JPH03101688A - Method for extracting of removing nucleic acid - Google Patents

Abstract

PURPOSE:To simplify extraction of nucleic acid and provide high yield in the field, etc., of genetic engineering by adding a protein modifying agent to a nucleic acid-containing sample, then adding an alcohol thereto and recovering formed precipitates. CONSTITUTION:A protein modifying agent (preferably 2.5-5.5M guanidine thiocyanate and >=6M urea) is converted into the form of a solution, etc., and added to a sample such as tissue, blood or sputum (if the sample has cell wall, etc., or is in the form of a lump, treatment such as homogenization, as necessary, is carried out) and an adequate amount of an alcohol (preferably ethanol, etc.) is then added to centrifuge formed precipitates, etc. Thereby, the objective nucleic acid is extracted or removed.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for extracting or removing nucleic acids from various samples containing nucleic acids, such as biological samples.

(Prior Art) In recent years, extraction of nucleic acids from biological samples has been actively carried out in various fields. For example, in genetic engineering and the production of DNA probes, the operation of extracting mRNA or DNA from cells that produce the target protein is also the process of extracting DNA.
In clinical diagnosis in which a probe is used to detect, for example, viral DNA (RNA), an operation is performed to extract the DNA (RNA) to be detected from a biological sample.

As described above, operations for extracting nucleic acids are extremely important in various fields.

Traditionally, nucleic acid extraction involves adding, for example, a caustic reagent to a sample;
Next, phenol or chloroform/phenol extraction is performed 1 to 3 times, and finally ethanol precipitation is performed,
A method is known in which cells are treated with a surfactant and proteinase K, followed by phenol extraction, and further ethanol precipitation. In addition, methods for extracting RNA include adding guanidine thiocyanate to cells and performing ultracentrifugation in a centrifugal solution prepared to a certain density, and methods using ion exchange force ram, gel filtration, or electrophoresis. It is being Furthermore, kits and extraction devices for extracting nucleic acids from biological samples are known. These methods include, for example, using proteinase K to lyse cells and simultaneously decomposing proteins, and extracting nucleic acids from the resulting solution using an ion exchange ram, or treating the sample with proteinase K.
Next, phenol extraction is performed, followed by alcohol precipitation.

(Problems with the Prior Art) The nucleic acid extraction method described above requires complicated operations, takes time, and requires treatment with dangerous organic solvents, and the amount of nucleic acid obtained is small. There are yield issues.

Specifically, a method in which a caustic reagent is added to a sample, then phenol or phenol/chloroform extraction is performed 1 to 3 times, and finally ethanol precipitation is performed, which requires complicated extraction operations and takes time to implement. , and requires the use of dangerous alkaline reagents and organic solvents such as chloroform.

The method in which a surfactant and proteinase K are allowed to interact, followed by phenol extraction and further ethanol precipitation has a problem in that protein degradation by proteinase K requires a patent. The problem with the method of adding guanidine thiocyanate to cells and performing ultracentrifugation in a centrifugation solution prepared to a certain density is that the centrifugation operation takes approximately one day.

Methods using an ion exchange force column, gel filtration, or electrophoresis have problems in that the operations are complicated and it takes time to implement. In the method explained above,
Due to the complexity of the operations, there is also the problem that, for example, it is very difficult to automate the operations. Kits and devices for extracting nucleic acids from biological samples also have problems, such as the fact that the reaction takes time because they use proteinase K.

(Means for Solving the Problems) As a result of intensive study on a method for extracting nucleic acids that can be carried out by simple operations and does not require complicated operations, the present inventors have solved the problems seen in conventional techniques. The method of the present invention was completed.

According to the invention, a method for removing nucleic acids from a sample is also particularly provided. That is, the present invention is a method for extracting nucleic acids, which comprises adding a protein denaturing agent to a sample containing nucleic acids, adding alcohol, and then collecting a precipitate.
This method of removing nucleic acids consists of adding a protein denaturant to a sample containing nucleic acids, then adding alcohol, and then removing the precipitate. The present invention will be explained in detail below.

The method of the present invention relates to a method for extracting or removing nucleic acids such as DNA and RNA from a sample.

Nucleic acid as used herein refers to DNA and/or RNA, which may be single-stranded or double-stranded, and is not limited by their genetic properties. For example, the DNA may be genomic DNA or DNA contained in mitochondria or chloroplasts, and the RNA may be messenger RNA or transfer RNA.

Examples of samples containing nucleic acids include biological samples such as tissues, cells, blood, bile, pus, cerebrospinal fluid, feces, saliva, and sputum. Regarding these samples, if the contained proteins or nucleic acids cannot come into contact with protein denaturants or alcohol, that is, if the samples have cell walls or cell membranes or are in the form of lumps,
For example, homogenization, surfactant treatment, or ultrasonic treatment may be performed as necessary.

Next, a protein denaturing agent is added to the sample to denature and solubilize the proteins in the sample. As the denaturing agent, any denaturing agent that can denature proteins can be used without particular restrictions, but guanidine salts or urea, which have a remarkable effect, are preferred.

As the guanidine salt, organic guanidine salts such as guanidine thiocyanate, guanidine hydrochloride, and guanidine carbonate can be used.

The protein denaturant can be added to the sample in solid form (powder, granules) or solution; however, if it is added in solid form, stirring must be performed to sufficiently denature the proteins in the sample. It is a good idea to make it possible.

The protein denaturant is added to a concentration such that it can denature the proteins in the sample at the time it is added to the sample, and at the same time, it can cause nucleic acid to precipitate when alcohol is added later.

For example, in the case of guanidine thiocyanate, if it is less than 1.5M, protein denaturation will not occur sufficiently, and if it is more than 6.5M, nucleic acid precipitation will not occur sufficiently when alcohol is added. Preferably 2.5M
Add to the sample so that the concentration becomes 5.5M. If guanidine hydrochloride is used, it may be added to a concentration of 2M or more, and there is no restriction on the upper limit. In the case of urea, 4
4. Protein denaturation does not occur sufficiently at M level.
Add to the sample so that the concentration is 5M or more, preferably 6M or more. The concentrations explained above are general conditions for carrying out the present invention, and are preferably determined in consideration of the amount of protein present in the sample when carrying out the present invention.

The concentration of the protein denaturant is as described above, but in addition to that, for example, first, add the protein denaturant to the sample to a concentration that makes it difficult for nucleic acids to precipitate when alcohol is added later. The object of the present invention can also be achieved by adding enough alcohol to dilute the denaturing agent to a concentration at which the nucleic acid precipitates during a subsequent alcohol addition step.

Next, alcohol is added to the sample solution containing the protein denaturant to precipitate the nucleic acids. Alcohols include ethanol, n-propanol, iSo-propanol, n-butanol, Sec-butanol, tert-butanol,
n-amyl alcohol, iso-amyl alcohol, t
Examples include ert-amyl alcohol.

Alcohol should be added at a concentration that will cause nucleic acid precipitation without precipitating proteins that are denatured by a protein denaturant and present in a solubilized state, but the specific amount to be added will depend on the type of alcohol used. Depends on. Alcohols with longer alkyl chains, that is, alcohols with higher hydrophobicity, can precipitate nucleic acids with smaller amounts added. Therefore, when carrying out the present invention, it is preferable to conduct a test using the alcohol to be used and investigate the appropriate amount to be added.

Among the specific alcohols that can be used in the present invention, alcohols other than ethanol and iso-propanol are known to cause phase separation from an aqueous phase such as a buffer solution. However, in the present invention, when these alcohols are added to a solution containing a guanidine salt, phase separation does not occur and a homogeneous phase is formed. In order to form phase separation in the present invention, a salt such as common salt may be added to the solution to reduce the solubility of alcohol in the solution.

The above operations cause the nucleic acids contained in the sample to precipitate, but on the other hand, the proteins remain in the solution in a dissolved state due to the action of the protein denaturant.

Therefore, it is possible to extract or remove nucleic acids by performing, for example, centrifugation or membrane separation after carrying out the method of the present invention.

There is no restriction on washing the extracted nucleic acid with an alcohol aqueous solution or the like that does not contain a protein denaturant after the present invention. Further, regarding the protein denaturant solution after nucleic acids have been removed according to the present invention, there is no restriction at all as to, for example, performing dialysis or the like after carrying out the present invention to reduce the concentration of the denaturant.

(Effects of the Invention) The present invention can be carried out without using dangerous organic solvents, and only by adding a protein denaturant and alcohol to a sample and then separating and removing the precipitate. Therefore, the time required to carry out the procedure is extremely short, and the nucleic acid extraction or removal operation, which conventionally required half a day to two days, can be carried out more quickly. Therefore, it is a particularly useful method in the field of genetic engineering, etc., where it is necessary to extract nucleic acids from a large amount or a large number of samples.

As described above, since the present invention can be carried out through extremely simple operations, it is also possible to automate the operations.

In addition, in the present invention, all operations can be performed in the same reaction vessel, and there is no need to separate or remove precipitates using a column, etc., so there is little loss of sample.
It is therefore possible to extract nucleic acids with high yields or to remove them without loss of protein. The fact that all operations can be performed in the same reaction vessel is useful, for example, when the nucleic acid to be extracted or removed is dangerous viral DNA, etc., and the sample is cells infected with the virus. This also means that contamination can be minimized.

(Examples) Examples will be described below to explain the present invention in more detail, but these examples are merely examples and do not limit the present invention.

Example 1 K562 cells, which are human leukocyte cancer cells (these cells are well-known and can be purchased from Dainippon Pharmaceutical Co., Ltd., for example), were treated with PRMI-164 containing 1% fetal bovine serum.
The cells were cultured in 0 medium. When 1 ml of the culture suspension reached 500,000 cells, 1 ml of the suspension was collected in a sampling tube, centrifuged at 500 rpa+ for 5 minutes,
Cells were collected in a precipitate.

5M guanidine thiocyanate, 1mM for cells
10o+MTris monohydrochloric acid buffer (p
ll8. After adding 0.4 ml of O), the mixture was stirred at room temperature for 1 minute, and then 1 ml of 99.5% ethanol was added. Subsequently, the solution was stirred at 1500 O rpm for 5 minutes.
Centrifugation was performed for a minute, and nucleic acids were extracted from the precipitate.

The supernatant was discarded, and the precipitate was washed with 70% ethanol and dried to obtain nucleic acids.

Restriction enzymes (Ba
m HISEco Rl, Alu I), RNa
As a result of the action of s eSDNa s e, the reactions by these enzymes were not inhibited.

Furthermore, the replication reaction by DNA polymerase using the extracted nucleic acid as a template was not inhibited. These results are
This indicates that restriction enzyme inhibitors such as histones have been removed.

After filtration, the membrane was washed with 70% ethanol,
After drying, nucleic acids were obtained in a buffer solution.

When the same tests as in Example 1 were conducted on the nucleic acids extracted as described above, all reactions were achieved without inhibition.

Example 2 K562m cells, which are human leukocyte cancer cells, were cultured in the same manner as in Example 1. When 500,000 cells per ml of culture suspension were obtained, a suspension of III11 was placed in a sampling tube, and the cells were collected by centrifugation at 500 rpI1 for 5 minutes.

After centrifugation, the supernatant was discarded, and 0.4 ml of 10 mM TrLs monohydrochloric acid buffer (pH 8.0) containing 5 M guanidine thiocyanate and 1 i MEDTA was added to the cells, stirred for 1 minute at room temperature, and then 99.5% ethanol was added. 1a+1
Added. Subsequently, the solution was poured into a polytetrafluoroethylene membrane (manufactured by NORTON, ZITEX 1-midum pore size).
) to collect nucleic acids.

Example 3 Escherichia coli strain JM109, which had undergone light transformation using plasmid plBI76 (manufactured by IB1), was cultured in LB medium.

When the number of cells reaches 100 million cells per ml of suspension, the 0.1I
1 ml was taken into a sampling tube, and 0.1 ml of 8M guanidine hydrochloride was added to the sampling tube. After adding 3 ml, it was stirred for 1 minute.

After stirring, 0. 2 ml of 99.5% isobrobanol was added, stirred, and centrifuged at 1500 rpm for 5 minutes to extract nucleic acids.

After discarding the supernatant and washing the precipitate twice with 70% ethanol, 10I! containing IIIMEDTA was added. 1M Tris-HCl buffer (pll8.O) 0. 4+Al and centrifuged at 1500 O rp- for 5 minutes to remove impurities.

Add 1 part of 99.5% ethanol to the supernatant obtained as above, centrifuge at 15000 rpII for 5 minutes, dry the obtained precipitate, and use the same method as in Example 1. When tests were carried out, all enzyme reactions were successfully achieved without being inhibited.

Example 4 K562 cells, which are human leukocyte cancer cells, were cultured in LB medium. When the culture suspension reached 500,000 cells per ml, 1 Ill of the suspension was obtained in a sampling tube.
Cells were collected by centrifugation at 500 rpI1 for 5 minutes.

10IIMTr containing 8M urea and 1mM EDTA in the precipitate
After adding 0.4 liters of is monohydrochloric acid buffer (pH 8.0), the mixture was stirred at room temperature for 1 minute, and then 99% secondary butyl alcohol was added. The solution was then filtered through a tetrafluoroethylene membrane similar to that used in Example 2. After filtration, the membrane was
% ethanol, drying was performed, and the nucleic acid was obtained in a buffer solution.

When the same tests as in Example 1 were conducted on the nucleic acids extracted as described above, all reactions were completed without being inhibited.

Example 5 B LV (Bov1ne LeukeIlia Vi
rus ; VIRO LOGY, Volume 138,
82, 1984) DNA of M13 phage (T
It was incorporated into the Bam HI site of Akara (manufactured by Akara). For reference, a portion of BLV DNA is shown in FIG. 1 for reference.

M13 phage having lng of DNA was added to 100 μl of bovine serum, 300 μl of 6M guanidine isocyanate was added to the serum, and the mixture was stirred for 1 minute.

Add 10 μl of 10 B/ml to the solution as a carrier.
of Salmon DNA and IIll of ethanol were added, stirred for 1 minute, and centrifuged at 1500 rpm for 5 minutes. Discard the supernatant, dry the precipitate, and
10mM Tris monohydrochloric acid buffer (pH 8.
0) Dissolved in 50 μl.

DNA synthesizer (Applied Biosystem)
Synthetic D having a sequence complementary to the above-mentioned base sequence consisting of the base sequence shown in FIG.
NA was prepared. The 5' end of the prepared DNA was aminated and alkaline phosphatase was attached. The above sequence was detected by the dot plot method using this DNA as a probe.

Although color development was observed for M13 phage DNA,
No color development was observed with the bovine serum used as a control. This means that according to the present invention, trace amounts of DNA (BLV DNA
This shows that A) can be extracted.

It is the meaning. Furthermore, in the figure, the underlined portions indicate portions that specifically pair with the DNA shown in FIG. 2.

FIG. 2 shows the base sequence of the DNA that specifically pairs with the underlined portion of the DNA shown in FIG. 1, which was used in Example 5 of the present invention.

Claims (3)

[Claims] (1) A nucleic acid extraction method consisting of adding a protein denaturant to a sample containing nucleic acids, then adding alcohol, and then collecting the precipitate. (2) A method for removing nucleic acids, which consists of adding a protein denaturant to a sample containing nucleic acids, adding alcohol, and then removing the precipitate. (3) The method according to claim (1), wherein the protein denaturant is a guanidine salt or urea. (4) The alcohol is at least one alcohol selected from ethanol, propanol, butanol, pentanol, or hexanol. The method according to claim (1) or (2), characterized in that