JPH04349892A - Method for isolating nucleic acid - Google Patents

Abstract

PURPOSE:To improve efficiency of operation such as genetic analysis requiring steps for isolating nucleic acid due to its isolation from a biological sample practicable in a shorter time than that in conventional methods by applying impact force to cells in chemically destroying the cells. CONSTITUTION:Cells are initially and chemically destroyed while applying impact force to the cells. Proteins of the destroyed cells are then denatured and insolubilized by preferably adding an acid and/or heat. The insolubilized proteins are subsequently removed. The cells are destroyed by placing small pieces for destruction such as beads made of glass in a cell solution to which a surfactant, e.g. saponin is added and vigorously stirring the cells.

Description

[Detailed description of the invention]

[Purpose of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a method for isolating nucleic acids from biological samples containing them.

0002

2. Description of the Related Art Among nucleic acids, genetic information imprinted in DNA (genes) is expressed as proteins or enzymes via messenger RNA. Various compounds are produced by the action of these proteins and enzymes, and living things exist as a collection of these compounds. The total number of human genes is said to be 50,000 to 100,000, and with recent developments in molecular biology, analysis of these genes is progressing rapidly at the molecular level. The Human Genome Project, which is currently being carried out worldwide, is a representative example. The Human Genome Project is an attempt to sequence all human genes. Furthermore, a technology called genetic diagnosis, which diagnoses diseases associated with genetic abnormalities by directly analyzing those genes, has been attracting attention in recent years. When analyzing human genes, it is first necessary to extract and purify nucleic acids containing DNA from tissue cells or peripheral blood.

[0003] A conventional method for isolating DNA from a whole blood sample of a living body will be described below. First, white blood cells are collected from whole blood that has been treated with heparin to prevent coagulation.
Cells in the sample are disrupted using a detergent in the presence of proteolytic enzymes. Surfactants destroy not only cell walls but also nuclear membranes inside cells, and proteolytic enzymes break down excess proteins inside cells. Next, by adding phenol and chloroform, the remaining protein is denatured and insolubilized. Ethanol is added to the aqueous layer from which insoluble materials and the phenol layer or chloroform layer have been removed to precipitate nucleic acids. Most of the obtained nucleic acids are DN
If further purification is required, RNase treatment is performed to degrade and remove the RNA contained in the nucleic acid together with DNA, and ethanol is added again.
Precipitate NA. Conventionally, purified DNA was obtained as described above.

0004

[Problem to be solved by the invention] In the above process, D
When obtaining NA, surfactants are used in the presence of proteolytic enzymes to destroy cells in the steps up to isolating nucleic acids, but this does not destroy the cell membrane or nuclear membrane to the point where nucleic acids can be isolated. This required at least 4 to 5 hours after adding the surfactant. For this reason, gene analysis work that requires analysis of a large number of samples is inefficient, and a method for isolating nucleic acids in a short time has been desired. Furthermore, in order to denature the remaining intracellular proteins, organic solvents such as phenol and chloroform, which are harmful to the health of experimenters, have traditionally been used.

[0005] In view of the above-mentioned problems, the first object of the invention is to provide a method for isolating nucleic acids from biological samples such as whole blood in a short time. . In addition, the second object of the invention is to
The present invention provides a method for isolating nucleic acids that can be performed safely. [Structure of the invention]

[0006]

[Means and effects for solving the problem] First aspect of the present invention
The invention relates to a nucleic acid isolation method comprising the steps of chemically destroying cells, denaturing and insolubilizing proteins in the destroyed cells, and removing the insolubilized proteins. This method of isolating nucleic acids is characterized by applying impact force to cells during destruction. Further, the second invention of the present invention provides a method for isolating a nucleic acid comprising a step of destroying cells, a step of denaturing and insolubilizing proteins in the destroyed cells, and a step of removing the insolubilized proteins. This is a nucleic acid isolation method characterized by denaturing and insolubilizing the proteins of the destroyed cells by applying at least either heat or heat.

[0007] In order to isolate nucleic acids from biological samples,
First, a step is required to destroy the cell membranes and nuclear membranes of cells derived from the biological sample. The sample containing cells destroyed in the above step contains residual substances other than nucleic acids. Most of the remaining substances in the sample are proteins that function as functional molecules in living organisms. Next, in order to isolate nucleic acids, it is necessary to first perform a step of denaturing those proteins, and then a step of removing the denatured proteins.

[0008] The first invention of the present invention relates to a process of destroying cells, in which an impact force is applied to the cells in addition to a chemical method in order to destroy the cells. Thereby, the cell destruction process can be performed in a shorter time than conventionally.

[0009] As a method of chemically destroying cells, a surfactant is used. Examples of surfactants include saponin, sodium dodecyl sulfate (SDS), Trit
onX-100 etc. is used.

[0010] As a method of applying impact force to cells, for example, a small piece for destruction of an appropriate size is added to a biological sample solution,
There is a method of stirring vigorously. The material of the destruction piece is not particularly limited, such as ceramics, glass, resin, etc., as long as it does not adversely affect the isolation of nucleic acids. There is no particular restriction on the shape of the breaking piece. The size of the breaking piece is preferably about 0.01 mm to 1 mm in diameter, for example, in the case of a spherical shape. This is because if the breaking piece is too small, the cells will be difficult to destroy even if the breaking piece collides with the cell, and if it is too large, the stirring efficiency will be poor. Other methods include a method of applying ultrasonic waves.

[0011] After the cell destruction step is carried out as described above, a step of denaturing and insolubilizing the protein is carried out. The step of denaturing and insolubilizing the protein may be carried out by adding an organic solvent such as phenol or chloroform as in the conventional method, or may be carried out according to the second aspect of the present invention described below.

[0012] In the second invention, at least one of acid and heat is added to a sample solution containing cells whose cell membranes and nuclear membranes have been destroyed in the previous step to denature and insolubilize impurity proteins. Since cell proteins are denatured by heat or acid, there is no need to use harmful organic solvents.

The above-mentioned acid is not particularly limited, such as hydrochloric acid, acetic acid, nitric acid, and sulfuric acid, as long as it does not adversely affect the isolation of nucleic acids. The acid concentration is preferably about 0.1N or more and 1N or less. By adding the above acid to the sample solution and allowing it to act, the proteins that were soluble in the sample become insolubilized and become colloidal. Further, the heat applied to the sample solution may be such that the sample solution boils. 5
By heating for more than a minute and continuing boiling, the proteins that were soluble in the sample denature and become insolubilized, becoming a colloid. Either acid or heat may be applied to the sample solution. Further, acid and heat may be used together.

[0014] Furthermore, in order to decompose proteins in cells and promote protein denaturation, a proteolytic enzyme (protease) may be added and activated during the step of destroying cells. Among proteolytic enzymes, proteinase K, which can decompose a wide variety of proteins, is particularly preferred.

Finally, a step of removing the insolubilized protein is performed. The insolubilized protein is removed by separation by centrifugation, suction, or filtration using a filter to obtain a supernatant in the sample solution. Since the obtained supernatant contains nucleic acids, it can be extracted using a solvent such as ethanol to obtain nucleic acids.

If the nucleic acid obtained as described above requires further purification, a method using glass beads such as silica that selectively adsorbs DNA or a method using RNase
There are various methods of processing. A method using glass beads that selectively adsorbs DNA is preferable because it is easy to operate and highly pure DNA can be obtained. As a procedure, DN
Glass beads that selectively adsorb A and an electrolyte that promotes adsorption are added at a high concentration, and the DNA is adsorbed onto the glass beads. After that, the DNA is washed with a buffer, and heated with water or a TE buffer (Tris-HCl, Elute in EDTA). The present invention will be explained in detail below with reference to Examples.

[0017]

【Example】

(Example 1)

[0018] Add 0 to 200 μl of whole blood collected with heparin.
．． Add 1 mm φ glass beads, 500 μl of 5% Sanipon, cell lysate (1% Triton
00 and 0.5% SDS 50mM EDTA) for 2m
1 tube and vortexed vigorously for 3 minutes to disrupt cells. Next, the mixture was incubated at 90°C for 15 minutes to denature and insolubilize the protein. To remove insolubilized proteins, filter (Pore Size 0.4
5 μm). 3M sodium acetate and 2.5 times the volume of ethanol were added to the filtrate, and filamentous DN was added.
A was wound up with a glass rod. The obtained DNA was washed with 70% ethanol and treated with TE (10mM Tris-HCl).
, 1mM EDTA (pH 8.0)) 200μl
A DNA sample solution was obtained. Approximately 3-4 μg of DNA was obtained from 200 μl of whole blood and could be digested with restriction enzymes. The time required was approximately 1 hour. (Example 2)

A sample solution was obtained by destroying cells in 200 μl of heparinized whole blood in the same manner as in Example 1. Then, 500 μl of 0.5N HCl was added and the mixture was incubated at 37°C.
The protein was incubated for 5 minutes to denature and insolubilize the protein. Thereafter, the precipitate was filtered. filtrate with NaO
After neutralizing with H and adding ethanol to precipitate the DNA, the DNA was obtained by centrifugation. The obtained DNA was washed with 70% ethanol and treated with TE (10mM Tris-HCl, 1
A DNA sample solution was obtained by dissolving in 200 μl of mM EDTA (pH 8.0). Approximately 3 from 200μl whole blood
~4 μg of DNA was obtained and could be cut with restriction enzymes. The time required was approximately 1 hour. (Example 3)

Proteins were denatured and filtered to 200 μl of heparinized whole blood in the same manner as in Example 1. Three times the amount of sodium iodide and DNA-adsorbing beads were added to the filtrate containing DNA, and the mixture was incubated at room temperature. It was left for 10 minutes. Furthermore, washing buffer (40mM Tris-HCl,
The DNA-adsorbed beads were washed with 4mM EDTA (pH 7.5) and TE (10mM Tris-HCl,
Add 1mM EDTA (pH 8.0)) and incubate for 50 minutes.
The DNA was eluted by standing at ℃ for 5 minutes. 200μl
Approximately 2 μg of pure DNA was obtained from whole blood. It could be cut with restriction enzymes, and the required time was about 1.5 hours. (Comparative example 1)

[0021] Add 2 to 200 μl of heparinized whole blood.
Three times the amount of ice-cold 0.2% NaCl was added to destroy the red blood cells, and the mixture was centrifuged at 5000 rpm at 4°C for 5 minutes to obtain a precipitate. This operation was repeated four times to completely remove red blood cells. 1 ml of 10 mM phosphate buffer (pH 7.5) was added to the obtained sediment and centrifuged in the same manner to wash white blood cells. Ice-cold cell lysate (0.32M Suc
rose, 1% TritonX-100, 5mM M
gCl2, 10mM Tris-HCl (pH 7.5)
), and after suspending, add 0.5ml of
I left it for a minute. Next, centrifugation was performed at 5000 rpm for 15 minutes at 4°C, and the resulting precipitate was added with ice-cold 25mM EDTA.
(pH 8.0) (contains 75mM NaCl) 0.5
After adding 1ml, 50 μl of 10% SDS, and 20 μl of proteinase K (20 mg/ml), the mixture was left at 37° C. for 2 hours. The process of disrupting the cells took a total of 2 hours and 45 minutes. Equal amount of TE (10mM Tr
is-HCl, 1mM EDTA (pH 8.0)) saturated phenol was added and mixed slowly for 10 minutes.
Centrifuge at 00 rpm for 10 minutes, remove the aqueous layer, and add phenol.
Extraction was performed in the same manner with chloroform solution (1:1 mixture) and chloroform. Add 2.5 times the amount of ethanol to form filamentous D.
Roll up the NA with a glass rod, wash it with 70% ethanol,
Dry. The obtained DNA could be cut with restriction enzymes, and the time required to perform the entire process was about 3 hours and 45 minutes.

As can be seen by comparing Examples 1 to 3 and Comparative Example 1, the time required for the process of destroying cells was about 3 minutes in Examples 1 to 3, and about 2 hours and 45 minutes in Comparative Example. This shows that according to the present invention, nucleic acids can be isolated in a short time. Furthermore, it can be seen that according to the present invention, nucleic acids can be isolated without using an organic solvent.

[0023]

[Effects of the Invention] As described above, according to the method of the first invention, nucleic acids can be isolated in a short time, and can be used for genetic analysis etc. that require isolation of nucleic acids from biological samples. Work efficiency can be improved. Further, according to the method of the second invention, the work can be carried out safely without using organic solvents that are harmful to the human body, which is advantageous.

Claims (2)

[Claims] Claim 1: A nucleic acid isolation method comprising the steps of chemically destroying cells, denaturing and insolubilizing proteins in the destroyed cells, and removing the insolubilized proteins. 1. A method for isolating nucleic acids, which comprises applying impact force to cells when disrupting them using a conventional method. 2. A method for isolating a nucleic acid comprising the steps of destroying cells, denaturing and insolubilizing proteins in the destroyed cells, and removing the insolubilized proteins, wherein at least one of acid and heat is applied. 1. A method for isolating nucleic acids, which comprises denaturing and insolubilizing the proteins of disrupted cells.