KR20190033263A - Extracellular vesicles lysis buffer and Method for extraction nucleic acids using thereof - Google Patents

Abstract

The present invention relates to an extracellular vesicle lysis buffer for DNA extraction comprising 1 to 6 M of guanidine-HCl, Tris-HCl and a detergent; and a nucleic acid extraction method using the lysis buffer. Analysis of DNA extracted by the extracellular vesicle lysis buffer can be used not only for diagnosis of various diseases, but also in various fields such as development of new drugs, preliminary tests for viral or bacterial infections, and forensic medicine.

Description

Extracellular vesicle lysis buffer and method for extracting nucleic acids using the same [

The present invention relates to an extracellular vesicle dissolution buffer for DNA extraction comprising 1 to 6 M of guanidine-HCl, Tris-HCl and a detergent.

Recently, there has been an increasing demand for manipulation and biochemical analysis of biological samples for the purpose of healing or preventing human diseases by analyzing the causes of diseases at gene level based on the result of human genome research. In addition to the diagnosis of diseases, techniques for extracting and analyzing nucleic acids from biological samples or samples containing cells are required in various fields such as development of new drugs, preliminary examination for viruses or bacterial infections, and forensic medicine.

On the other hand, extracellular vesicles are nanosecomposites composed of a double lipid membrane and having a size of 50-1000 nm. These extracellular vesicles are composed of substances having biological activity such as DNA, RNA and protein. They are secreted from the cell membrane, It has been found that it plays a very important role in the communication of In particular, in cancer cells, the extracellular endoplasmic reticulum is secreted much more than normal cells, and the extracellular endoplasmic reticulum secreted from cancer cells is called extracellular endoplasmic reticulum derived from cancer. Recent studies have revealed that cancer- Inhibition, metastasis, and angiogenesis. In addition, the extracellular endoplasmic reticulum is surrounded by a double lipid membrane, and major biomaterials such as nucleic acid and protein such as RNA as well as DNA present in the inside are stored very stably to provide an innovative basis for the diagnosis of cancer and the development of biomarker It is attracting attention. The extracellular endoplasmic reticulum is known to be present in body fluids that occur in most of our bodies, such as blood, urine, saliva, breast milk, pleural fluid, ascites, cerebrospinal fluid, and the like. Therefore, its clinical usefulness is very high. Efforts to develop a method for diagnosing various diseases as well as cancer by separating and analyzing the extracellular defoaming agent are being actively carried out internationally.

Extracellular endoplasmic reticulum can be separated from body fluids such as blood, urine, pleural fluid, and bronchoalveolar lavage fluid by various methods such as ultracentrifugation centrifugation and aqueous two-phase system.

Korean Patent No. 10-0454869 relates to a cell lysis buffer for DNA extraction and a DNA extraction method using the same, wherein Tris-Cl, EDTA, a detergent, a salt of acetic acid, It has been found that the steps and time required for DNA extraction are reduced by using a cell lysis buffer for DNA extraction containing an RNA degrading enzyme.

However, there have been no reports on buffers for dissolving extracellular endoplasmic reticulum and nucleic acid extraction methods using them.

The present invention provides an extracellular vesicle dissolution buffer for DNA extraction comprising 1 to 6 M of guanidine-HCl, Tris-HCl and a detergent.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides an extracellular infant dissolution buffer for DNA extraction comprising 1 to 6 M of guanidine-HCl, Tris-HCl and a detergent.

The surfactant may be anionic or nonionic.

When the surfactant is an anionic surfactant, it may be prepared from the group consisting of ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SLS) and sodium dodecyl sulfate (SDS) And may be included at a concentration of 0.01 to 1%.

When the surfactant is a nonionic surfactant, it may be selected from the group consisting of NP-40, Tween 20 and Triton X-100, and may be contained at a concentration of 10 to 30%.

The extracellular ER lysis buffer may further comprise a protease K or a ribonucleolytic enzyme (RNase).

In one embodiment of the present invention, there is provided a method of treating a cancer cell, comprising: (a) separating extracellular endoplasmic reticulum from a cell culture fluid or body fluid; (b) adding the lysis buffer of any one of claims 1 to 6 to the extracellular vesicle to dissolve the cells; And (c) transferring the dissolved cells to a column to extract DNA; And a nucleic acid extracting method.

The body fluid of step (a) may be any one selected from the group consisting of blood, urine, pleural fluid, feces, sputum, and bronchoalveolar lavage fluid.

(d) using the DNA extracted in the step (c) for genome inspection.

The present invention relates to an extracellular vesicle dissolution buffer for DNA extraction comprising 1 to 6 M of guanidine-HCl, Tris-HCl and a detergent, wherein the dissolution buffer is used to transform extracellular vesicle DNA When extracted, there is an advantage that the quality and quantity of DNA is superior to that of extracting extracellular vesicle DNA using a cell lysis buffer. Therefore, by analyzing the DNA extracted using the extracellular ER lysis buffer according to the present invention, it can be efficiently used not only for diagnosis of various diseases, but also in various fields such as development of new drugs, preliminary examination for viruses or bacterial infection, and forensic medicine .

Fig. 1 is a schematic representation of the process of extracellular ER separation.
Figure 2 shows the size and amount of extracellular ER DNA of a cell line using an extracellular ER lysis buffer.
FIG. 3 shows the results of showing the size and amount of extracellular ER DNA of a cell line using a cell lysis buffer.
4 is a result of electrophoresis of extracellular endoplasmic reticulum DNA of a cell line.
Figure 5 shows the size and amount of extracellular ER DNA isolated from blood using an extracellular ER lysis buffer.
Figure 6 shows the size and amount of extracellular vesicle DNA isolated from blood using a cell lysis buffer.
7 is a result of electrophoresis of extracellular vesicle DNA isolated from blood.
8 shows the EGFR mutation amplification curve of the extracellular vesicle DNA of the cell line.
Figure 9 shows the EGFR mutation amplification curve of extracellular ER DNA isolated from blood.
FIG. 10 shows the results of comparing the amplification curves Ct values of the mutant gene according to the dissolution buffer.

The present inventors prepared a lysis buffer capable of extracting DNA present in the extracellular endoplasmic reticulum isolated from cell culture fluids or body fluids, and quantified the amount and quality of extracellular endoplasmic reticulum DNA extracted using the lysis buffer, , The present invention was completed.

The term "extracellular < / RTI > endoplasmic reticulum" in the present specification is an endoplasmic reticulum produced in a cell and secreted outside the cell, including, but not limited to, exosome, microvesicle, microparticle and the like.

The term " bronchoalveolar lavage fluid " in the present specification may be a liquid obtained by injecting, sucking, and washing physiological staining on the peripheral side in a regional bronchial bronchus using a bronchial fibrosisoscope. The bronchoalveolar lavage fluid is collected by analyzing and analyzing physiologically active substances and metabolites present in cellular components such as macrophages, lymphocytes, polynuclear leukocytes, and the like present in the bronchopulmonary system, and detecting metabolism of the lungs Can be used.

The term "cell lysate" as used herein refers to a mixture comprising components of cells obtained by destroying cells. The cell lysate can be prepared by adding a lysis buffer to the cell-containing solution. In the present invention, an extracellular lysate buffer was used, and a cell lysis buffer was used for comparison with the extracellular lysate buffer. The lysis buffer may be made of various materials such as a commercialized product. In addition to the dissolution buffer, proteinase K, a protein hydrolyzing enzyme, or RNase, a ribonucleolytic enzyme, may be further added.

Hereinafter, the present invention will be described in detail.

The present inventors prepared a lysis buffer capable of extracting DNA present in the extracellular endoplasmic reticulum isolated from cell culture fluids or body fluids, and quantified the amount and quality of extracellular endoplasmic reticulum DNA extracted using the lysis buffer, , The present invention was completed.

The term "extracellular < / RTI > endoplasmic reticulum" in the present specification is an endoplasmic reticulum produced in a cell and secreted outside the cell, including, but not limited to, exosome, microvesicle, microparticle and the like.

The term " bronchoalveolar lavage fluid " in the present specification may be a liquid obtained by injecting, sucking, and washing physiological staining on the peripheral side in a regional bronchial bronchus using a bronchial fibrosisoscope. The bronchoalveolar lavage fluid is collected by analyzing and analyzing physiologically active substances and metabolites present in cellular components such as macrophages, lymphocytes, polynuclear leukocytes, and the like present in the bronchopulmonary system, and detecting metabolism of the lungs Can be used.

The term "cell lysate" as used herein refers to a mixture comprising components of cells obtained by destroying cells. The cell lysate can be prepared by adding a lysis buffer to the cell-containing solution. In the present invention, an extracellular lysate buffer was used, and a cell lysis buffer was used for comparison with the extracellular lysate buffer. The lysis buffer may be made of various materials such as a commercialized product. In addition to the dissolution buffer, proteinase K, a protein hydrolyzing enzyme, or RNase, a ribonucleolytic enzyme, may be further added.

Hereinafter, the present invention will be described in detail.

Extracellular vesicle dissolution buffer for DNA extraction

The present invention provides an extracellular infant dissolution buffer for DNA extraction comprising 1 to 6 M of guanidine-HCl, Tris-HCl and a detergent.

In the dissolution buffer according to the present invention, the guanidine-HCl may be contained at a concentration of 1 to 6 M, preferably at a concentration of 3 to 5 M, but is not limited thereto. At this time, when the concentration of guanidine-HCl is less than the above range, the concentration and purity of the extracted DNA are lowered. Also, the dissolution buffer may contain Tris-HCl as a pH buffer, and the Tris-HCl is preferably contained at a concentration of 0.1 to 20 mM, but is not limited thereto.

In addition, in the dissolution buffer according to the present invention, the surfactant may be an anionic or nonionic surfactant for dissolving the membrane protein. Specifically, when the surfactant is an anionic surfactant, it may be selected from the group consisting of ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SLS), and sodium dodecyl sulfate (SDS) But it is not limited thereto. When the anionic surfactant is sodium dodecyl sulfate, the sodium dodecyl sulfate may have a concentration of 0.01 to 1%, preferably 0.7 to 0.9%, but is not limited thereto. If the concentration of sodium dodecyl sulfate is less than the above range, the concentration and purity of DNA may be lowered. If the concentration of sodium dodecyl sulfate is more than the above range, the concentration and purity of DNA may be lowered. When the surfactant is a nonionic surfactant, the surfactant may be selected from the group consisting of NP-40, Tween-20, and Triton X-100, preferably Triton X-100, but is not limited thereto. When the non-ionic surfactant is Triton X-100, the concentration of Triton X-100 may be 10-30%, preferably 15-25%, but is not limited thereto. If the concentration of Troton X-100 is less than the above range, the concentration and purity of the extracted DNA may be lowered. If the concentration of Troton X-100 exceeds the above range, the solubility of Triton X-100 may be lowered.

In addition, the lysis buffer according to the present invention may further comprise a protease K or a ribonucleolytic enzyme (RNase).

As described above, the extracellular vesicle dissolution buffer according to the present invention can extract the nucleic acid contained in the extracellular vesicle by dissolving the extracellular vesicles isolated from the cell culture fluid or body fluid, Is advantageous in that a DNA having a size of 500 bp or more can be extracted in a remarkably large amount as compared with the case where the nucleic acid is extracted using the DNA.

Nucleic acid extraction method

(A) separating the extracellular endoplasmic reticulum from the cell culture fluid or body fluid; (b) adding the lysis buffer of any one of claims 1 to 6 to the extracellular vesicle to dissolve the cells; And (c) transferring the dissolved cells to a column to extract DNA; And a nucleic acid extracting method.

First, the nucleic acid extraction method according to the present invention includes a step (a) of separating extracellular endoplasmic reticulum from a cell culture fluid or body fluid. Specifically, the body fluid may be selected from the group consisting of blood, urine, pleural fluid, feces, sputum, and bronchoalveolar lavage fluid, but is not limited thereto. At this time, the extracellular endoplasmic reticulum can be separated from the body fluids by centrifugation at 500 to 1,500 x g for 5 to 20 minutes, preferably at 100,000 to 300,000 x g for 30 to 90 minutes, but is not limited thereto .

Next, the nucleic acid extracting method according to the present invention includes a step of dissolving cells by adding the dissolution buffer of any one of claims 1 to 6 to the extracellular endoplasmic reticulum (step (b)). The details of the dissolution buffer are as described above.

Finally, the nucleic acid extraction method according to the present invention includes a step (c) of transferring the dissolved cells to a column to extract DNA. Specifically, the DNA is extracted by dissolving the extracellular endoplasmic reticulum with the extracellular endoplasmic reticulum buffer according to the present invention, transferring the dissolve extracellular endoplasmic reticulum to the column, centrifuging at 5,000 to 10,000 × g for 10 to 120 seconds, , And is preferably performed by further centrifuging at 10,000 to 20,000 xg for 10 to 60 seconds, but is not limited thereto.

In addition, the nucleic acid extraction method according to the present invention may include the step of using the extracted DNA for the genome screening (step (d)). In one embodiment of the present invention, EGRF mutant gene analysis was performed using DNA extracted from extracellular endoplasmic reticulum. The extracellular vesicle DNA extracted using the extracellular vesicle dissolution buffer according to the present invention and the extracellular vesicle DNA extracted using the dissolution buffer for cell lysis were amplified by PCR reaction and the cycle value at which the amplification was started was analyzed As a result, it was confirmed that the number of mutant genes amplified when extracted with the extracellular vesicle dissolution buffer according to the present invention was remarkably increased.

As described above, the nucleic acid extraction method according to the present invention extracts DNA using the extracellular vesicle dissolution buffer according to the present invention, and the DNA extracted by the method described above, rather than the conventional DNA lysis buffer, There is an advantage that the amount and quality of extracted DNA is excellent. Therefore, by analyzing the DNA extracted by the above method, it can be used efficiently in various fields such as development of new drugs, preliminary examination for virus or bacterial infection, and forensic medicine as well as diagnosis of various diseases.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Isolation of extracellular endoplasmic reticulum using ultrafast centrifugation

1-1. Cell culture medium

The cells were centrifuged at 1000 xg for 10 minutes to remove cell and cell debris from the cell culture. Cells and cell debris were removed from the supernatant by ultrafast centrifugation at 200,000 × g for 1 hour to precipitate the extracellular endoplasmic reticulum. The supernatant was removed and the precipitate was dissolved in 200 μl of PBS buffer.

1-2. blood

The procedure of Example 1-1 was repeated except that blood was used.

Example 2. Preparation of Extracellular Epidermal Lysis Buffer

2-1. Extracellular vesicle lysis buffer for DNA extraction 1

An aqueous solution containing 6 M guanidine-HCl, 10 mM urea, 10 mM Tris-HCl, 20% Triton X-100 and 0.8% SDS was prepared.

2-2. Extracellular vesicle dissolution buffer for DNA extraction 2

The procedure of Example 1 was repeated except that the sample contained 0.1% SDS.

2-3. Extracellular vesicle lysis buffer for DNA extraction 3

1% NP-40 in the same manner as in Example 1, except that it contained 1% NP-40.

2-4. Extracellular lysate buffer for DNA extraction 4

8% NP-40 in the same manner as in Example 1. The results are shown in Table 1 below.

2-5. Extracellular lysate buffer for DNA extraction 5

1% Tween 20 in the same manner as in Example 1.

2-6. Extracellular lysate buffer for DNA extraction 6

8% Tween 20 in the same manner as in Example 1. The results are shown in Table 1. < tb > < TABLE >

[Comparative Example] Preparation of cell lysis buffer

An aqueous solution containing 6M guanidine-HCl, 10 mM urea, 10 mM Tris-HCl and 20% Triton X-100 was prepared.

[Experimental Example]

Experimental Example 1. Preparation of a solution of extracellular endoplasmic reticulum

After preparing the dissolution buffer prepared in Example 2 and the dissolution buffer prepared in Comparative Example as shown in Table 1, 200 쨉 l of the extracellular extracellular medium isolated in Example 1 was added with 20 mg / ml protease K 40 And the mixture was slowly mixed so that the contents became homogeneous, and then the solution was left at 70 for 10 minutes.

Extracellular vesicle
(Μl) Dissolution buffer
(Μl) Protease K
(Μl) Example 2-1 200 200 40 Example 2-2 200 200 40 Example 2-3 200 200 40 Examples 2-4 200 200 40 Example 2-5 200 200 40 Examples 2-6 200 200 40 Comparative Example 200 200 40

Experimental Example 2. DNA Extraction

The dissolved extracellular endoplasmic reticulum was transferred to a glass fiber column, centrifuged at 8000 xg for 1 minute, and the supernatant discarded. 500 [mu] l of wash buffer was added to the column, centrifuged at 8000 xg for 1 minute, the solution was discarded, and the procedure was repeated twice. The column was centrifuged at 12,500 xg for 30 seconds and the supernatant discarded to completely remove the wash buffer. Then, 50 쨉 l of distilled water was added to the column, followed by centrifugation at 8000 xg for 1 minute. The concentration of the separated DNA was shown in Table 2 below.

DNA concentration (ng / l) Example 2-1 41.7 Example 2-2 13.4 Example 2-3 16.4 Examples 2-4 27.2 Example 2-5 13.2 Examples 2-6 20.0 Comparative Example 15.5

Experimental Example 3. DNA Analysis

The concentration of the extracellular ER DNA was measured using a NanoDrop instrument for concentration measurement and the size of the DNA was measured using a bioanalyzer machine for DNA analysis.

As a result, the amount of DNA having a size of 1000 bp or more in the extracellular endoplasmic reticulum of Example 1-1 using Example 2 was 85 ng / μl, the amount of extracellular endoplasmic reticulum DNA in Example 1-1 using the comparative example was 24 ng / L. ≪ / RTI > As shown in Figs. 2 to 4, in the case of extracellular ER DNA extracted using the extracellular ER lysis buffer of Example 2, DNAs larger than 1000 bp in size were extracted using a cell lysis buffer of the comparative example It can be confirmed that there exist more abundant than extracellular vesicle DNA.

The amount of DNA having a size of 1000 bp or more in the extracellular endoplasmic reticulum of Example 1-2 using Example 2 was 23 ng / μl, and the amount of extracellular endoplasmic reticulum DNA in Example 1-2 using the comparative example was 12 ng / Respectively. In addition, as shown in Figs. 5 to 7, in the case of the extracellular vesicle DNA extracted using the extracellular vesicle dissolution buffer of Example 2, the DNA having a size larger than 500 bp was extracted using the cell lysis buffer of the comparative example And more abundant than the outer endoplasmic reticulum DNA.

Experimental Example 3. Analysis of EGFR mutant gene

EGFR mutant gene analysis was performed to analyze the DNA quality and quantity according to the type of lysis buffer.

70 ng of the extracellular ER DNA extracted from Example 4 was mixed with a primer, a PNA probe for suppressing wild-type gene amplification, and a PCR mix containing a fluorescent material to prepare 20 uL of PCR reaction solution. The reaction was carried out in CFX96, a real-time analysis PCR machine, for 94 cycles of 94, 30, 70, 20, and 63 for 30 cycles, and fluorescence values were measured for each cycle. In this case, when 94 heat is applied, denaturation in which one DNA is separated into two strands, and then the temperature is lowered to 70, the primer and the polymerase are bound to a specific site of the single strand DNA, do. The amount of the extracted extracellular ER DNA to be amplified can be increased to an enormous amount, and the amplification curve is measured with the measured fluorescence value and the cycle value (Ct) at which the amplification is started is analyzed to determine the presence or absence of the mutation gene Respectively.

As a result, as shown in FIGS. 8 and 9, the Ct value of the extracellular ER DNA extracted with the extracellular ER lysis buffer was lower than the Ct value of the extracellular ER DNA extracted with the cell lysis buffer, The mutant gene was not amplified in the extracted extracellular vesicle DNA. That is, the Ct value of the extracellular ER DNA extracted with the extracellular ER lysate buffer was 28.58, the Ct value of the extracellular ER DNA extracted with the lysis buffer was 31.5, and the extracellular ER DNA extracted with the extracellular ER lysate buffer was mutated And 7.6 times more genes are present.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (10)

Extracellular vesicle dissolution buffer for DNA extraction comprising 1 to 6 M of guanidine-HCl, Tris-HCl and detergent.
The method according to claim 1,
Wherein the surfactant is anionic or nonionic
Extracellular vesicle dissolution buffer for DNA extraction.
3. The method of claim 2,
When the surfactant is an anionic surfactant, the surfactant is preferably selected from the group consisting of ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SLS), and sodium dodecyl sulfate
Extracellular vesicle dissolution buffer for DNA extraction.
3. The method of claim 2,
The surfactant is selected from the group consisting of NP-40, Tween 20 and Triton X-100 when the surfactant is a nonionic surfactant.
Extracellular vesicle dissolution buffer for DNA extraction.
The method of claim 3,
Wherein the surfactant is contained in a concentration of 0.01 to 1%
Extracellular vesicle dissolution buffer for DNA extraction.
5. The method of claim 4,
Wherein the surfactant is contained in a concentration of 10 to 30%
Extracellular vesicle dissolution buffer for DNA extraction.
The method according to claim 1,
Wherein the extracellular ER lysis buffer further comprises a protease K (proteinase K) or a ribonucleolytic enzyme (RNase)
Extracellular vesicle dissolution buffer for DNA extraction.
(a) separating extracellular endoplasmic reticulum from cell culture fluids or body fluids;
(b) adding the lysis buffer of any one of claims 1 to 6 to the extracellular vesicle to dissolve the cells; And
(c) transferring the dissolved cells to a column to extract DNA; / RTI >
9. The method of claim 8,
Wherein the body fluid of step (a) is any one selected from the group consisting of blood, urine, pleural fluid, feces, sputum, and bronchoalveolar lavage fluid
Nucleic acid extraction method.
9. The method of claim 8,
(d) using the DNA extracted in the step (c) for genome inspection
Nucleic acid extraction method.

Images