JPH06289016A - Dna extraction reagent from living thing sample, dna extraction method using reagent, and dna extraction kit - Google Patents

Abstract

PURPOSE:To extract DNA with a high yield and a high purity with a simple operation quickly by allowing an aqueous solution including at least one type selected from a group consisting of protein denaturant and a reducer and then a surface-active agent and chelator to be made of a protein dissolution agent. CONSTITUTION:The title DNA extraction reagent is an aqueous solution including a protein denaturant as an essential ingredient. A chaotropic agent is desirable as the protein modification agent. Further, the reagent includes at least one type selected from a group consisting of a reducer, a surface-active agent, and chelator. Thiol reducer which can cut disulfide coupling, one which does not cause settlement in the aqueous solution with a reagent composition, and one which has a capacity for chelating divalent metal ion are desirable as the reducer, the surface-active agent, and chelator, respectively. The reagent is used with its pH being from weak acid to weak alkali.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a reagent for extracting DNA from a biological sample useful in the field of biotechnology such as genetic engineering, biochemistry and clinical tests using DNA as a material, a method for extracting DNA using the reagent, And a DNA extraction kit using the reagent.

[0002]

2. Description of the Related Art In order to extract DNA from biological samples such as cells, tissues, bacteria and viruses, the following three-step operation has been most popular.

1. Stage 1: Cell lysis and DNA solubilization DNA solubilization begins with the lysis of cells using ionic or nonionic detergents. However, since cell lysis accelerates the decomposition of the extracted DNA by nuclease, etc., a chelating agent such as ethylenediaminetetraacetic acid disodium dihydrate (EDTA) is used in the cell lysate to simultaneously suppress the nuclease activity. There is a need to do.

Secondly, since DNA exists in the form of a DNA-protein complex, it becomes necessary to release the DNA from the protein. Proteinase K, which is a non-specific proteolytic enzyme, is generally used for this. At the same time, it is considered that the effect of this proteinase K is enhanced by simultaneously forming a micelle with the protein bound to the DNA by the surfactant and peeling the protein from the DNA.

2. Second step: Phenol treatment Phenol treatment is carried out for the purpose of denaturing and separating and removing proteins derived from the sample and enzymes (proteinase K, ribonuclease, etc.) added to the sample during the purification process.
Here, some nucleases and other proteins are denatured and appear as precipitates in the intermediate layer between the phenol and water layers. By this treatment, the DNA is extracted in the upper layer, that is, the aqueous layer, and the separation from the protein component is achieved.

Besides phenol, it is also possible to use chloroform, phenol-chloroform, mixtures of chloroform with other organic solvents.

For the purpose of destroying nuclear proteins, chaotropic agents represented by guanidine hydrochloride and guanidine thiocyanate are also known. These reagents are used at high molar concentrations and are widely used because the destruction of proteins also serves to suppress the activity of nucleases. However, it seems that the chaotrope is generally mainly used for RNA extraction as a ribonuclease inactivating agent.

3. Step 3: Alcohol precipitation Alcohol precipitation is carried out for the purpose of concentrating DNA in the solution and removing contaminants such as phenol, salts and nucleotides, under the presence of 0.1-0.5M monovalent cation. Done. As the alcohol, 2 volumes of ethanol or 0.6 volumes of isopropanol (2-propanol) are used. By this operation, DNA is recovered as a white precipitate.

[0009]

However, in such a conventional method, enzyme treatment is indispensable, and DN is used.
Since it is difficult to separate A from other proteins, column processing and the like took time, and the operation was complicated. In addition, the use of organic solvents such as phenol and chloroform is disadvantageous and harmful to the human body. Further, when such an organic solvent is used,
There is also a problem in that the extract has to be transferred to another container and a large number of tubes are used.

Therefore, the present invention can be carried out in a single tube capable of extracting high-yield and high-purity DNA by a simple operation in a short time of about 60-120 minutes, so that it can be used in the clinical field. It is economical because it can prevent contamination (contamination), uses a small number of tubes, and is relatively inexpensive to use reagents. It does not use dangerous organic solvents such as phenol, chloroform, or their mixtures that are harmful to the human body. It is an object of the present invention to provide a DNA extraction reagent from a novel biological sample that does not require enzyme treatment with an enzyme, a DNA extraction method using the reagent, and a DNA extraction kit containing the reagent.

[0011]

The present inventors have devised the composition and concentration of each component of the reagent of the present invention when extracting DNA from cells, tissues, bacteria, viruses, etc. The inventors have found that DNA can be easily and reproducibly formed from nuclei, virus particles, and the like, and that the number of tubes used can be reduced, and the present invention has been completed.

The present invention is an aqueous solution containing the following composition: (1) a protein denaturant and (2) a reducing agent, a surfactant and a chelating agent, which has a weak pH. Provided is a reagent for extracting DNA from a biological sample, which comprises a protein lysing agent that is acidic to weakly alkaline.

The present invention also includes the following operations: a) rough extraction of cells or nuclei from a biological sample, b) the following composition: (1) protein denaturant and (2) reducing agent, surfactant and chelating agent It is an aqueous solution containing at least one selected from the group consisting of, and denatures and solubilizes cells and nuclei and other components and inclusions by adding a protein solubilizer having a weakly acidic to weakly alkaline pH. And c) a method of extracting DNA from a biological sample, which comprises performing the alcohol precipitation by adding the lower alcohol as it is without performing the DNA extraction with the organic solvent.

The present invention is further selected from the group consisting of the following reagents: a) cell lysate, b) the following compositions: (1) protein denaturants and (2) reducing agents, detergents and chelating agents. An aqueous solution containing at least one kind, which is a protein solubilizer having a weakly acidic to weakly alkaline pH, and c) an extracted D consisting of a mixed solution of a chelating agent and a buffer solution
From a biological sample consisting of a solvent for redissolving NA, D
A kit for extracting NA is provided.

DNA extraction using the reagent of the present invention is carried out by the following procedure.

(A) Crude extraction of cells or nuclei from a biological sample is performed by a known method. When the biological sample is blood, RCLB (Red Cell Lysis) described later is used.
Buffer), a method using dextran, a method of removing a buffy coat and the like can be used. RCLB even when the biological sample is a cultured cell
Can be used. In addition, when it is considered that the target sample is a trace amount or the amount of contaminating protein component is a trace amount, or when DNA is extracted from virus particles in serum, this operation is not performed and the following (b) operation is performed. It can be carried out.

(B) Next, denaturing and solubilizing the isolated cells or nuclei is performed using the DNA extraction reagent of the present invention.

The DNA extraction reagent of the present invention is an aqueous solution containing a protein denaturant as an essential component. As the protein denaturing agent, a chaotropic agent is preferable, and guanidine hydrochloric acid and guanidine thiocyanic acid are particularly preferable.

The DNA extraction reagent of the present invention further contains at least one selected from the group consisting of a reducing agent, a surfactant and a chelating agent. The reducing agent is preferably a thiol-based reducing agent capable of cleaving a disulfide bond.
-Mercaptoethanol, dithiothreitol and the like are particularly preferred. The surfactant is preferably one that does not cause precipitation in the aqueous solution of the reagent composition of the present invention, or one that can be redissolved by any method even if precipitation occurs, such as sodium N-lauroylsarcosine, polyoxyethylene. (10) Octyl phenyl ether (Tr
iton X-100), polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene (9) octyl phenyl ether (NP-40).
Are particularly preferable. The chelating agent is preferably one having the ability to chelate a divalent metal ion,
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDT
A) and the like are particularly preferable. Which of these is used depends on the type of biological sample and the nature of the DNA obtained.

An example of the DNA extraction reagent of the present invention and a preferred concentration range (final concentration in the mixed solution) are shown below: Guanidine hydrochloride 3-8M 2-mercaptoethanol 0.05-0.4M N-lauroylsarcosine sodium 0.1-2% (W / V) ethylenediaminetetraacetic acid disodium dihydrate 1-100 mM The above reagent is used in a pH range from weakly acidic to weakly alkaline.

In order to denaturate and solubilize cells or nuclei using the extraction reagent of the present invention, the reagent of the above composition is added to the crude extract obtained in (a) to avoid physical cleavage of DNA. Do this only with gentle agitation. Alternatively, a protein denaturant, which is an essential component for the extraction reagent of the present invention, may be added, and then other necessary constituent components may be sequentially added.

After the stirring, if the incubation is carried out at about 55 ° C. for about 30 minutes, the potency of the reagent solution is further enhanced and the DNA releasing ability is enhanced.

(C) Next, a lower alcohol such as isopropanol or ethanol is added to precipitate the DNA. This operation is called alcohol precipitation. When isopropanol is used, an equal volume or more is added, and when ethanol is used, ethanol is added so that the final concentration is 65% or more. Isopropanol is more preferred because it is easy to operate.

In order to recover the precipitated DNA, centrifugation is performed and the supernatant is discarded, or when the liquid volume is large, the DNA is sucked and collected with a dropper or a pipette with a tip cut off.

Next, DN obtained by alcohol precipitation
A is washed with 70% ethanol, vacuum-dried for about 5 minutes to remove the alcohol content, and then redissolved in an appropriate solution and stored to be ready for use. A suitable solution for redissolving is, for example, a mixed solution of a chelating agent and a buffer solution, and a TE solution having the following composition is particularly preferable (concentration is the final concentration in the mixed solution): 10 mM Tris-HCl buffer (pH 8.0) ) 1 mM EDTA solution (pH 8.0) The above method according to the invention can also be used in combination with conventional techniques. For example, after being dissolved in the reagent according to claim 2, extraction with phenol or chloroform and alcohol precipitation are performed to extract genome D from blood.
Extract NA. See also Birnboi
m) H. C. And Dolly J. et al. Alkali-S
DS method (Birnboim HC and Doly J. (1979) Nucleic A
cids Res. 7 , 1513), extraction of plasmid DNA from Escherichia coli by adding the reagent according to claim 2 to the “plasmid crude solution” from which Escherichia coli DNA and high protein have been removed, and performing alcohol precipitation. The method of the present invention can also be applied to the above.

The kit of the present invention contains a cell lysing solution and a mixed solution of a chelating agent and a buffer solution in addition to the above protein lysing agent.

The cell lysate is obtained by isolating mainly nuclear components in blood, tissue or culture medium from other coexisting cell constituents.
Any known cell lysate can be used,
When the biological sample is blood, for example, RCLB solution [final concentration 0.32M sucrose, 1% (V /
V) Polyoxyethylene (10) octyl phenyl ether, 5 mM magnesium chloride, 12 mM tris-hydrochloric acid buffer solution (pH 7.5) in the composition] can be included in the kit.

As the mixed solution of the chelating agent and the buffer solution, for example, a TE solution having the above composition can be used.

The action of the present invention will be discussed from the action and effect of each component constituting the reagent. A chaotropic agent such as guanidine hydrochloride, which is one of protein denaturants, has a protein denaturing solubilizing action and a nuclease activity inhibiting action. Therefore, the isolated cell or nucleus is disrupted or solubilized in the membrane structure, resulting in DN in the nucleus.
A is released in the reagent solution.

A reducing agent typified by 2-mercaptoethanol reductively cleaves a disulfide bond, which is a bond between sulfur atoms in a protein, destroys the higher-order structure of the protein and enhances solubility, and this action also causes protein denaturation. It is considered to have a role of enhancing the effect of the agent.

Surfactants such as sodium N-lauroylsarcosine form micelles with proteins and lipids attached to DNA and solubilize them to give D.
It has the function of releasing NA.

The divalent chelating agent such as EDTA strongly functions to chelate a divalent metal ion such as Mg ²⁺ necessary for expressing the activity of nuclease, and has a function of preventing decomposition of extracted DNA by nuclease.

The assay of DNA extracted by the method of the present invention can be carried out by the following items.

(1) Extraction amount The amount of the extracted DNA is obtained by dissolving it in the above TE solution, measuring the absorbance at a wavelength of 260 nm with a spectrophotometer (optical path length: 1 cm), and calculating from the conversion formula of 1OD = 50 μg / ml. And Normally, 1-5 ml of healthy human blood is 20-5
About 0 μg of DNA is extracted.

(2) Determination of Purity [Protein Contamination] The extracted DNA is dissolved in a TE solution, and then the peak absorption wavelength of nucleic acid is 26 by a spectrophotometer.
0 nm, the peak absorption wavelength of protein is 280 nm
The absorbance at was measured and the ratio A ₂₆₀ / A ₂₈₀ was calculated. Usually, the value of A ₂₆₀ / A ₂₈₀ of a highly pure DNA solution is 1.8-2.0.

[Mixture of Polysaccharide] The extracted DNA was dissolved in a TE solution and then analyzed by a spectrophotometer at 260 nm which is the peak absorption wavelength of nucleic acid and 234n which is the peak absorption wavelength of polysaccharide.
The absorbance at m was measured and the ratio A ₂₃₄ / A ₂₆₀ was calculated. Generally, it is considered that the smaller the value of A ₂₃₄ / A ₂₆₀ is, the less the content of polysaccharides is.

[RNA contamination] 0.5 μl of extracted DNA
0.6g agarose gel electrophoresis, and use ethidium bromide to check for the presence of stained bands other than DNA (because RNA has a smaller molecular weight than genomic DNA, so it is detected as a band on the low molecular side). Do by observing. Whether the extracted DNA is treated with ribonuclease (mixed with DNA: ribonuclease (weight ratio) = 2: 1, incubated at 37 ° C. for 1 hour) and not incubated in the same manner, and then electrophoresed to determine whether the stained image changes By observing.

[Nuclease Contamination] The extracted DNA was incubated at 37 ° C. for 4 times with buffers for nucleases having several salt concentrations.
It is carried out by incubating for a period of time and performing electrophoresis in the same manner as in the method of [contamination of RNA], and observing whether or not there is a change in electrophoretic image with the unincubated one as a control.

(3) Measurement of molecular weight of extracted DNA 0.5 μg of extracted DNA was electrophoresed under the same conditions as the electrophoresis conditions described in the above [RNA contamination] and compared with the band of the molecular weight marker electrophoresed on the same gel. And measure. The DNA extracted by the method of the present invention is 20 Kb or more.

The DNA extracted by the method of the present invention has high purity and can be applied to various fields of genetic engineering. For example, D extracted by the method of the present invention
NA (PCR) is one of the gene amplification technologies.
erase chain reaction) and the like. Further, since the extracted DNA can be cleaved by various restriction enzymes, when a commercially available restriction enzyme is purchased, it can be cleaved by the restriction enzyme using various buffer solutions. Furthermore, it is possible to detect the extracted DNA directly or after electrophoresis, immobilized on a solid phase, hybridized with a probe complementary to the sequence of the extracted DNA, and detected. As described above, the application range of the present invention is extremely wide.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

[0042]

【Example】

Example 1. Pretreatment of Biological Sample The following method can be used to isolate cells, nuclei, etc. from various biological samples. It should be noted that if the target sample is in a very small amount, the amount of contaminated protein components, etc. is in a very small amount, or if the viral DNA is extracted from the serum, this operation is not performed and the process can proceed to the next step.

When the subject is not blood, RNA contamination may occur, so that ribonuclease treatment may be necessary.

(1) Method using RCLB a. When the subject is blood, RCLB having the above-mentioned composition is used. However, this operation is preferably performed in ice water to prevent decomposition of DNA by nuclease. The same applies to other methods when RCLB is used.

RCLB is used for the sample blood and 2 for the sample blood.
Add twice the volume and stir gently. Next, 2000g, 5
Centrifuge for 4 minutes at 4 ° C, discard the supernatant and collect the precipitate.

B. When the Subject is Cultured Cells Cultured cells in the culture solution are centrifuged at 2000 g for 5 minutes at 4 ° C., the supernatant is removed, and the precipitate is collected. 10 ⁵ -10 ⁷ is suitable as the number of cells.

The cells were redispersed in 0.2 ml saline, 0.4 ml RCLB was added and gently stirred,
Centrifuge at 2000 g for 5 minutes at 4 ° C. Remove the supernatant and collect the precipitate. This operation is repeated once again, and the precipitate is similarly collected.

C. When the target is a tissue, a tissue section is frozen and crushed with liquid nitrogen or the like, RCLB is added according to the amount of tissue used, and gently stirred, then 200
Centrifuge at 0 g for 5 minutes at 4 ° C. Remove the supernatant and collect the precipitate. This operation is repeated once again, and the precipitate is similarly collected.

(2) Method using dextran (subject: blood) A physiological saline solution (0.9% (W /
V) Aqueous sodium chloride solution, sterilization required) 1, 6%
0.4 of physiological saline containing (W / V) dextran is added, mixed gently and allowed to stand at room temperature for 30 minutes. Since this solution is separated into two layers, the supernatant is collected and
Centrifuge for minutes, remove the supernatant and collect the precipitate.

This sediment is washed with a physiological saline solution having the same volume as the volume of blood used, and then centrifuged to recover the sediment. In addition,
RCLB may be used at this stage instead of saline. This operation is performed twice.

(3) Method of removing buffy coat (target: blood) Blood containing anticoagulant was centrifuged at 500 × g for 5 minutes,
Collect the buffy coat. RCLB is added to this in an amount equal to the volume of blood used, gently stirred, and similarly centrifuged to collect the precipitate. Repeat this operation again and collect the precipitate in the same manner.

(4) Extraction of plasmid DNA from Escherichia coli A culture solution containing E. coli was placed in a 1.5 ml tube, centrifuged at 8000 g for 2 minutes at 4 ° C., the supernatant was removed, and E. coli was precipitated. Collect as. 100 μl of this
25 mM Tris-HCl buffer (pH 8.0), 50 m
Resuspend by adding M glucose, 10 mM EDTA. Add 200 μl of 0.2N sodium hydroxide,
Add 1% SDS, mix gently and leave on ice for 5 minutes. Centrifuge at 8000g for 10 minutes at 4 ° C and collect the supernatant. Then, 5 μl of ribonuclease solution (1 mg / ml) is added and mixed, and the mixture is incubated at 37 ° C. for 30 minutes.

Example 2. Release of DNA (denaturation of protein components, solubilization) As a protein solubilizer, a reagent consisting of an aqueous solution having the following composition was prepared: 4M guanidine hydrochloride 0.2M 2-mercaptoethanol 0.5% N-lauroylsarcosine sodium 50 mM ethylenediamine. Disodium tetraacetate dihydrate The above protein solubilizer is added to the cells, nuclei, etc. isolated in Example 1, and the precipitate is dissolved by gently stirring. In the case of plasmid and virus in serum, the above protein lysing agent is added to the extract and mixed.

The amount of proteolytic agent added will depend on the starting blood volume, regardless of isolation method, if the subject is blood. When the target is an organization, the amount used depends on the type of organization. In the case of plasmid DNA, 0.5-1 ml is used in the above method. Table 1 below shows the amounts of protein lysing agents used.

Table 1 Then incubate at 55 ° C. for 30 minutes. After incubation, the mixture is allowed to stand at room temperature, isopropanol of the same volume as the added protein lysing agent is added, and the mixture is gently stirred to precipitate and precipitate the DNA. The supernatant is removed, the precipitated DNA is washed twice with 1 ml of 70% ethanol, and the supernatant is removed, followed by vacuum drying for 5 minutes to obtain an extracted DNA. The obtained DNA is redissolved in an appropriate amount of TE solution.

Example 3. Amount and Properties of DNA Extracted from Human Blood The amount of DNA extracted from the blood of a healthy person (Sample No. 1-18) by the method of Example 2 is described in the section of Assay (1) Extracted amount of extracted DNA. It was calculated by the method described above. Also, by the method described in the section of (2) Purity determination of the extracted DNA, the protein contamination by A ₂₆₀ / A ₂₈₀ and the polysaccharide contamination by A ₂₃₄ / A ₂₆₀ were measured to extract the extracted DNA. Was assayed for purity.

In order to clarify the difference due to the difference in the pretreatment and the difference in the extraction method, Table 2 shows the extraction results by the various methods described below. The abbreviations in the table have the following meanings: R: Method using RCLB D: Method using dextran B: Method using buffy coat (in this method, the buffy coat part is strictly separated) P: Conventional method using phenol-chloroflum after collecting buffy coat Table 2 Sample pretreatment A ₂₆₀ / A ₂₈₀ A ₂₃₄ / A ₂₆₀ Extraction amount Extraction amount No. Blood volume Extraction method (μg) (μg / blood ml) 1 5ml R 1.865 0.453 124.4 24.9 25ml R 1.822 0.472 246.6 49.3 3 3ml R 1.861 0.429 105.8 35.3 4 3ml R 1.838 0.436 84.8 28.3 5 1ml R 1.868 0.439 37.4 37.4 6 1ml R 1.957 0.414 33.3 33.3 7 0.5ml R 1.695 0.429 14.1 28.2 8 0.5ml R 1.702 0.480 12.7 25.5 9 0.3ml R 1.841 0.376 9.72 32.4 10 0.3ml R 1.835 0.369 10.3 34.3 11 0.1ml R 1.908 0.336 3.00 30.0 12 0.1ml R 1.783 0.250 2.59 25.9 13 1ml D 1.913 0.491 23.1 23.1 14 1ml D 1.872 0.617 36 .3 36.3 15 1 ml B 1.801 --6.8 6.8 16 16 1 ml B 1.922 --2.6 2.6 17 2 ml P 1.887 0.440 80.8 40.4 18 6 ml P 1.847 0.445 54.7 9.1 As is apparent from the table, the DNA obtained by the present invention
Showed that the extraction amount and the purity were equal to or better than the conventional method.

Example 4. Molecular Weight of Extracted DNA, Assay of Various Contaminants and Treatment with Restriction Enzymes Using the DNA extracted by the method of Example 2 from blood (2 samples) of a healthy person, various buffers (H buffer, M buffer, L
Buffer) and then subjected to agarose gel electrophoresis. The obtained results are shown in FIG. In the figure, the left lane represents the test group, and the right lane represents the control group (the one to which a buffer solution is added but not incubated). If nuclease is mixed in the extracted DNA, DN
A should autolyze and the fragments should appear on electrophoresis. As is clear from the figure, no difference was observed between the test group and the control group (lane H-1, 2, lane M-1,
2, lane L-1, 2), DN obtained by the method of the present invention
It shows that nuclease was not mixed in A.

Furthermore, when the same experiment was carried out by adding the restriction enzyme EcoRI, as shown in FIG.
NA was cut and became a smear (lane E-1,
2). Usually, when Southern blotting is performed, it is cleaved with a restriction enzyme, but if the DNA is insufficiently purified and a protein is attached to it, the restriction enzyme will not work. Therefore, the above experiment shows that the purification of the DNA extracted by the method of the present invention was completely performed.

Next, in order to examine the contamination of RNA in the extracted DNA, agarose gel electrophoresis was carried out by the method described in the above-mentioned determination of purity [RNA contamination]. Thus, no difference was found between the test group and the control group (lanes R-1, 2). This indicates that the extracted DNA by the method of the present invention is not contaminated with RNA.

Example 5. Amplification of extracted DNA (application to PCR method) Human leukocyte antigen (HLA) class II antigen DRB1 was prepared using the DNA extracted from the blood of a healthy person by the method of Example 2.
Amplification and typing were performed using a primer specific to the DNA encoding (Sumitomo Metal Industries, Ltd .: Sumitest H
LA-DR group determination kit used). The results of acrylamide electrophoresis are shown in FIG. The band observed below the amplification band of β-globin is the band amplified by this typing.

Amplification of DNA by PCR usually requires strict conditions, but as is clear from FIG. 2, the DNA extracted by the method of the present invention is of high purity and can be used for PCR amplification. is there. This enables application to HLA type determination and diagnosis during transplantation.

Example 6. Application to genomic Southern hybridization using extracted DNA Southern hybridization was performed using the DNA from the human genome obtained by the method of the present invention. Extracted DNA
Was treated with a restriction enzyme and then subjected to agarose gel electrophoresis,
The results of hybridization after blotting on the membrane are shown in FIG. As described above, according to the present invention, a long DNA applicable to genomic Southern hybridization can be stably obtained.

[0064]

INDUSTRIAL APPLICABILITY According to the present invention, a shorter time than conventional DNA extraction methods such as a method using phenol-chloroform, a method using alkaline denaturation, a method by boiling, and a method using proteolytic enzyme. Thus, highly pure DNA can be obtained easily, safely, and inexpensively. In addition, D extracted and purified by the method of the present invention
NA is sufficiently usable as a material for various genetic engineering, and its application field is extremely wide.

[Brief description of drawings]

FIG. 1 shows the molecular weight of DNA extracted by the method of the present invention,
It is a figure of agarose gel electrophoresis showing the results of the assay of various contaminants and the restriction enzyme treatment.

FIG. 2 shows the results of P using DNA extracted by the method of the present invention.
It is a figure of acrylamide electrophoresis showing the result amplified by CR method.

FIG. 3 is a diagram showing the results of genomic Southern hybridization using DNA extracted by the method of the present invention.

Claims (6)

[Claims] 1. An aqueous solution containing at least one selected from the group consisting of (1) a protein denaturant and (2) a reducing agent, a surfactant and a chelating agent, wherein the pH is weakly acidic. A reagent for extracting DNA from a biological sample, which comprises a protein lysing agent that is weakly alkaline. 2. The protein denaturing agent is a chaotropic agent, the reducing agent is a thiol-based reducing agent, and the surfactant is not likely to cause precipitation in an aqueous solution of the composition, or some means even if precipitation occurs. The reagent according to claim 1, wherein the reagent is capable of being redissolved by the method, and the chelating agent has the ability to chelate a divalent metal ion. 3. The chaotropic agent is guanidine hydrochloric acid or guanidine thiocyanate, and the thiol reducing agent is 2.
-Reagent according to claim 2, which is mercaptoethanol or dithiothreitol, the surfactant is sodium N-lauroylsarcosine and the chelating agent is ethylenediaminetetraacetic acid disodium dihydrate. 4. The following operations: a) roughly extracting cells or nuclei from a biological sample, b) the following composition: (1) a protein denaturant and (2) a group consisting of a reducing agent, a surfactant and a chelating agent. An aqueous solution containing at least one selected from the group consisting of: a protein lysing agent having a pH of weakly acidic to weakly alkaline, thereby denaturing and solubilizing cells and nuclei and other components and contaminants; and c) A method for extracting DNA from a biological sample, which comprises performing alcohol precipitation by adding a lower alcohol as it is without performing DNA extraction with an organic solvent. 5. The protein solubilizer is added and stirred, and then incubated at about 55 ° C. for about 30 minutes,
The method of claim 4. 6. The following reagents: a) cell lysate, b) the following compositions: (1) at least one selected from the group consisting of protein denaturants and (2) reducing agents, surfactants and chelating agents. Extracted D consisting of an aqueous solution containing C, which is a protein solubilizer whose pH is weakly acidic to weakly alkaline, and c) a mixed solution of a chelating agent and a buffer solution.
DN from a biological sample containing a solvent for redissolving NA
A kit for extracting A.