JP4519247B2 - Nucleic acid extraction and purification reagents - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a reagent for extraction and purification of nucleic acid that can be used for purification and measurement of nucleic acid. More specifically, a method for extracting and purifying nucleic acid from a sample containing nucleic acid in a short time without a complicated operation using a solid phase carrier capable of binding nucleic acid in the presence of a polyanionic substance, and nucleic acid extraction It relates to a purification kit.
[0002]
[Prior art]
A method for extracting and purifying nucleic acid from a sample containing nucleic acid is indispensable in the fields of genetic engineering and clinical diagnosis. For example, when analyzing a certain nucleic acid, it is necessary to extract and purify DNA or RNA from a biological sample containing the nucleic acid. In diagnosis of infectious diseases, it is common to detect after extracting DNA or RNA from bacteria or viruses.
[0003]
In biological samples, nucleic acids such as DNA and RNA generally exist in shells such as cell membranes and cell walls composed of proteins, lipids, and sugars, and nucleic acids exist in a free state. Rather, it forms a complex with the protein. For this reason, when extracting and purifying nucleic acid from a biological sample containing nucleic acid, the nucleic acid is liberated by enzymatic treatment with protease, denaturation treatment using a surfactant, or ultrasonic treatment or heat treatment. It is necessary to purify the nucleic acid by extraction with an organic solvent such as chloroform or ultracentrifugation using cesium chloride. These techniques are combined and optimized in the fields of genetic engineering and clinical diagnosis depending on the use of the nucleic acid to be used. The current situation is that.
[0004]
The extraction operation using the organic solvent requires time and a complicated centrifugal operation, and the ultracentrifugation operation using cesium chloride similarly requires time, requires desalting, and is costly. is there.
[0005]
On the other hand, as a simple nucleic acid extraction and purification method, there is a method using magnetic silica particles containing a superparamagnetic metal oxide (Japanese Patent Laid-Open No. 9-19292). This method allows nucleic acid to be adsorbed nonspecifically from a biological sample containing nucleic acid without centrifugation and analyzed immediately after elution of nucleic acid adsorbed from magnetic silica particles using a low-concentration buffer such as water or TE buffer. Has the advantage of being able to. However, magnetic silica particles are the only magnetic particles that can adsorb nucleic acids in a state where the ionic strength is increased by a chaotropic substance, and general magnetic particles on the market, for example, silanol groups are introduced into carboxyl groups or carboxyl groups. The magnetic particles having a functional group cannot adsorb nucleic acid in a state where the ionic strength is increased by a chaotropic substance.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to solve the above-mentioned problems of conventional techniques, and a reagent for extracting and purifying nucleic acid in a short time without complicated operation from a sample containing nucleic acid, and the reagent are used. A nucleic acid purification method and a nucleic acid extraction and purification kit are provided.
[0007]
[Means for Solving the Problems]
As a result of diligent studies to solve the above-mentioned problems, not only those coated with silica as described above, but also, for example, a carrier in which a silanol group is introduced into a carboxyl group or a carboxyl group by using an appropriate polyanionic substance In addition, the present inventors have found that the nucleic acid can be adsorbed efficiently and the adsorbed nucleic acid can be eluted using a low concentration buffer such as TE buffer.
[0008]
That is, the present invention relates to nucleic acid extraction and purification characterized in that dextran sulfate is present in the liquid phase when nucleic acid is extracted and purified from a sample containing nucleic acid by using magnetic particles having nucleic acid binding ability. Reagent. Another aspect of the present invention is a method for purifying a nucleic acid using this extraction and purification reagent. Yet another aspect of the present invention is a kit for measuring a nucleic acid containing reagents used for these extraction and purification.
[0009]
The nucleic acid purification method of the present invention comprises the following steps:
(A) A dextran sulfate solution and magnetic particles are added to and mixed with a sample containing nucleic acid to bind the nucleic acid to the magnetic particles .
(B) washing the nucleic acid- magnetic particle complex in step (a) with a washing solution;
(C) The nucleic acid is eluted from the nucleic acid- magnetic particle complex washed in the step (b) according to the intended use.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the nucleic acid may include plasmid DNA including DNA and RNA. The length is not particularly limited, but preferably 8 or more base sequences are suitable.
[0011]
In addition, the sample containing the nucleic acid used in the present invention is not particularly limited as long as it contains a nucleic acid, and examples thereof include PCR products and biological samples. A biological sample means a sample derived from an organism, such as whole blood, plasma, serum, bone marrow, buffy coat, urine, body fluid, saliva, nasal discharge, tears, fecal matter, cell culture, cell lysate, culture medium Examples of biological samples that may contain nucleic acids such as.
[0012]
If necessary, for example, the biological sample may be dissolved in advance to extract the nucleic acid. Examples of the method for elution / extraction of nucleic acid from a biological sample include a method of adding a lysis solution, lysing cells, and adding an organic solvent to extract nucleic acid.
[0013]
The lysis solution used for elution of nucleic acid from a biological sample preferably contains a protease, and the protease is not particularly limited as long as it destroys cell membranes or decomposes proteins contained in the biological sample. Specific examples include pronase and proteinase K. Among these, proteinase K is preferably used. The concentration used is preferably in the range of 0.1 to 1000 U / mL. Moreover, it is also possible to contain a buffer solution in a solution, and the range of pH 6-12 is suitable. The buffering agent is not particularly limited as long as it is generally used, but it is desirable to have a buffering ability at any pH in the range of pH 6 to 12, for example, 1,3 bis [ Examples include tris (hydroxymethyl) methylamino] propane, tris [hydroxymethyl] aminomethane, and the use concentration thereof is preferably 1 to 500 mmol / L, and pH is preferably in the range of 6 to 12.
[0014]
Furthermore, the organic solvent used in the present invention is not particularly limited as long as it does not hinder the binding of a nucleic acid to a solid phase carrier having a nucleic acid binding ability. Specific examples of the organic solvent used in the present invention include water saturated phenol, buffer saturated phenol, chloroform and the like. Of these, water-saturated phenol or buffer-saturated phenol, or a mixture of these saturated phenol and chloroform in an appropriate ratio is preferable.
[0015]
Further, sodium chloride, SDS, glycogen and the like may be appropriately used during the nucleic acid lysis / extraction operation.
[0016]
The present invention provides a nucleic acid separation means characterized in that a polyanionic substance is present in a liquid phase when a nucleic acid is separated from a sample containing the nucleic acid by using a solid phase carrier having nucleic acid binding ability. It is. The nucleic acid fractionating means according to the present invention can be broadly divided into three stages: (a) an adsorption process, (b) a washing process, and (c) an elution process.
[0017]
(A) Adsorption step In this step, a polyanionic substance solution and a solid phase carrier having nucleic acid binding ability are added to and mixed with a sample containing nucleic acid, and the nucleic acid is adsorbed to the solid phase carrier having nucleic acid binding ability.
[0018]
The polyanionic substance used in the present invention includes a polyanionic substance that enhances the affinity between the nucleic acid and the solid phase carrier. The polyanionic substance is presumed to promote the adsorption of a nucleic acid to a solid phase carrier having a nucleic acid binding ability. The polyanionic substance is not particularly limited as long as it is a polyanionic substance and enhances the affinity between the nucleic acid and the solid phase carrier. Specific examples thereof include dextran sulfate, magnesium phosphotungstate, heparin, Examples include chondroitin sulfate, hyaluronic acid, dermatan sulfate and the like. Of these, dextran sulfate is preferably used, its molecular weight is 500 to 500,000, and the concentration used is preferably 0.5 to 30%.
[0019]
A salt is added to the polyanionic substance solution used in the present invention to increase the ionic strength, and it is presumed that the action of the polyanionic substance is promoted. Specific examples of the salt used include sodium chloride, potassium chloride, magnesium chloride and the like, but are not particularly limited as long as the ionic strength is increased. Of these, sodium chloride is preferably used, and the concentration used is preferably in the range of 0.01 to 3 mol / L.
[0020]
The solid phase carrier having nucleic acid binding ability used in the present invention is not particularly limited as long as it is a solid that can adsorb and hold nucleic acid in the presence of a polyanionic substance. Specifically, silicon dioxide, that is, silica is preferably used. Further, glass, diatomaceous earth, which is a carrier composed of silica, or a material obtained by subjecting these to surface treatment by chemical modification, and a complex with another substance such as a superparamagnetic metal oxide are also included. Furthermore, those having a surface modified with a functional group comprising a carboxyl group, a silanol group, or a combination thereof, and a complex with another substance such as a superparamagnetic metal oxide are also included. Among these, considering the efficiency of adsorption and elution, the solid support is preferably a particle, and its size is preferably in the range of 0.05 to 10 μm.
[0021]
In the present invention, an extraction solution composed of an organic solvent, a polyanionic substance solution, and a solid phase carrier having a nucleic acid binding ability may be added separately or simultaneously.
[0022]
(B) Washing step This step is a step of separating and washing only the solid phase carrier having the nucleic acid binding ability adsorbed with the nucleic acid from the mixture of the solution, the organic solvent, and the solid phase carrier having the nucleic acid binding ability. At this time, it is preferable to repeatedly wash about 1 to 3 times using a washing liquid.
[0023]
The specific means for separating the solid phase carrier having the nucleic acid binding ability in the present invention varies depending on the form of the solid phase carrier to be used. For example, in the case of the solid phase carrier having the nucleic acid binding ability, centrifugation and filtration are performed. When a superparamagnetic metal oxide-containing material is used as a solid phase carrier, it is more preferable because it can be easily separated by a magnetic force using a magnet or the like.
[0024]
The washing solution used in the present invention is not particularly limited as long as it prevents the nucleic acid held on the solid phase carrier from being released or prevents the protein from adsorbing to the solid phase carrier. Specifically, it is preferable to wash with 0.5 to 2.5 mol / L sodium chloride solution or 40 to 100% ethanol.
[0025]
(C) Elution step This step is a step of eluting nucleic acid from a solid phase carrier having nucleic acid binding ability to which the nucleic acid is adsorbed, and the eluent used in the present invention is to promote elution of nucleic acid from the solid phase. If there is, it will not be specifically limited. Specifically, water or TE buffer [10 mmol / L Tris-HCl buffer, 1 mmol / L EDTA, pH 8.0] is preferable. The nucleic acid recovered at this time can be directly used for an enzyme reaction using a restriction enzyme, a nucleic acid polymerase, or the like without performing desalting and concentration operations such as dialysis and ethanol precipitation.
[0026]
As described above, since the nucleic acid purification method according to the present invention is composed of simple steps, it can be easily applied to nucleic acid extraction and purification kits and nucleic acid extraction devices that automate solid phase carrier separation and reagent dispensing operations. It is clear that this is possible.
[0027]
【Example】
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[0028]
Example 1 Recovery of DNA added to serum (1) Preparation of DNA extraction material 1 μg of λ-DNA (Takara Shuzo) was weighed and added to serum to prepare 1 μg / 200 μL. did.
[0029]
(2) Extraction and purification of λ-DNA 200 μL of the DNA extraction material prepared in (1) above was placed in three 2 mL microtubes, and 200 μL of each solution [50 mmol / L Tris (hydroxyl) aminomethane- Hydrochloric acid (pH 8.0), 5 mmol / L EDTA, 0.25 mol / L NaCl, 1% SDS, 20 μg / mL glycogen, 120 U / mL proteinase K] are added, mixed with a vortex mixer, and heated at 56 ° C. for 30 minutes. did. After warming, 400 μL of TE buffer saturated phenol / chloroform was added to each tube and stirred vigorously.
[0030]
800 μL of polyanionic substance solution [20% dextran sulfate (average molecular weight 5000, manufactured by Sigma), 2.5 mol / L NaCl] was added to these and mixed, and 40 μL of different types of 0.1 g / mL magnetic particles [( Average particle diameter 3 μm, containing triiron ferric oxide 15%, specific surface area 0.17 m ² / g, functional group C ₃ COOH; SiO ₂ ... SiOH: micromer-M made by micromod), (average particle diameter 6.17 μm, four 43% iron trioxide particles, specific surface area 159 m ² / g, pore volume 279 mm ³ / g, surface pore diameter 3.2 nm: other magnetic particles 1 and 2)] were added to the tube and mixed for 10 minutes at room temperature .
[0031]
The tube was then placed on a magnetic stand and the magnetic particles were collected on the tube wall and the supernatant was removed. After removing the tube from the magnetic stand, 1 mL of a washing solution [70% ethanol] was further added and mixed well using a vortex mixer. Similarly, the tube was placed on a magnetic stand, the supernatant was removed, and this washing operation was repeated twice. After removing the supernatant, the microtube was placed on a heat block set at 56 ° C. and allowed to stand for 10 minutes to evaporate and remove the ethanol in the tube and dry the particles. 50 μL of sterilized water was added thereto and mixed at 56 ° C. for 10 minutes, and then placed on a magnetic stand to collect magnetic particles, and the supernatant was collected.
[0032]
5 μL of each of the collected liquids using three types of magnetic particles was applied to a 1.0% agarose gel, electrophoresed at 100 V for 40 minutes, and then stained with ethidium bromide, and fluorescence bioimage analyzer [FMBIO (registered) Trademark) II Multi-View: manufactured by Takara Shuzo Co., Ltd.] is shown in FIG.
[0033]
(3) Restriction enzyme digestion of recovered λ-DNA Take 10 μL of the recovered solution obtained in (2) extraction and purification of λ-DNA, 3 μL of 10 × M buffer [100 mmol / L Tris-HCl (pH 7. 5), 100 mmol / L magnesium chloride, 10 mmol / L dithiothreitol, 500 mmol / L NaCl], 1 μL of 15 U / μL Hind III (Takara Shuzo) and sterilized water were added to make a total volume of 30 μL, and at 37 ° C. for 16 hours. As a result, it was confirmed that the λ-DNA extracted and purified by the fractionation means according to the present invention was completely cleaved by the restriction enzyme Hind III and could be used immediately for restriction enzyme digestion.
[0034]
【The invention's effect】
According to the present invention, by using a solid phase carrier having a nucleic acid binding ability in the presence of a polyanionic substance, nucleic acid is adsorbed from a sample containing nucleic acid, and further, by using an appropriate eluate, it is complicated. Nucleic acids can be extracted and purified in a short time without manipulation.
[Brief description of the drawings]
FIG. 1 shows that λ-DNA was recovered by magnetic particles in the presence of a polyanionic substance from serum added with λ-DNA (Example 1). In the figure, lane 1: λ-Hind III marker, lane 2: recovery 1 with magnetic particle micromer-M (3 μm), lane 3: recovery 2 with magnetic particle micromer-M (3 μm), lane 4: with other magnetic particle 1 Recovery 1, Lane 5: Recovery 2 with other magnetic particles 1, Lane 6: Recovery 1 with other magnetic particles 2, Lane 7: Recovery 2 with other magnetic particles 2. For collections 1 and 2, the same λ-DNA collection experiment was performed twice for each magnetic particle, and the respective results are shown.

Claims (8)

A reagent for nucleic acid extraction and purification using a magnetic particle having nucleic acid binding ability, which is a liquid reagent containing dextran sulfate . Magnetic particles, magnetic particles comprising a silica or a carboxyl group on the surface, silanol groups or nucleic acid extraction and purification reagent of claim 1 is a magnetic particle having a modified functional group comprising a combination thereof. A nucleic acid extraction and purification kit comprising magnetic particles for adsorbing nucleic acid and a nucleic acid adsorption promoting reagent containing dextran sulfate . Described solution and a washing solution for nucleic acid washing the magnetic particles adsorbed, claim 3 nucleic acid further comprises a a eluate to elute the nucleic acid from the magnetic particles adsorbed to dissolve the biological sample Nucleic acid extraction and purification kit. The nucleic acid extraction and purification kit according to claim 4 , wherein the lysate is a lysate containing protease. The nucleic acid extraction and purification kit according to any one of claims 3 to 5 , wherein the nucleic acid extraction and purification kit is a nucleic acid extraction and purification kit used in an automatic nucleic acid extraction apparatus. Nucleic acid sample containing nucleic acid, a solution and magnetic particles dextran sulfate, a step of binding the nucleic acid to the magnetic particles, the magnetic particles nucleic acid has bound and washing with the washing solution, from the magnetic particles to which the nucleic acid is bound And a step of eluting the nucleic acid. The method for purifying nucleic acid according to claim 7 , further comprising a step of adding a lysis solution to a sample containing the nucleic acid and extracting the nucleic acid before the step of binding the nucleic acid to the magnetic particles .

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