KR20030006505A - Cell lysis buffer for extracting DNA and extraction method by using thereof - Google Patents

Abstract

PURPOSE: A cell lysis buffer for DNA extraction and a method for extracting DNA using the same are provided, thereby simultaneously carrying out the cell lysis, protein separation and RNA removal, to reduce the DNA extraction steps and time and to treat a large number of samples. CONSTITUTION: The cell lysis buffer for DNA extraction contains Tris-Cl, EDTA, a surfactant, acetic acid salt, protease and RNase, wherein the surfactant is selected from the group consisting of Triton X-100, sarcosyl sulfate and SDS; and the acetic acid salt is ammonium acetate or sodium acetate. The method for extracting DNA comprises the steps of: (1) adding the cell lysis buffer into a cell; (2) transferring the lysed cell onto a column to fix DNA; (3) washing the column; and (4) recovering DNA from the column, wherein the column is selected from glass fiber column and silica membrane column.

Description

Cell lysis buffer for extracting DNA and extraction method using the same {Cell lysis buffer for extracting DNA and extraction method by using}

The present invention relates to a cell lysis buffer for DNA extraction and a DNA extraction method using the same, more specifically Tris-Cl, EDTA, surfactants, salt of acetic acid, protease and RNA It relates to a cell lysis buffer for DNA extraction comprising a degrading enzyme and a DNA extraction method using the same.

As genetic engineering advances, experiments using cloning, restriction enzyme analysis, PCR, sequencing, and other DNA have increased dramatically. It is an important variable. Contamination of DNA inhibits or hinders chemical reactions such as hybridization reactions such as Southern blots, enzymatic reactions, and the like, and contaminants in the DNA also cause errors when digesting the DNA to be tested and measuring the amount of DNA Will commit. Such contaminants include low-molecular substances such as fats, low-molecular enzyme inhibitors, as well as enzymes such as proteins, polysaccharides, and polynucleotides.

Many methods have been developed to solve this problem in order to obtain contamination free DNA suitable for the application of molecular biology methods. The traditional method for obtaining DNA is to dissolve cells by enzymatic or chemical methods to dissolve the DNA to remove contaminant proteins, RNA and other substances (Sambrook et al., Molecular cloning; A laboratory Manual, 2nd, Ed. Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, 1989; F. Ausubel et al., Current Protocols in Molecular Biology, Wiley-Interscience, NY, 1993). However, the methods involve many steps, and their success depends largely on the skill of the operator. Therefore, applying this method repeatedly may cause a change in the result of manual operation of an inexperienced worker.

Conventional methods include the extraction of DNA from phenol / chloroform or phenol / chloroform / isoamyl alcohol and the ethanol (or isopropanol) precipitation to recover the DNA. Separating from Ethanol (or isopropanol) precipitation extracts DNA from a liquid solution of DNA extracted with phenol / chloroform and removes residual phenol and chloroform from the DNA. In addition, ethanol precipitation may remove some of the nucleoside triphosphate and short (up to 30 bases) oligonucleotides.

However, the phenol / chloroform extraction process requires many steps and time to extract DNA, and uses phenol and chloroform harmful to the human body, which is very dangerous. Phenolic is very dangerous to burn on skin and chloroform is very volatile, toxic and flammable. The use of these dangerous phenols and chloroform also has to be tested in a hood. In addition, the phenol / chloroform extraction process is to damage the DNA by the oxidation of phenol, so only freshly distilled (phenol) should be used.

Therefore, methods have been devised that are more effective, simpler and minimize DNA separation time than conventional DNA separation processes through phenol / chloroform extraction and ethanol precipitation. Examples of such methods include the use of chaotropic salts and silica resins, the use of affinity resins, the ion exchange resin chromatography, and the use of magnetic beads, in addition to salting with phenol / chloroform. (US Patent No. 5057426, US Patent No. 4923978, EP No. 012767A1, EP No. 0515484B, WO No. 97/10331, WO No. 96/18731).

In recent years, in order to overcome the inconveniences of phenol / chloroform extraction and ethanol precipitation, and to promote convenience, a kit using a column is the basis of genomic DNA extraction. The principle of extracting genomic DNA through such a column is the use of glass fibers or silica membranes that specifically bind to DNA, and thus the principle of structural interaction between water molecules and DNA. To use. Glass fiber or silica membranes do not bind to salts, proteins and other cellular substances, ultimately purely separating DNA.

In recent years, with the development of genetic engineering, many experiments have to deal with a large number of samples, and reproducible results can be obtained when repeated experiments are performed, and the results are reliable and from blood and other body fluids. In extracting genomic DNA, there is a need for a simpler and less time-consuming way to extract DNA.

Accordingly, the present inventors have prepared a novel cell lysis buffer for extracting DNA, and completed the present invention by revealing that DNA can be highly reproducible from various samples in a short step and time using the buffer.

It is an object of the present invention to provide a cell lysis buffer for DNA extraction and a DNA extraction method using the same.

1 is an electrophoresis picture of genomic DNA extraction results according to the number of cells using the cell lysis buffer of the present invention,

Lane 1: 5 x 10 ⁵ cells, lane 2: 3 x 10 ⁶ cells,

Lane 3: 5 x 10 ⁶ cells, lane 4: 1 x 10 ⁷ cells,

FIG. 2 is an electrophoretic photograph obtained by digestion of genomic DNA extracted with a cell lysis buffer of the present invention with restriction enzymes.

Lane 1: not treated with restriction enzyme, lane 2: BamHⅠ ,

Lane 3: EcoRⅠ, Lane 4: HincⅡ,

Lane 5: HindⅢ, Lane 6: NcoⅠ,

Figure 3 is an electrophoresis picture of the PCR reaction using genomic DNA extracted using the cell lysis buffer of the present invention,

Lane 1: B16, lane 2: HeLa,

Lane 3: NB4, lane 4: SNU1,

Lane 5: SNU601,

Figure 4 is an electrophoresis picture of genomic DNA extracted from Salmonella gram negative bacteria using the cell lysis buffer of the present invention,

Lane 1: 3 ml, lane 2: 3 ml, lane 3: 3 ml,

Lane 4: 3 ml, 3/20, 4 ° C (35 days have elapsed),

Lane 5: 3 ml, 3/20, room temperature (35 days elapsed),

Lane 6: 3 ml, 4/13, 4 ° C (after 14 days),

Lane 7: 3 ml, 4/13, room temperature (after 14 days),

5 is an electrophoresis photograph comparing and analyzing genomic DNA separation efficiency according to the amount of cell lysis buffer of the present invention,

Lane 1: 5 × 10 ⁵ cells, 150 μl, lane 2: 5 × 10 ⁵ cells, 300 μl,

Lane 3: 1 × 10 ⁶ cells, 300 μl, lane 4: 3 × 10 ⁶ cells, 300 μl,

Lane 5: 3 × 10 ⁶ cells, 600 μl, lane 6: 1 × 10 ⁷ cells, 600 μl,

Figure 6 is an electrophoresis picture comparing genomic DNA extraction using the cell lysis buffer of the present invention and genomic DNA extraction using a third-party product (DNeasy ^TM Tisssue Kit, Qiagen).

Lanes 1, 2 and 3: using the cell lysis buffer of the present invention,

Lane 1: 3 X 10 ⁶ cells, lane 2: 5 X 10 ⁶ cells,

Lane 3: 1 x 10 ⁷ cells,

Lanes 4, 5 and 6: using third party products (DNeasy ^TM Tisssue Kit, Qiagen)

Lane 4: 3 x 10 ⁶ cells, lane 5: 5 x 10 ⁶ cells,

Lane 6: 1 x 10 ⁷ cells

In order to achieve the above object, the present invention provides a cell lysis buffer for DNA extraction.

The present invention also provides a DNA extraction method using the cell lysis buffer.

Hereinafter, the present invention will be described in detail.

The present invention provides a cell lysis buffer for DNA extraction.

Cell lysis buffer for DNA extraction of the present invention is prepared in an aqueous solution in which Tris-Cl, EDTA, surfactant (detergent), salt of acetic acid, protease and RNAase are dissolved.

Among the components of the cell lysis buffer, the surfactant may be selected from the group consisting of Triton X-100, sarcosyl sulfate, and SDS, and it is preferable to use Triton X-100. In addition, as the acetate salt in the dissolution buffer, ammonium acetate, sodium acetate and the like may be used, and ammonium acetate is preferably used.

In the components of the cell lysis buffer, Tris-Cl preferably has a final concentration of 0.1 to 0.5 M, more preferably 0.25 M. EDTA is preferably in a final concentration of 0.01 to 0.05 M, more preferably 0.05 M. Triton X-100 is preferably in the final concentration of 0.5 to 3% by weight, more preferably 1% by weight. It is preferable that the ammonium acetate has a final concentration of 1.5 to 5.4 M, more preferably 3.5 M. As the protease, it is preferable to use protease K having a final concentration of 10 to 50 µg / ml, more preferably at a concentration of 50 µg / ml. As the RNA degrading enzyme, it is preferable to use RNA degrading enzyme A having a final concentration of 10 to 50 µg / ml, more preferably at a concentration of 50 µg / ml.

Cell lysis buffer for DNA extraction of the present invention has the advantage of reducing the time required to extract the DNA and the step than the conventional method in extracting DNA. In addition, by enabling simultaneous cell lysis and DNA extraction, and by simultaneously digesting protein and RNA in the cell lysis step, it is possible to reduce the steps and time for processing each enzyme.

The present invention also provides a DNA extraction method using the cell lysis buffer.

DNA extraction method using the cell lysis buffer of the present invention,

1) lysing the cells by adding the cell lysis buffer to the cells;

2) transferring the lysed cells of step 1 to a column to fix DNA;

3) washing the column of step 2; And

4) recovering DNA from the column.

In step 2, the column may be selected from glass fiber column and silica membrane column.

Conventional DNA extraction methods are divided into lysing cells using various solutions, separating proteins after lysis, and extracting DNA.In the step of removing RNA and separating proteins, each reactive enzyme It is time consuming because it contains the reaction to show the enzyme activity for a certain time by processing the process, and it has to go through several steps, which increases the possibility of contamination of impurities and it is difficult to obtain reproducible results when performing repeated experiments. there was. However, the DNA extraction method using the cell lysis buffer according to the present invention reduces cell extraction and DNA extraction by simultaneously allowing cell lysis, protein separation, RNA removal and DNA extraction with a single cell lysis buffer. You can save time. In addition, by allowing all of the steps to be divided in one step, it is possible to obtain reproducible results by reducing errors and errors that can be made by the operator.

The cell lysis buffer of the present invention is capable of extracting DNA even with a very small amount of cells, and the extraction efficiency is constant even with a large number of cells (see FIG. 1 ), and DNA extracted using the cell lysis buffer of the present invention is a restriction enzyme. It is precisely cleaved by, and is not contaminated by proteins, RNA, and other enzymes. Therefore, even if the DNA is used in other experiments involving enzymatic reactions, it is not a problem (see FIG. 2 ). It is not a problem (see Fig. 3 ), and the cell lysis buffer of the present invention is also suitable for genomic DNA extraction of Gram negative bacteria (see Fig. 4 ).

The cell lysis buffer of the present invention can be more effective DNA extraction by varying the amount of cell lysis buffer according to the number of cells (see Fig. 5 ), and also DNA extraction using the cell lysis buffer of the present invention DNA using a third party product Extraction efficiency is higher than extraction (see FIG. 6 ).

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 Preparation of Cell Lysis Buffer

<1-1> Preparation of Cell Lysis Buffer 1

We preferred 0.25 M Tris-Cl (pH 8.0), 0.05 M EDTA (pH 8.0), 1 wt% Triton X-100, 3.5 M ammonium acetate (pH 7.0) as preferred embodiments of cell lysis buffer to extract DNA. , An aqueous solution containing protease K at a concentration of 50 μg / ml and RNA enzyme A at a concentration of 50 μg / ml was prepared.

<1-2> Preparation of Cell Lysis Buffer 2

Composition of cell lysis buffer to extract DNA was 0.5 M Tris-Cl (pH 8.8), 0.05 M EDTA (pH 8.0), 0.5 wt% sacosyl sulfate, 3.5 M ammonium acetate (pH 7.0), 50 μg / ml concentration An aqueous solution containing a protease K of RNA, RNA degrading enzyme A at a concentration of 50 μg / ml was prepared.

<1-3> Preparation of Cell Lysis Buffer 3

Composition of cell lysis buffer for DNA extraction was 0.25 M Tris-Cl (pH 8.0), 0.05 M EDTA (pH 8.0), 0.25 wt.% SDS, 1.2 M sodium acetate (pH 5.2), 50 μg / ml protein An aqueous solution containing RNAase A at a concentration of 50 μg / ml of enzyme K was prepared.

Example 2 DNA Extraction Using Cell Lysis Buffer

The present inventors performed DNA extraction using the cell lysis buffer of the present invention prepared in Example 1 as follows.

1) After incubating the animal cells, a turbidity solution containing 5 X 10 ⁵ , 3 X 10 ⁶ , 5 X 10 ⁶ and 1 X 10 ⁷ cells per 1.5 ml tube was prepared at 10-20 at 13,000 rpm. Cells were harvested by centrifugation 2-3 times for a second. The remaining medium was completely removed when the cells were recovered.

2) The recovered cells were added with 300 µl of the cell lysis buffer of the present invention. At this time, in order to increase the efficiency of cell lysis, the bottom of the tube was lightly patted to completely release the cell precipitate, and then cell lysis buffer was added.

3) After adding the cell lysis buffer of the present invention and left for 5 minutes at 70 ℃.

4) Pipette the lysed cells 2-3 times to release the denatured protein and the like in the lysis step so that the protein can pass through the column well.

5) The lysed cells were transferred to a silica membrane column, and then centrifuged at 13,000 rpm for 1 minute to discard the solution and centrifugation once again.

6) Centrifuge by adding 500 μl of wash buffer to the column. In order to increase the efficiency, the solution was discarded and centrifuged once again to completely remove the washing buffer.

7) Centrifugation was performed by adding 200 μl of the elution buffer to the column, and the eluted solution was once again centrifuged in the column to obtain a greater amount of genomic DNA.

8) The extracted DNA was observed by electrophoresis on 1.2% agarose gel and stained with EtBr.

As a result, DNA concentration of 7.2 μg / μl was extracted at 5 X 10 ⁵ cells, 13.9 μg / μl at 3 X 10 ⁶ cells, 13.4 μg / μl at 5 X 10 ⁶ cells, and 1 X 10 ⁷ DNA at a concentration of 19.7 μg / μl was extracted. As a result, the cell lysis buffer of the present invention was able to extract DNA even with a small amount of cells, it can be seen that the extraction efficiency of DNA even if the number of cells is large ( Fig. 1 ).

Experimental Example 1 Restriction Enzyme Cleavage of Genomic DNA

In order to check whether the DNA extracted using the cell lysis buffer of the present invention is contaminated with proteins, RNA, and other enzymes, the extracted DNA was digested with restriction enzymes. Specifically, the DNA extracted in Example 2 was digested with restriction enzymes BamHI, EcoRI, HincII, HindIII, and NcoI, respectively, and electrophoresed in 1.2% agarose gel for staining with EtBr.

As a result, genomic DNA extracted using the cell lysis buffer of the present invention was cleaved by restriction enzymes and not contaminated with proteins, RNA, and other enzymes, so that the DNA could be used for other experiments involving enzymatic reactions. It can be seen that no problem ( FIG. 2 ).

Experimental Example 2 PCR Reaction

In order to check whether the DNA extracted using the cell lysis buffer of the present invention is abnormal in the PCR reaction, various cells were extracted in the same manner as in Example 2, and the PCR reaction was performed.

B16, HeLa, NB4, SNU1, and SNU601 cell lines were purchased from a cell line bank located at the Seoul National University Cancer Institute. Genomic DNA was extracted in the same manner as in Experiment 1, using the DNA as a template, and performing a PCR reaction using an actin primer. It was. The PCR reaction was denatured for 5 minutes at 94 ° C, repeated 30 times at 94 ° C 30 seconds, 58 ° C 30 seconds, and 72 ° C for 1 minute, and then further reacted at 72 ° C for 5 minutes. PCR products were observed after staining with EtBr by electrophoresis on 1.2% agarose gel.

As a result, when PCR was performed on DNA extracted from B16, HeLa, NB4, SNU1, and SNU601 cells using the cell lysis buffer of the present invention, the PCR reaction occurred accurately in the expected band. It was found that there was no abnormality ( FIG. 3 ).

Experimental Example 3 Isolation of Genomic DNA from Gram-negative Bacteria

It was confirmed whether the cell lysis buffer of the present invention can be used to extract genomic DNA of Gram-negative bacteria.

Salmonella was used as a Gram-negative bacterium, and a process of extracting DNA from the salmonella was as follows. The strain was cultured until the OD 600 value was 1.8, and the cell number at this time was 1 × 10 ⁸ / ml. 3 ml of the strain was collected and collected by centrifugation. After releasing the cells well, 300 µl of the cell lysis buffer was added thereto. In addition, incubated at the same temperature for 5 minutes at 70 ℃, and hung on a column and extracted DNA through a washing step and an extraction step. The extracted DNA was observed by electrophoresis on 1.2% agarose gel and stained with EtBr.

In the above, the present inventors used three Salmonella strains, and lane 1 of FIG. 4 extracted DNA using 3 ml of Salmonella enteritidis (Salmonella Enteritidis). 8.9 ㎍ / μl of DNA was extracted using 3 ml of Salmonella Gallinarum (Salmonella Gallinarum) and 3.56 ㎍ / μl of DNA extracted with 3 ml of E. coli DH-5α. Concentration of DNA was extracted. Lanes 4 to 7 of Figure 4 are to determine the temperature conditions suitable for the storage of the buffer.

As a result, it was found that the cell lysis buffer of the present invention was also suitable for genomic DNA extraction of Gram-negative bacteria, and the buffer activity did not change even when the buffer was stored at 4 ° C. and room temperature ( FIG. 4 ).

Experimental Example 4 Amount of Cell Lysis Buffer and Separation Efficiency According to the Number of Cells

The appropriate amount of cell lysis buffer of the present invention according to the number of cells and the separation efficiency at that time were confirmed.

Genomic DNA was extracted in the same manner as in Example 2 by varying the amount of the cell lysis buffer of the present invention in samples having 5 × 10 ⁵ cells, 1 × 10 ⁶ cells, 3 × 10 ⁶ cells, and 1 × 10 ⁷ cells. The extracted genomic DNA was observed by electrophoresis on 1.2% agarose gel and stained with EtBr.

As a result, as with lane 1 of 5 5 X 10 7.2 ㎍ / ㎕ concentration of the addition 150 ㎕ cell lysis buffer ^of the ^five cells were DNA is extracted, 5 X 10 when the 300 ㎕ added to the ^five cell 6.3 Μg / μl (lane 2), adding 300 μl to 1 × 10 ⁶ cells, 7.2 μg / μl (lane 3), adding 300 μl to 3 × 10 ⁶ cells, 13.9 μg / μl (lane 4), When 600 μl was added to 3 × 10 ⁶ cells, 13.4 μg / μl (lane 5) was added, and 600 μl to 1 × 10 ⁷ cells extracted 19.7 μg / μl (lane 6).

In the above results, when the number of cells is 5 X 10 ⁵ to 1 X 10 ^6, it is preferable to use 150 μl to 300 μl of the cell lysis buffer, and when the number of cells is 3 X 10 ⁶ to 5 X 10 ⁶ , the cell lysis buffer is 300. When the number of μL to 600 μL and the number of cells is 5 × 10 ⁶ to 1 × 10 ^7, it was found that 450 μL to 600 μL of the cell lysis buffer was preferably used ( FIG. 5 ).

<Comparative Example 1> Comparison of DNA Extraction Efficiency with Other Companies' Products

DNA extraction using the cell lysis buffer of the present invention and DNA extraction efficiency using other companies were compared.

3 X 10 ⁶ cells, 5 X 10 ⁶ cells, 1 X 10 ⁷ cells using a third-party product (DNeasy ^TM Tisssue Kit, Qiagen) as a comparison group compared with the case of using the cell lysis buffer of the present invention The DNA extraction efficiency in the case was compared. Genomic DNA extraction using KIAGEN's product was performed according to the product manual.

As a result, when the cell lysis buffer of the present invention was used for 3 × 10 ⁶ cells, 5 × 10 ⁶ cells, and 1 × 10 ⁷ cells, DNAs having a concentration of 13.7 μg / μl, 13.9 μg / μl and 16.6 μg / μl were obtained. When extracted from the same cells using the Kiagen company, the DNA concentrations of 11.5 μg / μl, 13.1 μg / μl, and 9.4 μg / μl were extracted ( FIG. 6 ).

In the above results, the DNA separation efficiency was higher when using the cell lysis buffer of the present invention than the KIAGEN product, especially when the KIAGEN product was used as compared with the cell lysis buffer of the present invention when the number of cells was large. It was found that this drastically lowered.

As described above, the cell lysis buffer of the present invention simultaneously performs cell lysis, protein separation, and RNA removal, thereby reducing the time required for extracting DNA, and thus reducing the time required to process a large amount of sample, thereby reproducible. You can get results.

Claims (7)

Cell lysis buffer for DNA extraction, including Tris-Cl, EDTA, surfactant, salt of acetic acid, protease and RNA degrading enzyme. The method of claim 1, wherein the surfactant is Triton X-100, sarcosyl sulfate (sarcosyl sulfate), cell lysis buffer for DNA extraction, characterized in that selected from the group consisting of. According to claim 1, wherein the acetate is ammonium acetate (ammonium acetate) or sodium acetate (sodium acetate) characterized in that the cell lysis buffer for DNA extraction. The method of claim 1, wherein the Tris-Cl is 0.1 to 0.5 M concentration, EDTA is 0.01 to 0.05 M concentration, surfactant is 0.5 to 3% by weight, ammonium acetate is 1.5 to 5.4 M concentration, proteolytic enzyme is A cell lysis buffer for DNA extraction, characterized in that the protease K at a concentration of 10 to 50 µg / ml, and the RNA degrading enzyme is RNAase A at a concentration of 10 to 50 µg / ml. The method according to claim 4, wherein Tris-Cl is 0.25 M concentration, EDTA is 0.05 M concentration, Triton X-100 is 1 wt%, ammonium acetate is 3.5 M concentration and protease K is 50 μg / ml concentration. , RNA lyase A is 50 ㎍ / ㎖ concentration cell lysis buffer, characterized in that. 1) lysing the cells by adding the cell lysis buffer for DNA extraction of any one of claims 1 to 5; 2) transferring the lysed cells of step 1 to a column to fix DNA; 3) washing the column of step 2; And 4) DNA extraction method comprising the step of recovering DNA from the column. 7. The method of claim 6, wherein the column of step 2 is selected from a glass fiber column and a silica membrane column.