lane numbers are shown in Table 5 below.

Electrophoresis Lane Number Examples or Comparative Examples One Comparative Example 1 2 Example 5 (zeolite 2 mg) 3 Example 6 (4 mg of zeolite) 4 Example 7 (zeolite 6 mg) 5 Example 8 (zeolite 8 mg) 6 Comparative Example 2 7 Size indicator

As can be seen in Figure 3, when 4-8 mg of activated carbon was added, it was confirmed that the product was purified in a similar yield as compared to commercialized products.

Experimental Example 3

In order to observe the effect of sorbent consisting of activated carbon and zeolite on nucleic acid purification by concentration, 2 μl of the eluted plasmid DNA solution of Examples 9-12 was taken and electrophoresed in 0.5 × TAE buffer using 0.9% agarose gel. It was isolated by analytical method, stained with ethidium bromide, and confirmed by ultraviolet irradiation. And for the comparative experiments, the eluted plasmid DNA solutions of Comparative Examples 1 and 2 were analyzed in the same manner. The electrophoretic analysis results are shown in FIG. 4, and the examples or comparative examples according to the lane numbers are shown in Table 6 below.

Electrophoresis Lane Number Examples or Comparative Examples One Comparative Example 1 2 Example 9 (0.5 mg of activated carbon + 2 mg of zeolite) 3 Example 10 (Activated Carbon 1 mg + Zeolite 4 mg) 4 Example 11 (1.5 mg of activated carbon 6 mg of zeolite) 5 Example 12 (Activated Carbon 2 mg + Zeolite 8 mg) 6 Comparative Example 2 7 Size indicator

As can be seen in FIG. 4, the adsorbent using both activated carbon and zeolite at the same time was confirmed to be purified in a similar or better yield when compared to commercialized products.

Experimental Example 4

In order to observe the effect of the concentration or relative ratio of both components of the adsorbent consisting of activated carbon and zeolite on nucleic acid purification, 2 μl of the eluted plasmid DNA solution of Examples 13 to 17 was taken and 0.5x using 0.9% agarose gel. It was isolated by electrophoretic analysis in TAE buffer solution, and stained with ethidium bromide and confirmed by UV irradiation. And for the comparative experiments, the eluted plasmid DNA solution of Comparative Example 2 was analyzed in the same manner. The electrophoretic analysis results are shown in FIG. 5, and the examples or comparative examples according to the lane numbers are shown in Table 7 below.

Electrophoresis Lane Number Example or Comparative Example Number One Example 13 (1 mg of activated carbon + 2 mg of zeolite) 2 Example 14 (2 mg activated carbon + 4 mg zeolite) 3 Example 15 (4 mg of activated carbon + 4 mg of zeolite) 4 Example 16 (1 mg activated carbon + 4 mg zeolite) 5 Example 17 (2 mg of activated carbon + 8 mg of zeolite) 6 Comparative Example 2 7 Size indicator

As can be seen in Figure 5 when using the same ratio of activated carbon and zeolite or using zeolite twice the activated carbon it can be seen that the yield is increased, especially when using 2 mg and 4 mg zeolite activated carbon It was confirmed that the purified in high yield.

Experimental Example 5

In order to observe the effect on the purification of nucleic acid according to the irradiation time of microwave, 2 μl of the eluted plasmid DNA solution of Examples 18 to 25, which were experimented by increasing the operating time of the microwave oven by 10 seconds, was used to prepare a 0.9% agarose gel. It was isolated by electrophoresis analysis in 0.5x TAE buffer solution, and stained with ethidium bromide and confirmed by UV irradiation. And for the comparative experiments, the eluted plasmid DNA solution of Comparative Example 3 which was not heated in the microwave was analyzed in the same manner. The electrophoretic analysis results are shown in FIG. 6, and the examples or comparative examples according to the lane numbers are shown in Table 8 below.

Electrophoresis Lane Number Example or Comparative Example Number One Comparative Example 3 2 Example 18 (Microwave 10 seconds heating) 3 Example 19 (Microwave 20 sec heating) 4 Example 20 (30 seconds of microwave heating) 5 Example 21 (Microwave 40 sec heating) 6 Example 22 (Microwave 50 sec heating) 7 Example 23 (microwave oven 60 seconds heating) 8 Example 24 (microwave oven 70 seconds heating) 9 Example 25 (microwave oven 80 seconds heating) 10 Comparative Example 2 11 Size indicator

As can be seen in Figure 6, in order for the cell lysis to occur sufficiently, it was necessary to heat for 30 seconds or more, and in excess of 60 seconds, the solution was cut off and overflowed, rather the yield decreased. Therefore, the operation time was found to show excellent purification yield from 30 seconds to 60 seconds.

Experimental Example 6

In order to observe the effect of lysozyme on nucleic acid purification according to the amount of lysozyme, 2 μl of the eluted plasmid DNA solution of Examples 26 to 28, which were experimented with different amounts of lysozyme, was taken, and 0.5x TAE buffer solution using 0.9% agarose gel. It was separated by electrophoresis analysis, and stained with ethidium bromide and confirmed by ultraviolet irradiation. And for the comparative experiments were also analyzed for the eluted plasmid DNA solution of Comparative Example 4 which was not added lysozyme at all. The electrophoretic analysis results are shown in FIG. 7, and the examples or comparative examples according to the lane numbers are shown in Table 9 below.

Electrophoresis Lane Number Example or Comparative Example Number One Comparative Example 4 2 Example 26 (Lysozyme 0.01 mg) 3 Example 27 (lysozyme 0.1 mg) 4 Example 28 (lysozyme 1 mg) 5 Size indicator

As can be seen in Figure 7, in Comparative Example 4, since the lysozyme was not added, the cell was not lysed and the nucleic acid was hardly purified. When the lysozyme was used at 0.1 mg or more, the nucleic acid was purified in a high yield.

Experimental Example 7

In order to confirm the purity of the purified nucleic acids according to Example 1 and Comparative Example 2, each plasmid DNA and DNA treated with the restriction enzyme EcoR1 were subjected to electrophoresis analysis. FIG. 8 shows the result of the analysis. Examples or comparative examples according to the lane numbers are shown in Table 10 below.

Electrophoresis result lane number Example or Comparative Example Number One Purified Plasmid DNA of Example 1 2 DNA treated with plasmid DNA of Example 1 with restriction enzyme 3 Purified Plasmid DNA of Comparative Example 2 4 DNA treated with plasmid DNA of Comparative Example 1 with restriction enzyme

As confirmed in Figure 8 it was confirmed that when purified using the adsorbent of the present invention can be obtained nucleic acid in a better yield and purity than the result of using a commercialized product.

Experimental Example 8

In order to confirm the sequence of the nucleic acid purified according to Example 1 and Comparative Example 2, the collected nucleic acid was commissioned by Genotech Co., Ltd. to analyze the gene sequence. In FIG. 9, (a) is a plasmid DNA gene sequence of Comparative Example 2 and (b) is a plasmid DNA gene sequence of Example 1. FIG. Both sequences were exactly matched to confirm that the same nucleic acid was purified.

The foregoing description is merely illustrative of the technical idea of the present invention and various changes and modifications may be made by those skilled in the art without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, and thus the scope of the technical idea of the present invention is not limited to this embodiment. The scope of protection of the present invention should be construed in accordance with the following claims and all technical ideas within the technical scope of the claims should be construed as falling within the scope of the present invention.

Claims (26)

delete delete delete delete delete delete delete delete delete delete delete A method of purifying nucleic acid from a biological sample, comprising: a) centrifuging a biological sample to recover cells and suspending them in a buffer; b) lysing the cells in a suspension containing the adsorbent and then passing the column to purify the nucleic acid; And c) recovering the purified nucleic acid.
The method according to claim 12, wherein in step b), lysozyme is added and microwaves are lysed to dissolve the cell suspension.
The method of claim 13, wherein the microwave is irradiated for 30 to 60 seconds.
The method of claim 13, wherein the lysozyme is added at 0.1-10 mg.
13. The method of claim 12,
The adsorbent comprises at least one selected from the group consisting of zeolite, activated carbon, bentonite, clay and loess.
17. The method of claim 16,
The adsorbent is a purification method characterized in that for adsorbing the non-nucleic acid material for separating the nucleic acid.
17. The method of claim 16,
Purification method characterized in that the particle size of the adsorbent is 50 ~ 250 mesh.
17. The method of claim 16,
The nucleic acid is at least one selected from the group consisting of genomic DNA, RNA, plasmid DNA, mRNA and rRNA.
20. The method of claim 19,
Purification method characterized in that the nucleic acid is plasmid DNA.
17. The method of claim 16,
The adsorbent is a purification method comprising a zeolite and activated carbon in a 1: 1 to 4: 1.
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