KR20010034995A - A Modified Quartz microfiber membrane and the Use there of - Google Patents

Abstract

PURPOSE: A modified quartz microfiber membrane is provided to have a suitable hydrophilicity and a proper electropositivity by treating surface with NaOH or the mixture of NaOH and C3P, so as to usefully extract DNA or RNA. CONSTITUTION: A quartz microfiber membrane is filled in 50 ml beaker, treated with 0.2N NaOH 20 ml and then let alone 25 deg.C for 12 hours after being left 35 deg.C for 12 hours. The Quartz microfiber membrane treated with NaOH is washed with acetone 4 times after being cleaned with a distilled water 4 times. And the Quartz microfiber membrane is dried in an oven of 100 deg.C for 1 hour after being let alone in air 1 hour and is kept in a desicator. And a circular of 7mm diameter made of the membrane is inserted in a spin mini column after being piled up with 3 membranes. And then the membrane is fixed with a ring and sterilized by etylene oxide gas.

Description

A modified quartz microfiber membrane and the use there of}

The present invention relates to a modified quartz fiber membrane and its use, and more particularly, to a hydrophilicity and electropositivity of the membrane surface by combining NaOH or NaOH with PCl ₃ to the quartz crystal fiber membrane. It relates to a modified quartz microfiber membrane modified to be useful for extracting RNA.

Generally, in molecular biology, high purity two-stranded chain plasmid DNA, single stranded phage DNA, lambda DNA, chromosomal DNA, PCR products, RNA and agarose gels or polyacrylamides The preparation of purified DNA fragments from is a very important task. Therefore, the method for purifying such nucleic acid should be fast and simple and better if no additional manipulation is required, and the purified DNA or RNA may be transformed, restriction enzyme analyzed, ligation, sequencing, transfected. Can be used immediately for transfection.

Methods for obtaining nucleic acids that can satisfy these characteristics currently include automated DNA purification apparatus. However, these devices have been expensive and expensive to maintain too much problem.

Therefore, many laboratories use the alcohol precipitation method that is traditionally used. This method also requires a lot of labor and time, and the purity of purified DNA is too low. Therefore, CsCl gradient, gel filtration, ion exchange chromatography and the like have been used to solve these problems and to purify DNA with higher purity. However, these methods require a separate purification process or have a very low purity since negatively charged cell components may be purified together with DNA to contain unwanted levels of impurities.

There is also a method for purifying DNA using a solid phase having a silica surface such as glass or celite. However, these methods not only can attach excess DNA to a solid phase surface or do not have a surface suitable for recovering DNA molecules by extraction, but this type of DNA binding is toxic and expensive and high concentration of chao You need chaotropes or alcohol. For example, DNA may be bound to a glass powder or glass fiber membrane in the presence of chaotropes, followed by alcohol removal of chaotropic ions, and DNA extraction with a low concentration of buffer or water. . However, glass powder has a low DNA binding ability and tends to be cut in the case of large DNA. Therefore, there is an urgent need for the development of a simpler, faster and cheaper method of separating DNA or RNA with high purity without impurities.

Thus, the nickname of the present invention, as a result of intensive efforts to solve the above problems, the surface of quartz microfiber membrane (quartz microfiber membrane) by treating with NaOH alone, or by combining NaOH and Cl ₃ P by DNA or RNA The present invention has been completed by adjusting to have hydrophilicity and electropositivity useful for extracting bays.

Accordingly, an object of the present invention is to provide a modified quartz fiber membrane treated with NaOH alone or with NaOH and PCl ₃ .

It is another object of the present invention to provide a method for using the modified quartz fiber membrane as a filler for nucleic acid purification.

1A is an exploded view of a modified quartz fiber membrane column.

1B is an exploded view of the collecting tube.

FIG. 2 is a graph comparing the yield of DNA in an unmodified microfibrous membrane and a modified microfibrous membrane modified using various modifiers. FIG.

Figure 3 is a picture of plasmid DNA purified using a modified quartz fiber membrane column.

4 is a diagram showing restriction enzyme analysis of plasmid DNA purified using a modified quartz fiber membrane column.

Figure 5 is a diagram purified from agarose gel after cleavage of plasmid DNA purified using a modified microfiber membrane column with restriction enzymes.

Figure 6a is a picture of genomic DNA purified from human blood using a modified quartz fiber membrane column.

6B is a diagram showing the results of PCR using genomic DNA purified from prokaryotic cells and human blood using a modified quartz fiber membrane column as a template.

7 is a diagram illustrating purification of various sizes of DNA and single-stranded M13 phage DNA using a modified quartz fiber membrane column.

Figure 8a is a purified image of the various PCR products used for the DNA chip using a modified quartz fiber membrane column.

8b is a diagram showing the removal of primers, salts, etc. from the PCR product using a modified quartz fiber membrane column.

FIG. 9A is a diagram of transfection using DNA purified using a modified quartz fiber membrane column. FIG.

9b is a diagram transfected with DNA purified using CsCl.

10 shows the results of manual sequencing of purified pCR2.1-AK3 plasmid DNA using a modified quartz fiber membrane column using an M13 reverse primer.

FIG. 11 shows the results of automatic sequencing of pCR2.1-AK3 plasmid DNA purified using a modified quartz fiber membrane column using a T7 primer.

The modified quartz fiber membrane treated with NaOH alone or with NaOH and PCl ₃ of the present invention will be described in more detail.

The present invention relates to a modified quartz microfiber membrane, by treating the quartz microfiber membrane with NaOH alone, or by combining NaOH and Cl ₃ P to adjust the electrical properties of the membrane surface to be useful for extracting DNA or RNA Modified quartz fiber membranes. In other words, the surface of the crystal microfibrous membrane is treated with NaOH alone or NaOH and Cl ₃ P are combined to control the hydrophilicity and electropositivity of the membrane surface, thereby making it easy to display a nucleic acid showing negative electrical properties. It is to be combined.

For surface treatment of the crystal microfiber membrane, the crystal microfiber membrane was placed in a NaOH solution and left to stand at a temperature of 15 to 37 ° C for about 2 hours. In order to increase the electrical conductivity after the NaOH solution surface treatment, it may be further left for 1 hour at a temperature of 15 to 37 ℃ in Cl ₃ P solution. In addition, the surface treatment is a solution consisting of 2M dichloromethane and 10 mM PCl ₃ after 0.2 hours of treatment with 0.2 N NaOH so that the quartz crystal fiber membrane is completely submerged or washed three times with 0.2 N NaOH quartz crystal fiber membrane with distilled water three times. After treatment for about 12 hours in acetone and distilled water is preferably washed.

When the modified microfibrous membrane modified by the above surface treatment method is brought into contact with the cell components, the modified microfibrous membrane can remove all cell components by washing, and the modified microfibrous membrane is modified with only DNA or RNA. The bound DNA or RNA can be extracted from the modified quartz fiber membrane.

On the other hand, the present invention includes a filler for nucleic acid purification to which the modified microfiber membrane described above is applied. That is, it includes using the modified quartz fiber membrane of the present invention as a column filler for separating and extracting only nucleic acids such as DNA or RNA.

1A is an exploded view of a column for nucleic acid purification in which a modified quartz fiber membrane is filled with a filler, and FIG. 1B is an exploded view of a collecting tube. After making the modified microfibrous membrane with 7 mm round shape, inserting the modified microfibrous membrane into the nucleic acid purification column of FIG. 1a and fixing the modified microfibrous membrane with the ring of 7.25 mm, and then combining with the collecting tube of FIG. It was.

The present invention comprises the steps of preparing a modified quartz microfiber membrane (modified quartz microfiber membrane) according to the following Examples and Experimental Examples, the production of a column filled with a modified quartz microfiber membrane and using the two-stranded chain plasmid DNA Purification of DNA fragments from single-stranded M13 phage DNA, lambda DNA, chromosomal DNA, PCR products and agarose gels or polyacrylamide gels, restriction enzyme analysis, transfection, manual sequencing of purified plasmid DNA (manual sequencing) and automatic sequencing are described in more detail.

However, these Examples and Experimental Examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these Examples to those of ordinary skill in the art. Will be self explanatory.

Example 1 Preparation of Modified Microfiber Membranes and Columns for DNA Purification Using NaOH

A quartz microfiber membrane was placed in a 50 ml beaker, treated with 20 ml of 20 ml of 0.2N NaOH, and left at 35 ° C. for 12 hours and then left at 25 ° C. for 12 hours. NaOH-treated microfibrous membranes were washed four times with distilled water and four times with acetone. The modified microfiber membrane was left in air for 1 hour and then dried in an oven at 100 ° C. for 1 hour and then stored in a desiccator.

Then, the modified quartz microfiber is made into a 7 mm diameter circle, and three of them are inserted in a spin mini column (FIG. 1A), and then the ring is inserted and fixed. It was prepared by sterilization with ethylene oxide (Etylene Oxide) gas. When 10 ml of E. coli suspension is used, the yield of DNA is no longer increased when three or more modified quartz fiber membranes are overlapped.

Example 2 Preparation of Modified Microfiber Membranes and DNA Purification Columns Using NaOH and PCl ₃

The modified NaOH-treated quartz fiber membrane prepared in Example 1 was immersed in a solution containing 2M dichloromethane in PCl ₃ at 25 ° C. for 12 hours, washed three times with acetone, and washed three times with distilled water. The fibrous membrane was made and then dried in a vacuum desiccator.

This modified quartz microfiber is made into a circular diameter of 7 mm, as shown in FIG. 1A, and then three of them are inserted into a spin mini column, and then the ring is inserted to fix the ethylene oxide. It was prepared by sterilization with (Etylene Oxide) gas. In addition, the DNA yield does not increase any more when three or more modified microfibrous membranes are overlapped when 10 ml of E. coli suspension is used.

Experimental Example 1 Isolation of Plasmid DNA

Plasmid DNA was isolated and prepared using the modified quartz fiber membrane DNA purification column prepared in Example 1. Top 10 E. coli strains containing plasmid DNA (pCR2.1) were cultured and pelleted by centrifugation of the strain culture solution. The pellet was suspended in solution A (30 mM Tris / HCl pH 7.5 containing 20 mM EDTA and 100 μg / ml RNAse A), followed by solution B (0.2 N NaOH, 1% sodium dodecyl sulfate (SDS) (w) / v)), the cells were lysed, neutralized with C solution (3.1 mM potassium acetate, acetic acid, 4 M guanidine thiocyanate pH 4.2), and then centrifuged to supernatant to the modified quartz fiber membrane column. After binding the DNA, washed with D solution (20 mM Tris / HC1 pH 7.4, 100 mM NaCl, 10 mM EDTA, 50% ethanol) and eluted with distilled water to obtain a high-purity plasmid DNA.

Experimental Example 2 Comparison of DNA Yield between Modified Microfibrous Membrane and Modified Microfibrous Membrane

In order to compare the yield of the plasmid DNA of the modified microfibrous membrane and the modified microfibrous membrane, the same method as in Example 1 and Example 2 using the modified modified microfibrous membrane prepared using the respective modifiers To prepare a column, and centrifuged for 10 minutes from 10 ml of E. coli culture at 3,000 rpm to recover the precipitate of E. coli, and plasmid DNA was prepared in the same manner as in Experimental Example 1 to compare the yield and purity. Analyzed. The results are summarized in Table 1 and FIG. 2.

As a result, as shown in Table 1 and FIG. 2, the modified microfibrous membrane, especially the modified microfibrous membrane treated with NaOH or NaOH / PCl ₃ , showed superior DNA binding ability, and the purity was modified. Both OD ₂₆₀ = 1.6 and 1.8 were found in both the modified and unmodified quartz fibers.

Purity and yield of plasmid DNA according to modified quartz fiber membrane Modifier E. coli TOP 10 medium ^* OD ₂₆₀ / OD ₂₈₀ (purity) Yield NON 10 ml 1.60-1.75 0.3 to 0.5 μg 0.2N NaOH 10 ml 1.65-1.82 10 to 12 ㎍ 10N NaOH 10 ml 1.59-1.81 4 to 6 μg PCl ₃ 10 ml 1.61-1.80 0.2 to 0.4 μg 0.2N NaOH / PCl ₃ 10 ml 1.60-1.80 11 to 13 ㎍ ^* E. coli TOP 10 medium: LB medium

Experimental Example 3: Preparation of Plasmid DNA from Various Colony Cultures

Nine Escherichia coli (E. coil TOP 10) transformed with pCR2.1-AK3 were grown at 250 rpm at 37 ° C. for 12 hours in 10 ml LB medium, respectively, and then the plasmids were prepared in the same manner as in Experiment 1 above. As a result of purifying the DNA, 15 µg or more of the plasmid DNA could be extracted from each colony.

Experimental Example 4 Restriction Enzyme Cleavage of Plasmid DNA

Open reading frame for AK3 was amplified using polymerase reaction (PCR) and combined with pCR 2.1 vector, transformed into E. coli TOP 10 and grown on empicillin medium. Extracting the plasmid DNA in the same manner as in Experimental Example 1 and digesting with restriction enzyme EcoRI showed effective DNA cleavage as shown in FIG. 4.

Experimental Example 5: Isolation of DNA Fragments from Agarose Gel

DNA was isolated by electrophoresis of DNA on a TAE or TBE agarose gel, the band was cut and placed in an Eppendorf tube and gel lysis solution [50 mM Tris / HCl pH 7.0, 6 M guanidine thiocyanate] 500 μl per 200 mg, the gel was completely dissolved at 42 ° C., and DNA was bound to the modified quartz fiber membrane column, followed by washing solution [20 mM Tris / HCl pH 7.4, 100 mM NaCl, 10 mM EDTA, 50% ethanol]. After washing with DNA eluted with distilled water. As a result, as shown in FIG. 5, excellent DNA separation performance was exhibited. At this time, the elution yield reaches 85 to 95%.

Experimental Example 6 Preparation of Chromosome DNA from Blood

After EDTA was added to the blood collected from humans to 7.5%, 300 μl of the blood was collected, treated with red blood cell digestion solution (0.5% NH ₄ Cl), and centrifuged to obtain only white blood cells. To this, 1% sodium dodecyl sulfate (SDS) solution was added to decompose leukocytes, and 2.5M ammonium acetate was added to precipitate proteins. chromosomal) DNA was attached and washed with 50% ethanol and then eluted with distilled water [FIG. 6a]. In addition, PCR was well generated when PCR was performed using the eluted chromosomal DNA as a template (FIG. 6B).

Experimental Example 7: Preparation of plasmid DNA and single stranded DNA of various sizes

Plasmid DNA was prepared from the transformed Escherichia coli containing various sizes of plasmid DNA in the same manner as in Experimental Example 1, and the purification efficiency of the plasmid DNA was good regardless of the size of the plasmid DNA, and M13 phage DNA After precipitation with 30% PEG, the phage was dissolved in 1 ml of distilled water, and 500 µl of 50 mM Tris / HCl pH 7.0 and 4 M guanidine thiocyanate was added thereto, and DNA was bound to the modified quartz fiber membrane column. After washing with washing solution [20 mM Tris / HCl pH 7.4, 100 mM NaCl, 10 mM EDTA, 70% ethanol], single-stranded DNA was eluted with distilled water. As a result, as shown in Figure 7, it showed an excellent single-stranded DNA separation performance.

Experimental Example 8: Purification of Polymerase Reaction (PCR) Reaction

5 μl of vector DNA containing genes of various sizes was used as a template, 8 μl of 2.5 mM dNTPs, 5 units / μl Ex taq DNA polymerase 1 μl, 10X Extech 10 μl of buffer (Ex taq buffer), 1.5 μl of T7 primer and T3 primer (100 pmol / μl) and 73 μl of distilled water were mixed to make 100 μl of the total volume. The reaction was denatured at 94 ° C. for 5 minutes, denatured at 98 ° C. for 10 seconds, at 30 ° C. for 30 seconds, and then subjected to 35 cycles of expansion at 72 ° C. for 40 seconds. . 500 μl of a DNA binding solution [50 mM Tris / HCl pH 7.0, 4 M guanidine thiocyanate] was added to the polymerase reaction product thus prepared, and DNA was bound to the modified quartz fiber membrane column, followed by a washing solution [20 mM Tris]. / HCl pH 7.4, 100 mM NaCl, 10 mM EDTA, 50% ethanol] and the polymerase reaction was eluted with distilled water. As a result, as shown in Figure 8a exhibited excellent DNA separation performance, as shown in Figure 8b was removed after the polymerase reaction, the remaining primers were not removed.

Experimental Example 9: Cell transfection

Reporter plasmid (GFP) was added to 293 cells of human embryo kidney cell line using calcium modified fibrous membrane column and CsCl gradient prepared in Example 1, calcium phosphate (Calcium Phosphate). 36 hours after the transfection was observed, and the results are shown in FIGS. 9A and 9B. As a result, as shown in FIGS. 9A and 9B, when transfecting plasmid DNA prepared with the modified modified microfibrous membrane of the present invention, effective protein expression in almost all cells was similar to that of plasmid DNA purified with a CsCl gradient. It was confirmed that it was done.

Experimental Example 10 ³⁵ S sequencing analysis of purified plasmid DNA

ATP was used to react as a SequenaseTM version 2.0 DNA sequencing kit, separated from the sequencing gel, and confirmed by autoradiography. As a result, as shown in Figure 10, the sequencing of the DNA prepared by the modified microfiber membrane of the present invention was confirmed that the DNA band is clearly separated.

Experimental Example 11: Automatic Sequence Analysis of Purified Plasmid DNA

Automatic sequencing analysis was performed using ABI PRIM ^™ Dye terminator cycle sequencing ready reaction kit (Perkin Elmer), 8 μl terminal ready reaction mix, 0.4 μg template DNA, and 3 pmole (primary primer). sense primer: M13 reverse primer anti-sense primer: T7 promoter primer in 20 µl reaction volume in Perkin Elmer thermal cycler for 96 ℃ / 30min, 50 ℃ / 15sec, 60 ℃ / 4min After 25 cycles, the mixture was precipitated with ethanol, dried in a rotary vacuum evaporator (speed vac.), And analyzed by an automatic sequencer (Applied Biosystem).

As a result, as shown in FIG. 11, a very effective DNA sequencing was performed, wherein the template used above was pCR2.1 containing 680 bp of DNA and the primer was an M13 reverse primer or a T7 primer.

As described in detail above, the present invention relates to a modified quartz fiber membrane and its use. According to the present invention, two-stranded chain plasmid DNA, lambda DNA, chromosomal DNA, and single strand of high purity are simpler and faster. It is possible to purify chain phage DNA, remove various cofactors, buffer components and primers from PCR products, as well as effectively separate DNA fragments from agarose gels or polyacrylamide gels. In addition, the effect of restriction enzyme analysis, transfection, manual sequencing and automatic sequencing of purified plasmid DNA can be obtained.

Claims (5)

Modified microfibrous membrane, characterized in that the modified microfiber membrane was modified by NaOH alone or NaOH and PCl ₃ treatment. The modified quartz fiber membrane of claim 1, wherein the quartz fiber membrane is modified for 1 hour to 24 hours using a 0.2 to 10 N NaOH solution. The modified quartz fiber membrane according to claim 1, wherein the quartz fiber membrane is modified using 0.2 to 10 N NaOH solution and then modified using 1 mM to 1 M PCl ₃ . The filler for nucleic acid purification, characterized in that the modified quartz fiber membrane of claim 1 is applied. 5. The nucleic acid purification filler according to claim 4, wherein the nucleic acid is DNA or RNA.