KR100805449B1 - New method for extraction of dna from agarose gel by using zeolite as a molecular sieve - Google Patents

Abstract

A method for purifying DNA is provided to purify conveniently the DNA with high yield from an agarose gel by one time centrifugation by using a column charged with an activated zeolite powder. A method for purifying DNA comprises the steps of: (a) after electrophoresing an agarose gel, cutting a portion including DNA with a desired size from the agarose gel; and (b) after putting the cut portion in a column prepared by using an activated zeolite powder, centrifuging it.

Description

New method for extraction of DNA from agarose gel by using zeolite as a molecular sieve}

1 is a diagram showing the structure and purification method of the DNA purification column provided in the present invention.

Figure 2 is a photograph comparing the results of purification using the method provided by the present invention and the existing product.

Lane 1: size marker

Lane 2: PCR product purified using the column of the present invention

Lane 3: DNA from lane 2 eluted with the restriction enzyme BamHI

Lane 4: PCR product purified using QIAGEN's column

Lane 5: DNA obtained by treating the eluted DNA of lane 4 with restriction enzyme BamHI

Lane 6: size marker

Figure 3 is a chromatogram showing the results of determining the base sequence of the purified gene using the method provided by the present invention and the existing product.

Figure 4 is a photograph comparing the results of purifying gene fragments of various sizes using the method provided by the present invention and existing products.

Lane 1: size marker

Lane 2: 9 kb gene purified using the column of the present invention

Lane 3: 9 kb gene purified using QIAGEN's column

Lane 4: 6 kb gene purified using the column of the present invention

Lane 5: 6 kb gene purified using QIAGEN's column

Lane 6: 3 kb gene purified using the column of the present invention

Lane 7: 3 kb gene purified using QIAGEN's column

Lane 8: 1.5 kb gene purified using the column of the present invention

Lane 9: 1.5 kb gene purified using QIAGEN's column

Lane 10: 0.5 kb gene purified using the column of the present invention

Lane 11: 0.5 kb gene purified using QIAGEN's column

Lane 12: size marker

The present invention relates to a method for preparing a column that can be used to purify DNA on an agarose gel, and more particularly, to a disposable column filled with activated zeolite powder. The column provided in the present invention has a feature that the DNA can be purified with high efficiency in agarose gel by only one centrifugation as compared to the conventional product.

For genetic manipulation, it is necessary to separate and purify the restriction fragment cleaved gene fragment or polymerase chain reaction (PCR) product from gene fragments of different sizes. In order to accomplish this purpose, various methods have been devised and used to release specific bends from agarose gel among the bends observed after separation by size by electrophoresis of DNA. This method is a very important technique for making recombinant DNA, and thus, a method of recovering, eluting, and purifying eluted DNA has been continuously developed. Among the many methods of purifying DNA in Gel, the representative method is as follows.

1) Electroelution in Dialysis Bags

2) Elution using low melting agarose gel

3) Extraction using DNA adsorbent

Initially, electrophoresis to purify only gene fragments of desired size, agarose gel fragments cut to contain only gene fragments of desired size are transferred to a dialysis bag, followed by electroelution, and precipitation with ethanol to recover DNA. Used. This method is the most effective method for separating DNA fragments larger than 5 kb and the purity of the eluted DNA is very high. However, it is rarely used because of its inconvenience and long time.

The second method is a method of electrophoresis using low melting agarose gel, which is dissolved at low temperature, and then the agarose gel slices are melted by raising the temperature and immediately used for cloning. This method has the advantage of shortening the experiment time, but the method of gel making and electrophoresis with low melting agarose is more difficult than the existing agarose method and is not used because of the limitation due to the low concentration of eluted DNA.

Recently, the cut agarose gel is immersed in a solution containing a chemical that helps dissolve agarose gel such as sodium azide, dissolved at 50-60 ° C., and then a substance adsorbed with DNA is added to the dissolved solution. The DNA is recovered from the agarose gel by recovering by precipitating only DNA or passing the dissolved solution through a disposable column filled with DNA adsorbent. The adsorbed DNA is washed with a solution containing ethanol to remove impurities and then dissolved by distilled water. This method has a disadvantage in that the recovery rate is different and centrifuged several times depending on the size of the most widely used method or the size of DNA because it can easily recover high-purity DNA.

Zeolite is an ore that was discovered in 1756 by Swedish mineralogist Cronsted and named "zeo stone." Zeolite is filled with water molecules in the nano-sized pores inside, and when the ore is heated, the contained water molecules evaporate to generate water vapor.

This led to Cronsted's naming of the monument as a 'boiling stone' in our country, where there is a large reserve of natural monuments called clinoptilolite near Yeongil Bay. It is also called fluorite. It is used as ion exchanger because it contains cation and easily exchanges with other cations. It has relatively large pores (3 ~ 10Å) inside, so it has adsorbent, nanoreactor, catalyst, etc. Used as In addition, it is used as an additive for livestock feed, which promotes the growth of livestock, and mixes it with soil to increase crop harvest. Especially, when used as a building material, it keeps the indoor temperature and humidity constant regardless of the change of season. have. As natural products, there are green sand and artificially monuments, and they are usually replaced with ion exchange resins because they are limited to raw water of pH 6.7 ~ 8.2. The zeolite skeleton is a three-dimensional inorganic polymer in which silicon (Si) and aluminum (Al) are connected through four cross-linked oxygen, respectively, and have a negative charge as the aluminum combines with four oxygens. Various cations (M +) are present to counteract this negative charge.

[Skeletal structure of the monument]

Using this feature of zeolite, when agarose gel containing DNA is passed through a column filled with zeolite powder by centrifugation, the agarose gel does not pass through the column and remains on the surface. Adsorbed into the pores of the zeolite, they do not pass through the column. On the other hand, large-molecules with negative charges such as DNA do not adsorb to the zeolite and pass through the gap between the zeolite powder, so that the single-use column filled with zeolite powder is activated to selectively adsorb only cations and small molecules without adsorption to DNA. Was used to purify DNA from agarose gel to develop a method for purely purifying DNA from agarose gel in a single centrifugation. Completion of the present invention by confirming that the purified DNA using this method and the purified DNA using the conventional method are similar in purity to those purified by the existing method when compared with restriction enzyme treatment, vector cloning efficiency and sequencing. It was.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for more conveniently recovering DNA from agarose gel by providing a method for preparing a disposable column from zeolite powder which has no defect ability with DNA and is activated to selectively adsorb only a cationic material.

In order to achieve the above object, the present invention provides a method for producing a column for DNA purification. In addition, the present invention provides a method for producing a zeolite powder for producing the column. Furthermore, the present invention provides a method for purifying DNA from agarose gel using the column.

Hereinafter, the present invention will be described in detail.

Since the column provided in the present invention is designed to be mounted and used in a 1.5 ml centrifuge tube, the column may be manufactured in plastic and does not require a specific form. It does not use ethanol solution to remove impurities or add 3 ~ 5 times the volume of agarose gel to dissolve agarose gel like conventional products. This is possible. The general structure of the column, as shown in Figure 1, the structure of the inlet portion is projected so as to hang over a 1.5ml centrifuge tube and the bottom portion is a structure with a small hole through which DNA solution can pass. To prevent the passage of filled zeolite powder through the hole in the bottom, filter paper of the same size as the floor area is attached to the floor. After activating and adding 0.2ml of the zeolite solution suspended in distilled water, the zeolite powder is allowed to settle for 10 minutes or more. Distilled water remaining in the upper column of the column is removed from the bottom using a vacuum suction or centrifuged for 1 minute at 12000rpm. The zeolite filled in the column maintains a relatively solid form but has the disadvantage of being re-powdered when all the moisture contained is evaporated. Therefore, the filled zeolite powder needs some moisture to remain in solid form or additional supplementation is required. In the present invention, 30-40 μl of 0.3-0.5% agarose solution was applied on the surface of the zeolite powder from which water was removed. Leave at least 10 minutes at room temperature to allow the added agarose solution to solidify, and then centrifuge for 1 minute at 12000 rpm to completely remove water. Since the added agarose solution penetrates into the cracks of the zeolite powder and coagulates to act as an adhesive, the zeolite powder can remain solid even after the water is completely removed, and the coagulated agarose does not pass through the column even after centrifugation.

The activation method of zeolite used in the present invention is as follows. Zeolite used in the present invention is a natural zeolite of 230 mesh size. The size of the zeolite powder is suitable between 150 mesh and 250 mesh. If it is larger than 150 mesh, the purity of the passed DNA solution is decreased, and if it is smaller than 250 mesh, the DNA recovery rate is reduced. Zeolite powder Suspended zeolite powder in distilled water solution of 2 volumes, and then add ammonium salt 1/1000 of the zeolite weight. After stirring for 10 minutes or more, the zeolite and the supernatant are allowed to separate, and then the supernatant is removed. This process is repeated twice to remove the dissolved ammonium salt without reacting with the substituted ions. The supernatant was removed, and the dried zeolite powder was reacted in an oven at 500 ° C. for 1 hour to evaporate sodium ions and substituted ammonia, followed by adding two volumes of 0.1 M EDTA (Eyhylene Diamine Tetraacetic Acid) to increase the cation binding capacity of the zeolite powder. Add and stir for at least 10 minutes. After allowing the zeolite and the supernatant to separate, remove the supernatant, add 2 volumes of distilled water, stir for at least 10 minutes, and leave it again to remove the supernatant twice to remove the EDTA remaining in the solution. do. Finally, the zeolite powder suspended in 2 volumes of distilled water is sterilized at 120 ° C. for 20 minutes by autoclave and then used for column production.

Method for recovering DNA from agarose gel using a column provided in the present invention is as follows. After electrophoresis, the site containing the DNA of the desired size is cut out of the agarose gel. At this time, in order to prevent the concentration of the purified DNA solution from being lowered, the surface of the gel was wiped with Kimwipes, and then the size of the gel was minimized and cut out. The cut pieces of agarose gel were placed in a column, mounted in a 1.5 ml centrifuge tube, and centrifuged at 10,000 rpm for 3 minutes (Figure 1). DNA purified by this method is ready for cloning, restriction enzyme processing and sequencing.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Purification and Cloning Efficiency Comparison of PCR Products

In order to verify the cloning efficiency using the column provided by the present invention, the inventors purified the same amount of PCR products using QIAGEN's gel elution kit and the column provided in the present invention, and then added distilled water to the same volume. Adjusted. The PCR product used in this experiment was 1.8Kb in size and separated into 1Kb and 0.8Kb when treated with restriction enzyme BamHI. FIG. 2 shows the results of electrophoresis of 5 μl of purified DNA by each method (lanes 1 and 3). When DNA was purified from the agarose gel by the method provided by the present invention, it could be confirmed that a similar amount of DNA was eluted when using KIT of QIAGEN. In addition, in order to confirm the purity of the purified DNA, each treatment with restriction enzyme BamHI and electrophoresis, and the result of the comparison was confirmed that the same efficiency as when using the KIA KIT KIT (Lane 2 and 4 of Figure 2). In order to compare the purity of the purified gene, the sequence was analyzed using the primers used for each purified DNA fragment. Figure 3 shows the chromatogram of the sequencing results performed by each method. The sequencing efficiency was similar for both methods. In order to compare the cloning efficiency of the purified gene fragments, the same amount of eluted DNA was linked with T-vector (Invitrogen), and then transformed into E. coli DH5a, and then to LB solid medium containing X-Gal and ampicilline. Smear was compared to compare cloning efficiency. In both cases, similar numbers of colonies were formed and the cloning efficiencies were similar at 60-70%.

Example 2 Comparison of Cloning Efficiency by Size of Gene Fragments

The present inventors provide the same amount of 9 kb, 6 kb, 3 kb, 1.5 kb, 0.5 kb gene fragments in QIAGEN gel elution kit and the present invention to verify DNA purification efficiency from agarose gel according to gene fragment size. After each purification using a column was adjusted to the same volume by adding distilled water. As a result of comparing the same volume of eluted DNA by electrophoresis, it was confirmed that the DNA was eluted with a constant efficiency regardless of the gene size when using the method provided in the present invention compared to the gel elution kit of QIAGEN (Fig. 4).

As described above, the gel elution column provided in the present invention is very fast, inexpensive and simple to purify DNA from agarose gel compared to the conventional method, it can be very useful in genetic recombination experiments.

Claims (3)

A method of activating zeolite powder for use as a molecular sieve for DNA purification. DNA purification using the zeolite powder prepared by the method of claim 1. The method of claim 2, wherein the DNA is purified from an agarose gel.

Images