KR102344395B1 - Method for Isolating and Preparing Nucleic Acid Using Magnetic Parcticle - Google Patents

Abstract

The present invention relates to a method for separating and purifying nucleic acids using magnetic particles, and more particularly, (a) adding magnetic particles having an average particle size of 50 nm to 1 μm to a biological sample containing nucleic acids to obtain insoluble aggregates or nucleic acid molecules and the binding magnetic particles; and (b) separating the magnetic particles bound to insoluble aggregates or nucleic acid molecules using a magnetic field to obtain nucleic acids.
The nucleic acid separation method using the magnetic particles of the present invention can significantly shorten the nucleic acid separation time compared to the conventional method, and even after the nucleic acid or protein separation process, the purification is completed within a short time using the silica magnetic particles, so that the centrifugation method or gravity separation method It has the advantage of being able to complete the entire process in a much shorter time compared to others, and improving the yield.

Description

Method for Isolating and Preparing Nucleic Acid Using Magnetic Particles

The present invention relates to a nucleic acid separation and purification method using magnetic particles, preferably hydrophobic magnetic particles or silica magnetic particles, and more particularly, (a) magnetic particles having an average particle size of 50 nm to 1 μm in a biological sample containing nucleic acids by adding, binding the insoluble aggregates or nucleic acid molecules to the magnetic particles; and (b) separating the magnetic particles bound to insoluble aggregates or nucleic acid molecules using a magnetic field to obtain nucleic acids.

A method for rapidly and rapidly isolating and purifying a high-purity nucleic acid or protein from a biological material is of utmost importance in biochemical research and diagnostic processes. To this end, various studies have been made on a method for selectively effective and reproducible separation of only nucleic acids or proteins from various types of substances included in the cell lysis solution.

Recently, research on separating nucleic acids or proteins from cell mixtures using magnetic particles is being actively conducted. In the method using magnetic particles, by adding magnetic particles to a cell mixture, binding between the nucleic acid or protein and the magnetic particle is induced to form a binder, and then only the nucleic acid or protein bound to the magnetic particle is selectively separated using an external magnetic field. way to do it In general, it is known that the particle size of magnetic particles used for selectively separating and purifying only nucleic acids or proteins is about 500 to 2000 nm.

US Patent No. 5665554 discloses a method using magnetic beads and a magnet as a method of separating plasmid DNA from genomic DNA material. Specifically, it is a method of isolating only plasmid DNA by adding magnetic beads that bind to plasmid DNA, binding magnetic beads and plasmid DNA to precipitate, and removing the supernatant except for binding magnetic beads and plasmid DNA using a magnet.

U.S. Patent No. 6027945 discloses a method for separating a target material by providing a magnetic field after forming a combination of a biological target material and magnetic silica particles using silica magnetic particles, and at least 2ug of a target material per 1 mg of magnetic silica particles are combined, and a yield of more than 60% is presented.

In US Patent No. 7078224, magnetic particles having a size of 1 to 15 μm are used, and the magnetic particles are separated using pH-dependent ion exchange particles and silica magnetic particles coated with silica oxide. However, these silica-coated magnetic particles have problems in that the manufacturing process is complex and it is difficult to uniformly control the particle size distribution.

The present inventors have developed a new manufacturing method that can more easily manufacture silica magnetic particles and uniformly control the particle size (Korean Patent No. 10-1053023), and the magnetic silica particles produced by the method have a function on the surface It has a spherical shape and has the advantage of uniform particle size distribution. Its particle size is also prepared from 1 to 20 μm.

In order to quickly separate only nucleic acids or proteins bound to magnetic particles from a biological sample, various products are being released to provide a magnetic field from the outside more easily. As an example, Maglisto (Bioneer) has a tube into which a biological sample can be injected and a tube fixing part to which the tube is fixed, and a magnet fixing part capable of providing a magnetic field at an external position corresponding to the tube. The magnet fixing unit provided at this time is also designed to be easily detachable with a magnet from the tube fixing unit, so that the user can easily separate nucleic acids or proteins from biological materials.

However, the magnetic particles used in the above methods do not have sufficient binding force with the insoluble aggregate or the target nucleic acid, so the development of magnetic particles with improved binding force for the binding material is required.

Accordingly, the present inventors confirmed that, when nucleic acids are separated using magnetic particles, preferably hydrophobic magnetic particles or silica magnetic particles, insoluble aggregates including protein-denatured aggregates or nucleic acid molecules can be separated more quickly and efficiently. and completed the present invention.

An object of the present invention is to provide a method for separating nucleic acids from a biological sample using magnetic particles capable of more rapidly and efficiently separating insoluble aggregates or target nucleic acids from biological samples.

Another object of the present invention is to provide a fully automatic nucleic acid separation apparatus for fully automatically separating nucleic acids from a biological sample using the magnetic particles.

Another object of the present invention is to provide a fully automatic nucleic acid separation method using a fully automatic nucleic acid separation device for fully automatically separating nucleic acids from a biological sample using the magnetic particles.

Another object of the present invention is to provide a kit for separation and purification of nucleic acids from a biological sample including magnetic particles having an average particle size of 50 nm to 1 μm.

In order to achieve the above object, the present invention comprises the steps of (a) adding magnetic particles having an average particle size of 50 nm to 1 μm to a biological sample containing a nucleic acid, thereby binding the magnetic particles to an insoluble aggregate or nucleic acid molecule; and (b) separating the magnetic particles bound to insoluble aggregates or nucleic acid molecules using a magnetic field to obtain nucleic acids.

The present invention also includes a pipette block installed to be movable in a horizontal direction and a vertical direction, and for mounting a plurality of pipettes to be detachable; and a magnetic field applying unit for applying and releasing a magnetic field to a specific unit well of a multi-well plate, wherein the biological sample and magnetic particles having an average particle size of 50 nm to 1 μm are added to the multi-well plate. Provided is a fully automatic nucleic acid separation apparatus that fully automatically separates nucleic acids from a sample.

The present invention also provides a method for isolating a target nucleic acid from a biological sample using the fully automatic nucleic acid separation device,

1) adding magnetic particles to a specific unit well of a multi-well plate to bind insoluble aggregates or nucleic acid molecules excluding the target nucleic acid to magnetic particles;

2) obtaining a mixture containing a target nucleic acid excluding insoluble aggregates or magnetic particles bound to nucleic acid molecules;

3) adding hydrophilic magnetic particles to the obtained mixture and binding to the target nucleic acid;

4) removing the mixture except for the hydrophilic magnetic particles to which the target nucleic acid is bound; and

5) separating the target nucleic acid from the hydrophilic magnetic particles bound to the target nucleic acid;

It provides a fully automatic nucleic acid separation method comprising a.

The present invention also provides a kit and apparatus for separation and purification of nucleic acids from a biological sample containing magnetic particles having an average particle size of 50 nm to 1 μm.

The nucleic acid separation method using the magnetic particles of the present invention, preferably hydrophobic magnetic particles or silica magnetic particles, can significantly shorten the nucleic acid separation time compared to the conventional method, and use silica nanoparticles magnetic particles in the process after nucleic acid or protein separation. By completing the purification within a short time, the entire process can be completed in a much faster time than the centrifugal separation method or the gravity separation method, and there is an advantage in that the yield can also be improved.

1 is a diagram showing the result of electrophoresis on an agarose gel after restriction enzyme treatment of plasmid DNA isolated from E. coli cells using the magnetic particles of the present invention.
2 is a diagram showing the results of electrophoresis on an agarose gel by extracting nucleic acid DNA from blood using the method of the present invention.
3 is a diagram showing the results of electrophoresis on an agarose gel by extracting nucleic acid DNA from a bacterial culture using the method of the present invention.
4 is a diagram showing the results of electrophoresis on an agarose gel by extracting nucleic acid DNA from cells cultured using the method of the present invention.
5 is a diagram showing the results of electrophoresis on an agarose gel after extracting nucleic acid DNA from animal tissue cells using the method of the present invention.
6 is a diagram showing the results of electrophoresis on an agarose gel after extracting nucleic acid DNA from plant tissue cells using the method of the present invention.
7 is a diagram showing the results of electrophoresis on an agarose gel by extracting nucleic acid RNA from various cells cultured using the method of the present invention.
8 is a schematic diagram of an automatic refining apparatus.
9 is a state diagram of a base plate in use.
10 is a state diagram in which the tube rack is mounted on the base plate.
11 is a diagram illustrating a state of using a base plate.
12 is a magnet member device for separating magnetic particles to which the method of Example 6 of the present invention is applied.
13 is a view showing a state of use in which a magnet member is disposed on a base plate.
14 is a cross-sectional view in which a multi-well plate is inserted into a base plate on which a magnet member is disposed.
15 is a perspective view of a magnet mounting unit provided with a magnetic field applying unit.
16 is a flowchart of a method for separating a target nucleic acid using magnetic particles.
17 is a result of electrophoresis of the extracted plasmid on an agarose gel by performing extraction of several of the same sample with a fully automatic nucleic acid extraction equipment to which the same method as in Example 2 is applied.
FIG. 18 shows the results confirmed on electrophoresis after the plasmid nucleic acid was extracted and treated with a restriction enzyme capable of separating a specific nucleotide sequence around the inserted target gene, appropriately cut to the size of the target gene.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

In one aspect, the present invention provides the steps of: (a) adding magnetic particles having an average particle size of 50 nm to 1 μm to a biological sample containing a nucleic acid, and binding the magnetic particles to an insoluble aggregate or nucleic acid molecule; and (b) separating the magnetic particles bound to insoluble aggregates or nucleic acid molecules using a magnetic field to obtain nucleic acids.

Also, after the above step

(c) binding the target nucleic acid to the hydrophilic magnetic particles by adding the hydrophilic magnetic particles having an average particle size of 50 nm to 1 μm to the mixture obtained in step (b); and

It relates to a method for isolating a nucleic acid from a biological sample, further comprising the step of (d) separating the hydrophilic magnetic particles bound to the target nucleic acid using a magnetic field to obtain the target nucleic acid.

The nucleic acid is preferably DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), genomic DNA, plasmid DNA, phage DNA, recombinant DNA, mRNA, rRNA, tRNA, recombinant RNA, micro RNA, etc. may be exemplified, The present invention is not limited thereto. The final target nucleic acid obtained in the present invention may be preferably selected from the group consisting of plasmid DNA, bacterial genomic DNA, blood and animal and plant tissue DNA, and animal cell line DNA and RNA.

In the present invention, the nucleic acid separation method may be variously applied to a centrifugal separation method, a vacuum manifold type, a filter separation method, a gravity separation method or a chromatography method, and a separation method using a magnetic field. This is obvious to those of ordinary skill in the art, and a detailed description thereof will be omitted.

In the present invention, the magnetic particles may be hydrophobic magnetic nanoparticles or silica magnetic nanoparticles.

The magnetic particles may be made of one or more materials selected from the group consisting of iron, cobalt, nickel and oxides or alloys thereof, and have an average size of 50 nm to 1 μm, preferably 50 to 700 nm, most preferably 200 It can be prepared and used at ~ 500 nm.

When the diameter of the magnetic particles is 1 μm or more, it is not preferable because the sedimentation rate of the magnetic particles is increased, which causes inconvenience in use. In addition, when the diameter is 50 nm or less, the magnetic properties are weak, and there may be a problem in separation from the target material. As for the shape of the particle, all of various types of particles such as round, square, needle-shaped, etc. can be used.

In the present invention, the hydrophilic magnetic particles used may be used by coating the magnetic particles with silica. In addition, AccuNanoBead ^TM manufactured by Bioneer can be used, but is not limited thereto, and the average size is 50 nm to 1 μm, preferably 50 to 700 nm, most preferably 200 to 500 nm. A wide range of magnetic particles used by technicians to isolate and purify nucleic acids are available.

The magnetic particles may be used by themselves or dispersed in an aqueous solution, but dispersion in an aqueous solution is more preferable in terms of not only reducing aggregation of magnetic particles but also experimental convenience. In this case, the aqueous dispersion may further contain a dispersing agent to prevent agglomeration and sedimentation of the magnetic particles themselves, and may be stored and used. Lycoside, polyacrylate, polymethacrylate, polyvinylpyrrolidone, polyethyleneamine, polyvinylamine, betaine, glycinate, and at least one dispersant selected from the group consisting of imidazoline and glycerol may be used. .

The step of providing the magnetic particles is

i) providing directly to the biological sample

ii) providing a biological sample by mixing it with a preservation solution for preservation treatment

iii) providing a preservation solution for preserving the biological sample after treatment

iv) providing the biological sample by mixing it with a buffer solution for dissolution

ⅴ) providing a buffer solution after treatment for dissolution of the biological sample

ⅵ) mixing and providing a buffer solution for forming protein-denatured aggregates in the dissolved biological sample

ⅶ) providing a solution in which protein-denatured aggregates are formed

It can be selectively added and used in any one or more steps selected from ii) The most effective way to separate from nucleic acids because magnetic particles are evenly distributed in insoluble aggregates to mix and provide a biological sample with a preservation solution. desirable.

In addition, in applying the step of adding the magnetic particles, it can be used by applying to a nucleic acid or protein extraction kit comprising the magnetic particles according to the present invention.

Another object of the present invention is to provide a method using a material having a magnetic field in a method for separating and separating nucleic acids from insoluble protein-denatured aggregates and cell debris particles bound to magnetic particles. This is a method that utilizes the magnetic properties of magnetic particles, and has the advantage of easily separating the aggregates by providing a magnetic field to the outside of the magnetic particles combined with insoluble protein-denatured aggregates and cell debris particles.

In another aspect, the present invention is a pipette block that is installed to be movable in the horizontal and vertical directions, and for mounting a plurality of pipettes to be detachable; and a magnetic field applying unit for applying and releasing a magnetic field to a specific unit well of a multi-well plate, wherein the biological sample and magnetic particles having an average particle size of 50 nm to 1 μm are added to the multi-well plate. It relates to a fully automatic nucleic acid separation device for fully automatically separating nucleic acids from a sample.

The present invention can automatically separate and purify nucleic acids or proteins from biological samples by using an automatic purification apparatus that is a fully automatic system. The automatic purification device may use the device described in Korean Patent No. 10-25135, Korean Patent Publication No. 2011-0121588, and Korean Patent No. 14-00675 or Exiprep / Exiprogen manufactured by Bioneer (FIG. 8) 15), but is not limited thereto, and is generally applicable without limitation to an automated or semi-automated system capable of separating and purifying nucleic acids or proteins.

The automatic purification device includes: a pipette block installed to be movable in a horizontal direction and a vertical direction, and for detachably mounting a plurality of pipettes through which a fluid material is sucked and discharged; It relates to an automatic biological sample purification apparatus equipped with a magnetic field applying unit, which is mounted on a base plate and includes a magnetic field applying unit for applying and releasing a magnetic field to a specific unit well of a multi-well plate located below the pipette block. .

The present invention may further include a heating unit for heating the specific unit well of the multi-well plate, wherein the magnetic field applying unit includes a magnet mounting unit mounted with a magnet and positioned below a specific unit well of the multi-well plate; a lifting unit for raising and lowering the magnet mounting unit to apply and release a magnetic field to a specific unit well of the multi-well plate; Including, the heating unit may be installed in the magnet mounting unit, the heating unit may be a heating film installed in contact with the magnet mounting unit.

The present invention provides a fixed body for supporting the pipette block; a solution receiver installed to be movable in a horizontal direction by a solution receiver moving means installed on the fixed body and positioned below the plurality of pipettes mounted on the pipette block when the pipette block is moved in the horizontal direction; may include, and is in close contact with the solution holder located under the plurality of pipettes so that parts of the plurality of pipettes soaked in the solution containing the target nucleic acid are blocked from the outside, and the target nucleic acid of the plurality of pipettes It may include an aerosol barrier formed to surround the parts wetted with the containing solution.

In the present invention, the base plate has a pipette rack in which a plurality of pipettes for mounting on the pipette block are inserted and accommodated, and a plurality of target nucleic acid storage tubes for accommodating the separated target nucleic acids are inserted and received in the first tube rack. And a waste liquid container for accommodating waste liquid discarded from a plurality of pipettes mounted on the pipette block may be mounted, and a cooling block for cooling the first tube rack may be mounted on the base plate.

The magnetic field applying unit may additionally include a magnet member to which a magnetic rod is mounted. The magnet member can be mounted under a specific unit well of the multi-well plate, so that the mixture bound to the magnetic particles formed in the specific unit well of the multi-well plate can be agglomerated and separated.

The automatic purification apparatus is characterized in that it uses a multi-well plate manufactured for the purpose. The multi-well plate is prepared by containing one or more solutions selected from a solution for separation and purification of nucleic acids, that is, DEPC distilled water, cell lysis solution, expression solution, magnetic particle suspension, washing solution, protease, and the like. The solutions for isolation and purification of nucleic acids are not limited thereto, and may include a wide range of solutions used by those skilled in the art for isolation and purification of nucleic acids.

In another aspect, the present invention provides a method for separating a target nucleic acid from a biological sample using a fully automatic nucleic acid separation device,

1) adding magnetic particles to a specific unit well of a multi-well plate to bind insoluble aggregates or nucleic acid molecules excluding the target nucleic acid to the hydrophobic magnetic particles;

2) obtaining a mixture containing a target nucleic acid excluding insoluble aggregates or magnetic particles bound to nucleic acid molecules;

3) adding hydrophilic magnetic particles to the obtained mixture and binding to the target nucleic acid;

4) removing the mixture except for the hydrophilic magnetic particles to which the target nucleic acid is bound; and

5) separating the target nucleic acid from the hydrophilic magnetic particles bound to the target nucleic acid;

It relates to a fully automatic nucleic acid separation method comprising a.

In step 2) or 4), the insoluble aggregate or magnetic particles bound to nucleic acid molecules or hydrophilic magnetic particles bound to target nucleic acid are aggregated at the bottom of the multi-well plate by the magnetic field applying unit included in the automatic purification device to be separated. can

In another aspect, the present invention relates to a kit for separation and purification of nucleic acids from a biological sample comprising magnetic particles having an average particle size of 50 nm to 1 μm, preferably hydrophobic magnetic particles or silica magnetic particles.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of hydrophobic magnetic particles and hydrophilic magnetic particles

The hydrophobic magnetic particles used in the present invention were purchased and used with an average of about 400 nm (Cosmo Advanced Materials, SMT-01S). The magnetic particles have a surface area of 7.87 m ² /g, a pore volume of 0.01608 cm ³ /g, and a pore size of 8.17 nm.

The hydrophilic magnetic particles used in the present invention were used ^{after purchasing a silica-coated magnetic particle product (Bioneer, AccuNanoBead TM} TA-1010) having an average of about 400 nm. The hydrophilic magnetic particles have a surface area of 15.50 m ² /g, a pore volume of 0.02277 cm ³ /g, and a pore size of 5.87 nm.

Example 2: Nucleic acid extraction using hydrophobic magnetic particles

To separate nucleic acids from biological samples, protein-denatured aggregates, cell debris particles, and chromosomal DNA were aggregated together with the hydrophobic magnetic particles of Example 1, and then the nucleic acids were separated using a magnet, and then the silica magnetic particles and After purifying the plasmid DNA using a magnet, the yield and purity were confirmed by measuring the absorbance.

As a biological sample, a 3.0 kb pGEM-B1 vector (Bioneer, Korea) into which the ampicillin resistance gene was inserted was used in the DH5α E. coli strain. After inoculation in LB broth containing 100 μg/ml of ampicillin, incubated with shaking at 37° C. for 16 hours to obtain an O.D600 value of 2.0. 2ml of the cultured E. coli culture medium was centrifuged to separate the culture medium and E. coli cells, and only E. coli cells were obtained by removing the supernatant.

To each of the obtained E. coli cells, a buffer solution for preservation treatment containing hydrophobic magnetic particles and RNase A was added, the cells were well released, and then a buffer solution for cell lysis was added and mixed well. Finally, a neutralization buffer solution was added, mixed well, and the solution and insoluble aggregates were separated using a magnet.

After adding and mixing guanidine hydrochloric acid and magnetic silica particles to the separated solution, only the supernatant was removed using a magnet and washed again with ethanol. After removing all remaining ethanol through a hot air blower, dry heat dryer, or natural drying, the nucleic acid was recovered by dissolving it in water or TE buffer.

The recovered nucleic acid was checked for DNA yield and purity using a spectrophotometer, and the results are shown in Table 1.

Nucleic acid concentration (ng/ul) OD260/OD280 OD260/OD230 sample 1 207.4 1.96 2.18 sample 2 195.5 2.03 2.33

In general, nucleic acids in a well-purified and clean state refer to when OD260nm/280nm is 1.8 or more and OD260nm/230nm is 2.0 or more.

As shown in Figure 1 and Table 1, when the method using magnetic hydrophobic particles and silica magnetic particles was tested, OD260nm/280nm values were 1.8 or more in all samples, and OD260nm/230nm values were 2.0 or more. Confirmed.

Example 3: Comparison of nucleic acid separation using hydrophobic magnetic particles and nucleic acid separation method using a filter

The total experimental time was compared between the method of separating nucleic acids by treating hydrophobic magnetic particles and the method of separating nucleic acids without using the hydrophobic magnetic particles.

According to Example 2, 2 ml of E. coli cells were prepared, and after the same experiment, the total required time was calculated and compared with the commonly used filter method.

the present invention filter separation method Biological sample dissolution and aggregation, nucleic acid and separation process
time taken 2 minutes 11 minutes Refining process time required 2 minutes 4 minutes Nucleic acid recovery time 2 minutes 2 minutes Total time required 6 minutes 17 minutes

In addition, the process of dissolving and aggregating biological samples to separate them from nucleic acids is performed by centrifugation, and since centrifugation is required for about 10 minutes to separate, this process takes a lot of time.

On the other hand, in the method using hydrophobic magnetic particles, the process of dissolving and aggregating the biological sample and separating it from the nucleic acid is completed in a short time (within 1 minute) using a magnet, and the purification process can be completed quickly using a magnet without using a machine. can

As shown in Table 2, the nucleic acid separation process according to the present invention exhibited the effect of reducing the time required by about 1/3 compared to the filter separation method.

Example 4: Confirmation of inhibition of enzymatic activity of nucleic acids isolated from biological samples using an experimental method treated with hydrophobic magnetic particles

1 μg of the plasmid isolated in Example 2 was cut with Eco RI purchased from Bioneer at 37° C. for 1 hour, and the size of the plasmid DNA cut by restriction enzymes was 3.0 kb through electrophoresis on a 1.0% agarose gel. It was confirmed, and the result of the nucleic acid separation method containing the hydrophobic magnetic particles prepared in Example 1 was confirmed to contain enzyme activity inhibitory factors, and the results are shown in FIG. 1 .

M lane of FIG. 1 is a size marker (size marker, Bioneer Inc., 1 kb ladder), and lanes 1 to 3 are experimental groups in which the plasmid DNA isolated by the method according to the present invention is treated with restriction enzyme (EcoRI).

As can be seen from the electrophoresis picture of FIG. 1 , it was found that the plasmid DNA isolated by the method according to the present invention did not have any inhibitory elements on enzyme activity.

Example 5: Nucleic Acid Extraction Method of Blood Sample Using Silica Magnetic Nanoparticles

The method of Example 2 was applied to animal blood samples in a simple manner as follows.

Add guanidine hydrochloride, surfactant, and buffer solution to the obtained blood sample and mix well. Next, proteinase K is added and subjected to proteolysis at 60° C. for 10 minutes. Next, add ethanol to the solution and mix, add and mix silica magnetic nanoparticles, and then use a magnet to remove only the supernatant. give. After removing all remaining ethanol through a hot air blower, dry heat dryer or natural drying, the nucleic acid was recovered by dissolving it in water or Tris buffer solution.

M in FIG. 2 is a 1kb ladder product purchased from Bioneer, which is a size marker, and lanes 1 to 4 are electrophoresis of nucleic acid DNA isolated by the method according to the present invention to confirm that the concentration and purity of the nucleic acid are constant. did

Example 6: Nucleic Acid Extraction Method of Cultured Bacterial Sample Using Silica Magnetic Nanoparticles

The method of Example 2 was applied to cultured bacterial samples as follows.

A culture sample containing the cultured bacteria is centrifuged at high speed to obtain a bacterial precipitate. This is similar to the process of obtaining E. coli cells from a biological sample in the method of Example 2. In the case of gram-positive among bacterial types, the resulting precipitate is mixed with a lysozyme enzyme and a gram-positive buffer solution and reacted at 37°C for 30 minutes. make it Among the gram-negative bacteria, add a cell lysis buffer from the precipitate and mix well. Next, proteinase K is added and subjected to proteolysis at 60° C. for 10 minutes. Next, add guanidine hydrochloride, a surfactant, and a buffer solution to the solution and mix. After that, in the same way for both gram-positive and negative bacteria, ethanol was added and mixed, and then silica magnetic nanoparticles were added and mixed, and then only the supernatant was removed using a magnet. Then, guanidine hydrochloric acid and ethanol mixture, sodium chloride and ethanol mixture, and ethanol were sequentially added. to wash the silica magnetic particles. After removing all remaining ethanol through a hot air blower, dry heat dryer or natural drying, the nucleic acid was recovered by dissolving it in water or Tris buffer solution.

M in FIG. 3 is a 1kb ladder product purchased from Bioneer and is a size marker, and lanes 1 and 2 are electrophoresis of the nucleic acid DNA isolated by the method according to the present invention to confirm that the concentration and purity of the nucleic acid are constant. did

Example 7: Nucleic acid extraction method of cultured cells using silica magnetic nanoparticles

The method of Example 2 was applied to the cultured cell sample in the following way.

A sample mixed with cultured cells and culture medium is centrifuged at a speed of 3000 times the acceleration of gravity for 10 minutes to remove all of the supernatant and resuspended by adding phosphate buffer (PBS). Proteinase K, guanidine hydrochloric acid, surfactant, and buffer solution are added, and proteolytic reaction is performed at 60° C. for 10 minutes. Next, add ethanol to the solution and mix, add and mix silica magnetic nanoparticles, and then use a magnet to remove only the supernatant. give. After removing all remaining ethanol through a hot air blower, dry heat dryer or natural drying, the nucleic acid was recovered by dissolving it in water or Tris buffer solution.

M in FIG. 4 is a 1kb ladder product purchased from Bioneer, which is a size marker, and lanes 1 to 4 are electrophoresis of the nucleic acid DNA isolated by the method according to the present invention to confirm that the concentration and purity of the nucleic acid are constant. did

Example 8: Nucleic acid extraction method from animal tissue using magnetic silica nanoparticles

The method of Example 2 was applied to the collected animal tissue samples in the following way.

The collected animal tissue samples are rapidly frozen in liquid nitrogen, and then crushed to as powder as possible using a tissue shredder or mixer. Proteinase K and tissue lysate are added to 20 mg of the powdered tissue sample and subjected to proteolysis at 60° C. for 10 minutes. Next, add a mixture of guanidine hydrochloric acid, surfactant, and buffer solution to the solution, mix as much as possible, add ethanol to mix, and then add magnetic silica nanoparticles to mix, remove only the supernatant using a magnet, Wash the silica magnetic particles sequentially using a mixture of sodium chloride, ethanol, and ethanol. After removing all remaining ethanol through a hot air blower, dry heat dryer or natural drying, the nucleic acid was recovered by dissolving it in water or Tris buffer solution.

M in FIG. 5 is a 1kb ladder product purchased from Bioneer, which is a size marker, and lanes 1 to 4 are electrophoresis of the nucleic acid DNA isolated by the method according to the present invention to confirm that the concentration and purity of the nucleic acid are constant. did

Example 9: Nucleic Acid Extraction Method of Plant Tissue Using Magnetic Silica Nanoparticles

The method of Example 2 was applied to the collected plant tissue samples in the following way.

The collected plant tissue samples are rapidly frozen with liquid nitrogen, and then crushed to as powder as possible using a tissue shredder or mixer. A tissue lysate containing proteinase K and surfactant is added to 100 mg of the powdered tissue sample and subjected to proteolysis at 60° C. for 10 minutes. Next, add acetic acid and buffer solution to the solution, mix as much as possible, and place on ice for 10 minutes, centrifuge at high speed and transfer the supernatant to a new tube. Add and mix guanidine hydrochloric acid and ethanol mixture to the supernatant, then add magnetic silica nanoparticles to mix, remove only the supernatant using a magnet, and sequentially use guanidine hydrochloric acid and ethanol mixture, sodium chloride and ethanol mixture, and ethanol to silica Wash the magnetic particles. After removing all remaining ethanol through a hot air blower, dry heat dryer or natural drying, the nucleic acid was recovered by dissolving it in water or Tris buffer solution.

M in FIG. 6 is a 1kb ladder product purchased from Bioneer and is a size marker, and lanes 1 to 4 are electrophoresed on nucleic acid DNA isolated by the method according to the present invention to confirm that the concentration and purity of the nucleic acid are constant. did

Example 10: Nucleic acid RNA extraction method of cultured cells using silica magnetic nanoparticles

The method of Example 2 was applied to the cultured cell sample in the following way.

Collect all samples of the cultured cells and culture solution, centrifuge at a speed of 300 times the acceleration of gravity for 5 minutes to remove all of the supernatant, add guanidine hydrochloride, surfactant, and buffer solution, and mix evenly for 1 minute or more. through the crushing process. Next, add ethanol to the solution and mix, add and mix silica magnetic nanoparticles, and then use a magnet to remove only the supernatant. give. After removing all remaining ethanol through a hot air blower, dry heat dryer or natural drying, the nucleic acid was recovered by dissolving it in water or Tris buffer solution.

In lanes 1 to 4 of FIG. 7, it was confirmed that the concentration and purity of the nucleic acid were constant by electrophoresis of the nucleic acid RNA of various cultured cells isolated by the method according to the present invention.

Example 11: Nucleic Acid Extraction Method of Various Samples Using Silica Magnetic Nanoparticle Treatment

The method of Example 2 was applied as a simpler method to the method for extracting nucleic acids from various samples.

Add the pretreatment cell lysis buffer to the obtained blood sample, animal cell lines cultured in liquid medium, animal tissue, and plant tissue, and mix well. At this time, the tissue cells of animals and plants may proceed with a protein degradation step such as proteinase in addition to the buffer solution. Then, add guanidine hydrochloric acid, magnetic silica nanoparticles, and ethanol to the solution, mix them, and then use a magnet to remove only the supernatant, and then sequentially use the guanidine hydrochloric acid and ethanol mixture, sodium chloride and ethanol mixture, and ethanol washed away After removing all remaining ethanol through a hot air blower, dry heat dryer or natural drying, the nucleic acid was recovered by dissolving it in water or Tris buffer solution.

As a result of confirming through agarose gel electrophoresis whether the result of the nucleic acid separation method was well extracted without impurities, it could be confirmed that the result was well extracted without impurities as shown in FIGS. 2 to 7 . M lanes of FIGS. 2 to 7 are size markers (size marker, Bioneer Inc., 1 kb ladder), and lane numbers are sample DNA extracted by the method according to the present invention.

Example 12: Fully automated extraction method using hydrophobic magnetic particles and silica magnetic particle treatment applied to nucleic acid extraction equipment

The nucleic acid extraction method of Example 2 was fully automated using a nucleic acid extraction equipment.

Equipment for protein synthesis purification and nucleic acid extraction ExiProgen ( Bioneer Inc., Korea Patent Publication Nos. 2011-0041126 and 2011-0085824) All of the nucleic acid extraction methods of Example 2 are put into the sample injection compartment of the dedicated kit used, etc. The procedure is fully automated within the machine.

The equipment for nucleic acid extraction ExiProgen is shown schematically and state diagrams in FIGS. 8-15.

Except for the first cell lysis buffer, all solutions are included in a separate kit, and experiments can be carried out by moving and mixing solutions from one sample to a maximum of 16 samples at the same time.

To each E. coli cell obtained by the method of Example 1, add a buffer solution for preservation treatment containing RNase A, loosen the cells, add a buffer solution for cell lysis, mix well, inject a sample into a dedicated kit, and operate the equipment do. A solution in which the neutralization buffer solution and hydrophobic magnetic particles were mixed inside the equipment was attached to some wells of the kit using a magnetic device installed in the equipment, and the solution was separated. The installed magnet device is mounted in the ExiProgen device with the structure shown in FIG. 12 .

After adding and mixing guanidine hydrochloric acid and magnetic silica particles to the separated solution, only the supernatant was removed using a magnetic field applying unit driven in the equipment, and then washed again with ethanol. After all ethanol was removed by the heating device of the magnetic field applying unit, the nucleic acid was recovered by dissolving it in water or TE buffer.

The recovered nucleic acid was checked for DNA yield and purity using a spectrophotometer, and the results are shown in Table 3.

Nucleic acid concentration (ng/ul) OD260/OD280 OD260/OD230 sample 1 95.5 1.92 2.23 sample 2 94.6 1.88 2.2 sample 3 108.9 2 2.46 sample 4 81.2 1.99 2.43 sample 5 73.2 1.97 2.45 sample 6 73.3 2.02 2.46 sample 7 106.9 2 2.43 sample 8 78.6 1.82 2.4

In general, nucleic acids in a well-purified, clean state refer to when OD260nm/280nm is 1.8 or more and OD 260nm/230nm is 2.0 or more.

Equipment for protein synthesis purification and nucleic acid extraction ExiProgen ( Bioneer, Korean Patent Laid-Open Nos. 2011-0041126 and 2011-0085824) If all tasks are performed within the equipment by using the automation system, up to 16 units can be operated simultaneously. The /230nm value was maintained at 2.0 or higher, confirming that the nucleic acid was cleanly purified.

As a result of electrophoresis of the isolated nucleic acids in Table 3 on a 1.0% agarose gel, it was confirmed that plasmid DNA was clearly purified among the nucleic acids purified in the fully automated device. Lane M of FIG. 17 is a size marker (size marker, Bioneer Inc., 1 kb ladder), and lanes 1 to 8 are nucleic acid samples recovered in Table 3.

Example 13: Confirmation of inhibition of enzymatic activity of nucleic acids isolated from biological samples using a fully automated device

1 μg of the plasmid isolated in Example 12 was cut with Eco RI purchased from Bioneer at 37° C. for 1 hour, and the size of the plasmid DNA cut by restriction enzymes was 3.0 kb through electrophoresis on a 1.0% agarose gel. Confirmed, and the result separated by the fully automated device is shown in FIG. 18 .

18, M lane is a size marker (size marker, Bioneer Inc., 1 kb ladder), U lane is plasmid DNA not cut by restriction enzyme, and C lane is an experimental group in which plasmid DNA is treated with restriction enzyme (EcoRI) . As can be seen from the electrophoresis results, when treated with a restriction enzyme capable of separating a specific nucleotide sequence around the target gene inserted into the plasmid nucleic acid, it was appropriately cut to the size of the target gene, and it was confirmed that it appeared clearly on the electrophoresis.

As the content of the present invention has been described in detail above, for those of ordinary skill in the art, it will be clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. . Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

120: piston 130: piston guide part
140: pipette part 141: first row pipette 142: second row pipette
150: piston guide support plate
200: fixed body
231: vertical movement motor 232: vertical movement belt
233: vertical movement screw 241: forward and backward movement slider
300: casing
310: forward and backward movement support rod 311: forward and backward guide guide
320: forward and backward movement motor 330: forward and backward movement belt
340: UV lamp for sterilization
400: base plate 401: handle
420: multi-well plate 420': multi-well plate
430: pipette rack 440: tube rack for sample storage
440-1: tube insertion hole for target nucleic acid storage 440-3: tube insertion hole for target nucleic acid diagnosis
442-1: tube for storing target nucleic acid 442-3: tube for diagnosis of target nucleic acid
450: waste liquid container 460: high temperature reaction block
472: tube for biological sample
700: magnetic field applying unit
710: magnet mounting part 711: magnet
713: unit well inlet groove
720: magnet mounting part support 730: guide rod
740: guide block 750: tension spring
760: elevating unit 761: elevating motor
762: first lifting shaft 763: lifting cam
764: second lifting shaft
770: sensing unit mounting plate
780: height detection sensor
781: sensing unit 782: sensing target unit
810: heating unit 812: heating unit fixing plate

Claims (18)

A method of separating nucleic acids from a biological sample using magnetic particles comprising the following steps
(a) adding hydrophobic magnetic particles having an average particle size of 50 nm to 1 μm to a biological sample containing a nucleic acid, and binding the particles to an insoluble aggregate or nucleic acid molecule other than a target nucleic acid;
(b) separating the hydrophobic magnetic particles bound to insoluble aggregates or nucleic acid molecules other than the target nucleic acid using a magnetic field to obtain a mixture containing the target nucleic acid;
(c) binding the target nucleic acid to the magnetic silica particles by adding magnetic silica particles having an average particle size of 50 nm to 1 μm to the mixture obtained in step (b); and
(d) separating the magnetic silica particles bound to the target nucleic acid using a magnetic field to obtain the target nucleic acid
A nucleic acid isolation method comprising a.
delete delete According to claim 1,
The method for separating nucleic acids, characterized in that the average particle diameter of the hydrophobic magnetic particles is 50nm ~ 700nm.
According to claim 1,
The method for separating nucleic acids, characterized in that the silica magnetic particles have an average particle diameter of 50 nm to 700 nm.
The method of claim 1, wherein the magnetic particles are at least one selected from iron, cobalt, nickel, and oxides or alloys thereof.
delete According to claim 1,
The step of adding the magnetic particles is
i) providing directly to the biological sample;
ii) providing a biological sample mixed with a preservation solution for preservation treatment;
iii) providing a preservation solution for preserving the biological sample after treatment;
iv) providing a mixture with a buffer solution for dissolution of the biological sample;
v) providing a buffer solution after treatment for dissolution of the biological sample;
ⅵ) mixing and providing a buffer solution for forming protein-denatured aggregates in the dissolved biological sample; and
iv) providing a solution in which the protein denatured aggregate is formed;
Separation method of nucleic acid, characterized in that added to the step selected from.
The method according to claim 1, wherein the target nucleic acid to be isolated is selected from the group consisting of plasmid DNA, bacterial genomic DNA, blood and animal and plant tissue DNA, and animal cell line DNA and RNA.
a pipette block installed to be movable in the horizontal and vertical directions, and for mounting a plurality of pipettes to be detachably;
a magnetic field applying unit for applying and releasing a magnetic field to a specific unit well of the multi-well plate;
a magnet mounting part positioned below a specific unit well of the multi-well plate;
a lifting unit for raising and lowering the magnet mounting unit;
a magnet member for separating magnetic particles; and
a heating unit for heating a specific unit well of the multi-well plate;
including,
Method of separating a target nucleic acid from a biological sample using a fully automatic nucleic acid separation device for automatically separating nucleic acids from a biological sample, characterized in that a biological sample and magnetic particles having an average particle size of 50 nm to 1 μm are added to the multi-well plate As,
1) adding hydrophobic magnetic particles to a specific unit well of a multi-well plate to bind magnetic particles to insoluble aggregates or nucleic acid molecules except for the target nucleic acid;
2) separating the hydrophobic magnetic particles bound to insoluble aggregates or nucleic acid molecules other than the target nucleic acid using a magnetic field to obtain a mixture containing the target nucleic acid;
3) adding magnetic silica particles having an average particle size of 50 nm to 1 μm to the mixture obtained in step 2) and binding to the target nucleic acid;
4) removing the mixture except for the magnetic silica particles bound to the target nucleic acid; and
5) separating the target nucleic acid from the magnetic silica particles bound to the target nucleic acid;
Fully automatic nucleic acid separation method comprising a.
delete delete delete delete delete 11. The method of claim 10,
In separating the material bound to the magnetic particles in step 2) or step 4), a fully automatic nucleic acid separation method, characterized in that the separation is performed by attaching it to the inner wall of the lower part of a specific unit well of the multi-well plate using a magnetic field applying unit
11. The method of claim 10,
The target nucleic acid to be isolated is selected from the group consisting of plasmid DNA, bacterial genomic DNA, blood and animal and plant tissue DNA, and animal cell line DNA and RNA.
A kit for separation and purification of nucleic acids from a biological sample containing magnetic silica particles and hydrophobic magnetic particles having an average particle size of 50 nm to 1 μm.

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