KR100286896B1 - Glass microfiber column, method of preparation and purification for plasmide DNA using the same - Google Patents

Abstract

PURPOSE: Provided are a glass microfiber column and methods for purification and preparation of plasmid DNA by using it. The glass microfiber column enables preparing plasmid DNA simply and quickly and mass production thereof. The plasmid DNA can be used in various reactions without other purification process. The column kit can separate DNA from agarose gel or polyamide gel efficiently and can be applied to quantitative analysis of isotope ion, preparation of RNA, purification of isotope probe and PCR reactant and prepared plasmid DNA, and is thus useful to biotechnology. CONSTITUTION: The glass microfiber column includes Borosilicate glass microfiber particle having a size of 3mm or less and resin comprising guanidine hydrochloride mainly. DNA is separated from agarose gel or polyacrylamide gel by using glass microfiber column kit. DNA is purified by removing RNA and impure protein mixed with DNA.

Description

Glass microfiber column, method of preparation and purification for plasmide DNA using the same

The present invention relates to a glass microfiber (GF) column and a method for preparing plasmid DNA and DNA purification using the same. Specifically, the present invention relates to a plasmid DNA production and purification column kit composed of an active borosilicate microfiber membrane and a plasmid DNA production and purification method using the same. More specifically, the present invention forms a guanidine hydrochloride film on borosilicate or silicate glass microfibers having a weak DNA adsorption capacity, and thus, a column kit for plasmid DNA production and purification using a material having improved DNA adsorption capacity as a resin. And it relates to a method for producing and purifying a large amount of high purity plasmid DNA in a short time using the same.

Preparation and purification of plasmid DNA is the most widely required technique in molecular biology research. Conventionally, methods for preparing plasmid DNA are known. In many laboratories, plasmids are manufactured according to the manual method, which is time-consuming and unreliable, and the laboratories with good research conditions often use products from Qiagen (Germany) or Promega (US). These conventional products mainly use a method in which plasmid DNA is bound to silica gel. On the other hand, according to Vogelstein and Gillespie's thesis (1978), the molten glass is made of large flint glass particles, medium flint glass particles, and powdered molten glass particles. A method of separating DNA from agarose gel is known as a principle of binding to molten glass by making DNA particles in NaI solution and measuring DNA bound under NaI solution. The product based on this principle is already used as a product called 'glass milk' and is useful for separating DNA from agarose gel. However, the DNA separation effect according to this product showed the most effective DNA binding ability in the powdered molten glass powder, but the effect rapidly decreased in the medium molten glass particles, and the large molten glass. Particles (large flint glass particles) has a problem that the effect is further reduced significantly. In addition, large borosilicate glass particles of different glass materials are not as effective in binding DNA as in large molten glass particles, and glass fiber filters have very low adsorption power. The yield was very low problem. And when using porous glass beads (porous glass bead) showed excellent DNA binding activity, but it takes a long time to bind DNA, degradation of DNA occurs when obtained from beads, yield is also not suitable for quantitative analysis There was a problem. In addition, the silica gel shows a very effective DNA binding effect as in powdered molten glass, but has a problem of hand mechanically and a low yield. Silica gel is used as a column kit for plasmid DNA production, and 'Qiagen' and 'Promega' products are commercially available. However, the products are required for a long time by using a column flow method using a drop flow (gravity flow) in the manufacturing process of the plasmid DNA, and a separate purification process is required because the resin slurry is contained in the manufacturing DNA, Endoinase (endonuclease) contains self digestion when stored for a long time, and protein impurities such as endotoxin cannot be removed, causing cytotoxicity during transfection of mammalian cells In this case, or in animal experiments, problems causing an immune response are required, requiring high purity DNA purification. In addition, since importing these foreign products is expensive, there is an urgent need for the development of a low-cost plasmid DNA production column kit capable of obtaining a large amount of high-purity plasmid DNA by a simple process.

On the other hand, DNA purification removes protein impurities such as purification of isotopically labeled probes, purification of PCR reactions, RNA and endonuclease, and endotoxin of already purified plasmid DNA to increase purity. It is a way. In addition, when an experiment for cell proliferation assay is performed, an isotope is administered to the cultured cells, and the cell-containing culture solution is passed through the column by spin. If the cell is not inserted into the cell, the isotope is inserted into the cell and inserted into the cell. Only isotopes are present in the column to quantify intracellular insertion isotopes without additional expensive equipment. Therefore, the present inventors have completed the present invention by preparing a novel column kit made of a borate free microfiber resin so that the above DNA quantification can be obtained in high purity in a short time. That is, the present inventors are physiologically inert materials, resistant to weak acid weak alkalinity, excellent absorption of moisture in 1.0 μm particles, loading capacity is at least accurate, and even microorganisms even for long-term storage It has the advantage that there is no problem of contamination by and it contains the guanidine hydrochloride in borosilicate, a boride compound of silicate which has a very good ability to remove impurities. Thus, the present invention was completed by using this as a resin in a column kit for preparing and purifying plasmid DNA. Borate containing guanidine hydrochloride had 2-4 times higher DNA adsorption capacity than silica gel.

An object of the present invention is to provide a column kit for preparing and purifying plasmid DNA composed of an active borosilicate microfibrous membrane. Another object of the present invention is to provide a method for preparing the plasmid DNA and a column kit for purification. Still another object of the present invention is to use a plasmid DNA preparation and purification column kit to purify DNA from agarose gel or polyacrylamide gel, to purify isotope labeled probe, to purify PCR reactant, and to purify the already prepared plasmid DNA. DNA production, RNA production and intracellular insertion isotopic quantification method, and the like.

The object of the present invention is to prepare a plasmid DNA using a column kit prepared by preparing a borosilicate glass microfiber membrane multilayer column and a glass microfiber membrane / particle column for small, medium and large column kits of glass fibers (galss fiber (GF)). Prepared and purified plasmid DNA with restriction enzyme, cell transfection and transduction, 35S sequencing, auto sequencing, sequencing of PCR reaction product, protein expression of purified DNA product Achieved by demonstrating the functional excellence of the plasmid DNA preparation and purification column kits of the invention.

1A is an exploded view of a borosilicate glass microfiber membrane multilayer column kit (GF mini column kit).

FIG. 1B is an assembled sectional view of the column kit shown in FIG. 1A.

Figure 2 is a graph showing the change in plasmid DNA yield with increasing borosilicate glass microfiber membrane.

Figure 3a is an exploded view of a large borosilicate glass microfiber membrane multilayer column kit.

3B is a cross-sectional view of the column kit shown in FIG. 3A.

4A is an exploded view of a glass microfiber membrane / particle column kit (GF midi column kit).

4B is an assembled cross sectional view of the column kit shown in FIG. 4A.

Figure 5 is a graph showing the change in plasmid DNA yield with increasing the glass microfiber particles.

Figure 6 is a graph comparing the yield of plasmid DNA according to the material of borosilicate glass microfibers and silicate glass microfibers.

Figure 7 shows the restriction enzyme cleavage results of the plasmid DNA prepared by the column kit of the present invention.

8 is a 35S dATP sequencing analysis of the plasmid DNA prepared by the column kit of the present invention.

Figure 9a is a photograph showing the staining reaction by β-galactosidase protein production in the transfection of the plasmid DNA prepared with a conventional column kit.

Figure 9b is a photograph showing the staining reaction by β-galactosidase protein production during transfection of the plasmid DNA prepared by the column kit of the present invention.

10 is an analysis table of automatic sequencing of pGEM 7Z plasmid DNA prepared by the column kit of the present invention using a T7 primer.

FIG. 11 is an analysis table of automatic sequencing of pGEM 7Z plasmid DNA prepared by the kit of the present invention using an Sp6 primer.

Figure 12 is a photograph of the reaction on the agarose gel after performing a PCR reaction by mixing the primers in a template with ?? rasmid DNA prepared as a column kit of the present invention.

Figure 13 after the reaction of the RNA contained in the plasmid prepared by omission of the ethanol washing process in the plasmid manufacturing process using the column kit of the present invention as RNAse, the reaction passes through the column of the present invention RNA and protein impurities in the plasmid Will remove and increase the purity.

Figure 14 is a photograph of the DNA band cut from the agarose gel and separated using a GF small column kit and electrophoresis on the agarose gel.

The present invention is a glass microfiber multilayer column for borosilicate glass fiber (GF) small column kit, glass microfiber multilayer column for borosilicate glass fiber large column kit, glass microfiber membrane / particle column for borosilicate medium column kit, Borosilicate glass microfiber and silicate glass microfiber multilayer column and silica gel / borosilicate glass microfiber bilayer column for small and medium column kits were prepared, and the plasmid DNA was purified by the prepared column to obtain a yield. Investigating; Purifying the E. coli strain culture with GF small column kit, GF medium column kit or GF large column kit to prepare high purity plasmid DNA; The prepared plasmid DNA was digested by restriction enzyme digestion, 35S sequencing, comparison of protein fermentation effect by column kit and cell transfection and transduction of Qiagen, Germany, and sequencing of purified DNA using an automatic sequencing analyzer. Analyze and amplify the DNA amplified by PCR, and obtain the DNA in the other unpurified, and then electrophoresed on a gel to investigate the removal of excess primer, using the GF matrix large column of the present invention plasmid DNA with strain culture medium After washing with WA solution, passing through the column, precipitation with ethanol without washing with WA solution, dissolved in DW solution, passed through column, dissolved in DW solution without washing with WA solution, and then inactivated by adding Rnase. Separation and purification by passing through a column to compare the yield and purity and PCR-amplified DNA Separation is performed by electrophoresis and the separation solution is purified by GF small column to examine DNA separation performance by examining the DNA separation performance of the column of the present invention.

Hereinafter, specific examples of the present invention will be described with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Preparation of Glass Microfiber Column and Obtaining Plasmid DNA

Experimental Example 1 Preparation and Use of Glass Microfiber Membrane Multilayer Column for GF Small Column Kit

A small spin mini column (13 mm in diameter and 31 mm in length) is made of borosilicate glass microfibers with a diameter of 8 mm and a plurality of layers 13 of 2 to 10 mm. After insertion into 11), a 7.5 mm ring 12 was inserted to fix the contents and sterilized by gas (FIG. 1A). When using the prepared glass fiber column was used in the collection tube (10) (Fig. 1b). Multilayer columns of borosilicate glass microfiber membranes dramatically increased the thickness of the membrane by 2 mm to improve the purity and yield of plasmid DNA.

As a result of separating plasmid DNA using a GF small column composed of multilayered borosilicate glass microfiber membranes, the plasmid DNA yield was about 200 μg at 8 mm as shown in FIG. 2. However, at 10 mm or more, no further yield was increased.

Experimental Example 2: Preparation and Use of Glass Microfiber Membrane Multilayer Column for GF Max Column Kit

A glass microfiber membrane multilayer column for GF large column kit was prepared in the same manner as in Experimental Example 1. As shown in FIG. 3A, the glass microfiber membrane 33 was cut into a circular column having a diameter of 22 mm in a large column 31 having a diameter of 22 mm and a length of 86 mm, and filled with about 1 g to the maximum column and fixed with a cross ring 32. When using the prepared large column, 50 mL falcon tube 30 was used as a collecting tube (FIG. 3B). The column was used to obtain about 1.2 mg of plasmid DNA from about 250 mL of culture.

Experimental Example 3: Preparation and Use of Glass Microfiber Membrane / Particle Column for GF Medium and Small Matrix Column Kits

Borosilicate glass microfibers and 10 mM Tris / HCl pH7.5, 1 mM EDTA solution were mixed in a blender to form a glass microfiber particle suspension. Into the medium 15 mm diameter, 80 mm polypropylene medium column 22 shown in Fig. 4a, a 8 mm diameter glass microfiber particle 24 having a diameter of about 2 mm was put therein, followed by a suspension of glass microfiber particle and a swing rotor centrifuge. Spinning in a centrifuge) made a 5-10 mm glass microfiber membrane / particle double column layer and fixed the contents with a 8 mm diameter ring or 8 mm diameter molten glass and gas sterilized. When using the prepared glass fiber column was supported by the Eppendorf tube 21 was used in the collection tube 20 (Fig. 4b). The plasmid DNA yield with increasing free microfiber membrane and the plasmid DNA yield with increasing capacity of free microfiber particles were increased as shown in FIG. 5, respectively. The borosilicate glass microfiber film was cut to a diameter of about 3 mm or less by a paper cutter, filling about 0.1 g of resin into a small column and about 0.42 g of resin into a medium column, loading 4.2 M guanidine HCl, and passing through a spin. To prepare an active-column. About 100 ug plasmid DNA was obtained using a small column and about 200 ug using a mid column. Plasmid DNA yield and purity using these columns were shown in Table 1 below.

Plasmid DNA Purity and Yield by Small and Medium Columns Type of column E. coli JM109 culture solution OD ₂₆₀ / 0D ₂₈₀ Yield GF matrix small column 10 mL 1.7 to 1.9 To 100 μg GF Matrix Medium Columns 100 mL 1.7 to 1.9 ~ 200 ㎍

Experimental Example 4: Comparison of plasmid DNA yield by borosilicate glass microfiber column and silicate glass microfiber column

In order to compare the yield of plasmid DNA according to the material of borosilicate glass microfiber and silicate glass microfiber particles, a column was prepared in the same manner as in Experimental Example 1 and E. coli Plasmid DNA was prepared from the cultures, and the yield and purity were compared. As a result, as shown in FIG. 6, the yield in the silicate glass microfiber column was reduced by about 20% compared to the borosilicate glass microfiber column, and the purity was also OD ₂₆₀ / OD ₂₈₀ =. The plasmid DNA purity OD ₂₆₀ / OD ₂₈₀ = 1.7 to 1.8, which was prepared by borosilicate glass microfiber column as 1.5, was not reached.

Experimental Example 5: Comparison of yield and purity of plasmid DNA prepared by silica gel column and silica gel / borosilicate microfiber bilayer column

As in Example 3, five medium columns were filled with silica gel of about 8 mm, and the other five medium columns had a borosilicate glass microfiber membrane / particle layer of about 2 mm. After preparing a silica gel layer of about 6mm layer on top again, plasmid DNA was prepared as in Experiment 2 to analyze purity and yield. As a result, as shown in Table 2, the yield and purity of the borosilicate glass microfiber layer introduced into the silica gel column were significantly increased compared to the silica gel alone. Hereinafter, the plasmid DNA provided in all the experiments used what was manufactured by the borosilicate glass microfiber column.

Purity and yield of plasmid DNA by column Type of column E.coli JM109 culture OD ₂₆₀ / OD ₂₈₀ (purity) Yield Silica gel 200 ml 1.2 to 1.5 40 to 80 µg Silica Gel / Borosilicate Glass Microfiber 200 ml 1.5 to 1.8 70 to 90 µg

Example 2: Plasmid DNA Preparation

Plasmid DNA was prepared using the GF small column kit, GF medium column kit, or GF large column kit prepared in Example 1 above. E. coli strains containing plasmids were incubated and the strain culture was centrifuged to obtain pellets. This pellet was dissolved in Buffer A solution (50mM Tris, 10mM EDTA pH8.0, 100µg / ml RNase A.), and then cell lysed with Buffer B solution (20mM NaOH, 1.0% SDS (W / V)). It was neutralized in C solution (3.2mM Potassium acetate, pH5.0). The neutralized solution was centrifuged to bind the supernatant to DNA in the guanidine HCl-activated GF column kit, followed by removal of nuclease, washing and eluting to obtain high purity plasmid DNA. At this time, 4.2M guanidine hydrochloride was used to remove nuclease, and 10 mM Tris / HCl (pH 7.5), 50 mM NaCl, 0.1 mM EDTA, 70% ethanol was used as a washing solution.

Example 3: Molecular Biology Evaluation and Analysis of Prepared Plasmid DNA

Experimental Example 1: Restriction Enzyme Treatment

As a result of cleaving the plasmid DNA prepared in Example 2 with restriction enzymes EcoRI and Hind III, effective DNA cleavage was achieved as shown in FIG.

Experimental Example 2: 35S Sequencing Analysis

10 μg of the plasmid DNA prepared in Example 2 and 10 μg of the plasmid DNA produced by Qiagen, Germany were separated from the sequencing gel by using an α35S d-ATP in a Sequenase ^™ version 2.0 DNA sequencing kit (United State Biochemical). After autoradiography was performed. As a result, as shown in Figure 8, the sequencing analysis of the DNA prepared by the column kit of the present invention (sequencing analysis) is clearly separated from the DNA band, while in the sequencing analysis of the DNA produced by Qiagen, Germany, DNA band effectively No separation was made.

Experimental Example 3: Cell Transfection and Transduction

Cell transfection and transduction were performed using the column kit of the present invention prepared in Example 1 and the existing column kit (Qiagen, Germany). As a result, as shown in Figures 9a and 9b, when transfected plasmid DNA prepared by the column kit of the present invention, effective protein expression was made in almost all cells stained (Fig. 9b) When the DNA prepared by the product's column kit (Qiagen) was transfected, no effective protein expression (FIG. 9A) was achieved.

Experimental Example 4: Automatic Sequencing

Automatic sequencing in this experiment was performed using the ABI PRIM ^TM dye-terminal cycle sequencing ready reaction kit ^{(ABI PRIM TM Dye terminator cycle sequencing} ready reaction kit) (Perkin Elmer), terminal ready reaction mixture (terminal ready reaction mix ) 8 μl, template DNA 0.4 μg, primer 3 pmole at 20 μl reaction volume for 96 ° C./30 min, 50 ° C./15 sec, 60 ° C./4 min for 25 cycles in Bio Rad Gene Cycler ^™ After drying in (speed vac) it was analyzed by an automatic sequencer (Applied Biosystem). As a result, very effective sequence analysis was performed as shown in FIGS. 10 and 11. In this case, the template used above was a 1.2 kb cloned DNA containing pGEM7Z, and the primer was a T7 primer or an SP6 primer.

Experimental Example 5: Purification of the PCR reaction and the PCR reaction.

PCR reaction was performed using the CMV-TNF plasmid DNA obtained from the column prepared in Example 1 as a template. Into 20 ng CMV TNF plasmid, 1uM dNTP 2uL, 10x binding buffer 5uL, Tag 2U, and TNA antisense primer mixture, each of a neighboring promoter primer or a distant promoter primer was added. After the reaction for 30 cycles in the condition of 95˚C-1 min, 55˚C-1 min, 72˚C-1 min and 72˚C-10 min. Dividing the reaction into 2 groups, one group was mixed with the same volume of 4.2M guanidin HCl (Guanidinw HCl) and passed through a micro DNA purification column, washed with WA solution to obtain DNA. The two groups were electrophoresed on an agarose gel, respectively. As a result, excess primers were completely removed from the PCR reaction product purified in a micro DNA purification column as shown in FIG. 12.

Experimental Example 6: DNA Purification

Plasmid DNA was prepared from three groups of 250 mL cultures using the GF matrix maxi column prepared in Example 1. The first method is a general method of washing with WA solution, the second method is ethanol precipitation without washing with WA solution and dissolved in 500 uL DW solution. The third method divides the plasmid DNA prepared by the second method by 250 uL. Each 20 uL RNase A (3mg / mL) was added for 30 minutes at 37 ° C and then inactivated for 10 minutes at 65 ° C and the reaction was passed through a micro column (micro column). This was isolated from each plasmid DNA on an agarose gel as summarized in Table 3. This result is shown in FIG. Plasmid DNA without ethanol wash showed high yield but RNA contamination. On the other hand, when the plasmid DNA was passed through a micro column after RNase A treatment, RNA impurities were completely removed, and the DNA purity (OD ₂₈₀ / OD ₂₆₀ ) also increased from 1.72 to 1.90. Therefore, the above method was able to completely eliminate the RNA contamination problem, which is a chronic problem in plasmid preparation, and increase the DNA yield and purity.

DNA Purification Using GF Matrix Large Columns OD ₂₈₀ / OD ₂₆₀ density Full DNA WA wash 1.44 7.60 760 µg Do not wash 1.68 10.80 1.08 µg Column cleaning without washing 1.90 11.75 1.175mg

Experimental Example 7: DNA Separation from Agarose Gel or Polyacrylamide

DNA was isolated by electrophoresis from agarose gel / TAE or TBE, the bands were cut and placed in a dialysis bag, filled with TAE solution and electrophoresed to separate DNA from the gel. Take a solution, add the same volume of 4.2M guanidine hydrochloride, spin into GF small column of the present invention, spin and wash with WA solution (10 mM Tris / HCl pH7.5, 30 mM EDTA, 70% ethanol), then 50 μl DW Was added to spin to separate. Experimental results showed excellent DNA separation performance as shown in FIG. The results are summarized in Table 4. In the column kit of the present invention, first, a simple and rapid plasmid DNA was prepared. Second, the DNA yield was extremely good, and thus, in the GF small column kit, the amount of DNA up to about 50-100 times (˜200 μg) was higher than that of the conventional spin column kit. In the case of the GF medium column kit, a DNA amount of about 400 µg was obtained, and in the case of the GF large column kit, 2.2 mg of plasmid DNA was obtained. Third, the plasmid DNA made of glass microfibers was used for the measurement of spectrophotometer. The high purity plasmid DNA of purity (OD ₂₆₀ / OD ₂₈₀ ) = 1.7-1.9 was obtained. Maximum plasmid yield and efficient conditions are shown in Table 5. Fourth, it effectively blocks nuclease activity because it removes nucleases commonly found in E. coli and carbohydrates found in JM109 strains.

Efficiency of Plasmid DNA Prepared Using GF Medium Column Kit with Chaotropic Salt and Guanidine HCl Manufacturing method E. coli JM109 culture solution Plasmid type OD ₂₆₀ / OD ₂₈₀ (purity) Maximum DNA Yield Chaotropic salt 200 ml pGem 7z 1.7 to 1.8 50-100 ㎍ Guanidine HCl 200 ml pGem 7z 1.7 to 1.9 200-400 µg

Efficient Plasmid Yields and Conditions by GF Column Kits Manufacturing method E. coli JM109 culture solution Plasmid type OD ₂₆₀ / OD ₂₈₀ (purity) GF Resin DNA yield GF small column kit 10 mL pGem 7% 1.7 to 1.8 0.1g ~ 70 ㎍ GF Medium Column Kits 100 mL pGem 7% 1.6 to 1.9 0.42 g ~ 200 ㎍ GF large column kit 250 mL pGem 7% 1.7 to 1.9 1 to 1.34 g 832 µg to 2.2 mg The GF small column and the GF medium column were prepared by the GF matrix column method, and the GF large column was used by the GF multilayer column method.

As described in detail in the above embodiment, the glass microfiber column of the present invention has an effect of simply and quickly preparing plasmid DNA to obtain a large amount of high purity plasmid DNA, and the prepared plasmid DNA has excellent purity and is further purified. There is an effect that can be used for various reactions without a process. In addition, the column kit of the present invention can be used to effectively separate DNA from agarose gels or polyamide gels, and for intracellular insertion isotope quantification, RNA preparation, isotope probe purification, purification of PCR reactions, and already prepared plasmid DNA. It is a very useful invention in the biopharmaceutical industry because of the excellent effect that can be applied to various applications such as tablets.

Claims (5)

A method for producing a glass fine fiber column, characterized in that it contains a resin made mainly of borosilicate glass fiber particles having a size of 3 mm or less and 4.2 M guanidine hydrochloride. Glass microfiber column kit, characterized in that prepared by the method of claim 1. A method for producing plasmid DNA using the glass microfiber column kit according to claim 3. A method for separating DNA from agarose gel or polyacrylamide gel using the glass microfiber column kit according to claim 3. The DNA purification method of increasing the purity of the DNA by removing the RNA and the impure protein mixed in the DNA using the glass microfiber column kit according to claim 3.