JP2014030364A - Dna extraction method - Google Patents

Abstract

A novel DNA extraction method is provided.
A sample containing deoxyribonucleic acid (DNA) is mixed with a lysate containing a chaotropic substance and a nucleic acid-binding solid phase carrier to adsorb the DNA on the carrier, and the carrier on which the DNA is adsorbed. The step of washing with a washing solution not containing an organic solvent and the step of eluting the DNA adsorbed on the carrier into the eluate at a temperature higher than 70 ° C. are performed.
[Selection figure] None

Description

  The present invention relates to a method for extracting DNA.

Boom et al. Reported a method for more easily extracting nucleic acids from biomaterials using a nucleic acid-binding solid phase carrier such as silica particles and a chaotropic agent (see Non-Patent Document 1). Including the method of Boom et al., A method of adsorbing and extracting a nucleic acid on a carrier using a nucleic acid-binding solid phase carrier such as silica and a chaotropic agent mainly includes (1) a nucleic acid-binding solid phase in the presence of a chaotropic agent. A step of adsorbing nucleic acid to the phase carrier (adsorption step), (2) a step of washing the carrier adsorbed with nucleic acid with a washing solution (washing step) to remove non-specifically bound impurities and chaotropic agents, and ( 3) It consists of three steps of eluting nucleic acid from the carrier with water or a low salt concentration buffer (elution step). Here, as the washing liquid of (2), in order to dissolve the chaotropic agent and prevent elution of the nucleic acid from the carrier, conventionally, a water-soluble organic solvent, particularly ethanol, is used in an amount of 50 to 8.
Water or a low salt concentration buffer containing about 0% is used.

However, when this water-soluble organic solvent remains in the step (3), the enzyme reaction is inhibited when the extract is treated with an enzyme, and therefore, usually after washing with an aqueous solution containing ethanol, as necessary. An operation of washing with 100% ethanol or acetone with higher volatility and then drying to completely remove the organic solvent from the system is performed. Not only does this drying take time, but if the drying time is insufficient, ethanol will remain, and if it is excessive, the nucleic acid will be too dry and solidified, making it difficult to elute, resulting in nucleic acid. It is known that this leads to a decrease in the recovery amount and reproducibility. In this way, the use of organic solvents not only makes it difficult to determine the degree of drying, but organic solvents such as ethanol and acetone are flammable and volatile. There is also a danger.

Therefore, after the nucleic acid is adsorbed on the carrier, the carrier is washed with water or a low salt concentration buffer solution containing no organic solvent such as ethanol in the washing step (2), and 50% in the elution step (3). ~ 70
A method has been developed for extracting ribonucleic acid (RNA) by eluting the nucleic acid with water or a low salt concentration buffer at 0 ° C. (see Patent Document 1).

Japanese Patent Laid-Open No. 11-146783

J.Clin.Microbiol., Vol.28 No.3, p.495-503 (1990)

An object of the present invention is to provide a novel method for extracting deoxyribonucleic acid (DNA).

Until now, after adsorbing a nucleic acid on a carrier in the Boom method, in the washing step (2), the carrier is washed with water or a low salt concentration buffer that does not substantially contain an organic solvent such as ethanol. ) Is released from the carrier prior to ribonucleic acid (RNA), and therefore, in the elution step of (3), the nucleic acid is eluted with water or a low salt concentration aqueous solution at 50 to 70 ° C.
It was thought that RNA could be isolated without contamination of NA (Japanese Patent Application Laid-Open No. 11-146783). However, the present inventors have found that DNA can be extracted by elution of nucleic acid with water or a low salt concentration aqueous solution at 80 ° C. or higher in the elution step (3), and the present invention has been completed.

In one embodiment of the present invention, a sample containing DNA is mixed with a lysate containing a chaotropic substance and a nucleic acid-binding solid phase carrier, and the DNA is adsorbed on the carrier;
A method for extracting DNA, comprising: a step of washing the carrier on which A is adsorbed with a washing solution not containing an organic solvent; and a step of eluting the DNA adsorbed on the carrier in an eluent at a temperature higher than 70 ° C. . The cleaning liquid may be water or a low salt concentration aqueous solution, but the salt concentration of the low salt concentration aqueous solution is preferably 100 mM or less. Moreover, although the eluate may be water or a low salt concentration aqueous solution, the salt concentration of the low salt concentration aqueous solution is preferably 100 mM or less. The DNA adsorbed on the carrier may be eluted at less than 100 ° C. The lysate is 3-7 M guanidine salt, 0-5% nonionic surfactant, 0-0.2 mM E.
It may be a neutral solution containing DTA, 0 to 0.2 M reducing agent. The carrier may be magnetic particles.

  The present invention has made it possible to provide a novel DNA extraction method.

In one Example of this invention, it is a graph which shows the correlation of the eluted DNA yield and elution temperature.

Unless otherwise described in the embodiments and examples, MR Green & J. Sambrook (Ed.
), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Pres
s, Cold Spring Harbor, New York (2012); FM Ausubel, R. Brent, RE Kingston,
DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in M
A method described in a standard protocol collection such as olecular Biology, John Wiley & Sons Ltd., or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Reagent for DNA extraction, etc. ==
In the method for extracting deoxyribonucleic acid (DNA) according to the present invention, a sample containing DNA is mixed with a lysate containing a chaotropic substance and a nucleic acid-binding solid phase carrier, and the DNA is adsorbed on the carrier (adsorption step). ), A step of washing the carrier on which the DNA is adsorbed with a washing solution not containing an organic solvent (washing step), and a step of eluting the DNA adsorbed on the carrier into the eluate at a temperature higher than 70 ° C. (elution step) ,including.

The sample from which DNA is extracted is not particularly limited as long as it contains DNA, a biological sample such as a cell or a cell mass such as a tissue, a virus, a synthetic DNA, or a sample in which impurities or impurities are mixed into DNA once isolated It may be.

The chaotropic substance generates chaotropic ions (monovalent anions having a large ionic radius) in an aqueous solution and has an action of increasing the water solubility of hydrophobic molecules, contributing to adsorption of DNA to a solid phase carrier. If it is a thing, it will not specifically limit. Specific examples include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, sodium perchlorate and the like. preferable. The concentration of these chaotropic substances used varies depending on each substance. For example, when guanidine thiocyanate is used, it is in the range of 3 to 5.5M, and when guanidine hydrochloride is used, it is used at 5M or more. Is preferred.

The lysing solution is not particularly limited as long as it contains such a chaotropic substance, but it may contain a surfactant for the purpose of disrupting cell membranes or denaturing proteins contained in cells. The surfactant is not particularly limited as long as it is generally used for nucleic acid extraction from cells or the like. Specifically, a Triton surfactant such as Triton-X or Tw
Nonionic surfactants such as tween surfactants such as een20 and anionic surfactants such as sodium N-lauroyl sarcosine (SDS) can be mentioned. It is preferable to use it in the range of 1 to 2%. Furthermore, it is preferable that the solution contains a reducing agent such as 2-mercaptoethanol or dithiothreitol. The lysis solution may be a buffer solution, but is preferably neutral at pH 6-8. In consideration of these matters, specifically, it contains 3 to 7 M guanidine salt, 0 to 5% nonionic surfactant, 0 to 0.2 mM EDTA, 0 to 0.2 M reducing agent and the like. It is preferable to do.

The nucleic acid-binding solid phase carrier is not particularly limited as long as it is a solid having a hydrophilic surface capable of adsorbing nucleic acid in the presence of chaotropic ions, that is, holding the nucleic acid by reversible physical binding. Specifically, a substance containing silicon dioxide, for example, silica, glass, diatomaceous earth, or those subjected to surface treatment by chemical modification is preferable, and a complex with a magnetic substance or a superparamagnetic metal oxide is used. More preferred. When surface treatment is performed by chemical modification, an appropriate positive charge may be imparted to the extent that reversible binding to nucleic acids is not hindered.

Specific examples of the nucleic acid-binding solid phase include particles, filters, bags, dishes, reaction vessels, and the like, but are not particularly limited. Among these, the particle form is more preferable in consideration of the efficiency of adsorption and elution. The particle size in that case is not particularly limited,
0.05-500 micrometers may be sufficient, Preferably it is 1-100 micrometers, Most preferably, it is 1-10 micrometers.

The washing liquid is substantially free of organic solvents such as ethanol and isopropyl alcohol and chaotropic substances, and is preferably water or a low salt concentration aqueous solution. In the case of a low salt concentration aqueous solution, a buffer solution is preferable. . The salt concentration of the low salt concentration aqueous solution is 100
mM or less is preferred, 50 mM or less is more preferred, and 15 mM or less is most preferred. The lower limit of the low salt concentration aqueous solution is not particularly limited, but is preferably 0.1 mM or more, more preferably 1 mM or more, and most preferably 10 mM or more. Moreover, this solution may contain surfactants, such as Triton, Tween, and SDS, and pH is not specifically limited. The salt for making the buffer is not particularly limited, but Tris, Hepes, Pipes,
A salt such as phosphoric acid is preferably used.

The eluate is not particularly limited, but water or an aqueous solution having a low salt concentration is preferable, and an eluent substantially free of an organic solvent such as ethanol or isopropyl alcohol and a chaotropic substance is more preferable. In the case of a low salt concentration aqueous solution, a buffer solution is preferred. The salt concentration of the low salt concentration aqueous solution is preferably 100 mM or less, more preferably 50 mM or less, and most preferably 15 mM or less. Although there is no particular lower limit of the low salt concentration aqueous solution, it is preferably 0.1 mM or more,
It is more preferably 1 mM or more, and most preferably 10 mM or more. The salt for making the buffer is not particularly limited, but salts such as Tris, Hepes, Pipes, and phosphoric acid are preferably used, and TE buffer (10 mM Tris-HCl buffer, 1 mM EDTA, pH 8.
0) is most preferred. Note that the washing solution and the elution solution may be the same or different.

== DNA extraction method ==
Specifically, DNA may be extracted as follows.

First, as a lysis step, a sample for extracting DNA and a nucleic acid-binding solid phase carrier are mixed in an appropriate amount of lysate, and the sample is crushed by a homogenizer, a vortex mixer, or the like.
NA is adsorbed on the carrier.

Next, as a washing step, in order to remove non-specifically adsorbed impurities on the carrier, it is preferable to wash the carrier on which the DNA is adsorbed with an appropriate amount of lysis solution. Although the number of times of washing is not particularly limited, it may be washed once to several times.

The term “washing” as used herein refers to an operation of removing a non-specifically bound substance other than DNA from the carrier by bringing the carrier to which the DNA is bound into contact with a washing solution and separating it again. The specific separation method varies depending on the form of the carrier to be used, but when the carrier is in the form of particles such as beads, centrifugation, filtration separation, column operation, and the like can be used. When the carrier contains a magnetic material, the carrier can be easily washed using a magnet or the like.

Then, as an elution step, an appropriate amount of eluate is added to the carrier on which the DNA is adsorbed and mixed by a vortex mixer or the like to elute the DNA from the carrier. At this time, DNA extraction is promoted by heating the eluate. Although heating temperature is not specifically limited, What is necessary is just to be higher than 70 degreeC, 80 degreeC or more is preferable and 95 degreeC or more is more preferable. The upper limit of the heating temperature is not particularly limited, but is preferably less than 200 ° C, more preferably less than 150 ° C, further preferably less than 125 ° C, and further preferably less than 110 ° C. In particular, when it is desired to isolate double-stranded DNA, the temperature is preferably less than 100 ° C. As a heating method, the carrier may be added to the preheated eluate, or may be heated after the carrier is added to the eluate. The heating time is not particularly limited, but is preferably about 30 seconds to 10 minutes.

In order to prevent RNA contamination, after adding an appropriate amount of eluate, heat it to below 70 ° C to elute the RNA, remove the eluate, wash the carrier with a washing solution, and then add a new appropriate amount of elution. The DNA may be eluted from the carrier by adding a solution and heating to a temperature higher than 70 ° C.

Alternatively, in order to remove RNA, RNase may be added to any step in the DNA extraction step. However, in order to efficiently decompose RNA, it is preferable to contain it in the eluate. Ribonuclease A may be contained at 10 to 20 μg / mL. Usually R
PCR and other operations can be performed while Nase is contained. However, when it is desired to remove RNase, the adsorption step-washing step-elution step may be repeated again, or DNA may be purified by other known methods. .

The DNA eluted in this way can be directly used for enzymatic reactions such as PCR without performing desalting and concentration operations such as dialysis and ethanol precipitation.

To 50 μL of whole blood sample in a 1.5 mL Eppendorf microcentrifuge tube, 375 μL of lysate (8 M guanidine hydrochloride, 50 mM)
Ethylenediaminetetraacetic acid dihydrogen dihydrate (EDTA · 2H ₂ O), 10% polyoxyethylene sorbitan monolaurate (Tween 20)] was added and dissolved. To the obtained cell lysate, 20 μL of magnetic silica particles (NPK-101, Toyobo Co., Ltd.) was added, and the mixture was stirred at room temperature.
Stir for a minute with a vortex mixer. Thereafter, the microtube was placed on a magnetic stand (MGS-101, Toyobo Co., Ltd.) to collect magnetic silica particles, and the supernatant was removed.

Next, remove the microtube from the magnetic stand, add 450 μL of Washing Solution I (8M guanidine hydrochloride), mix well, then place it on the magnetic stand again and remove the supernatant to wash the magnetic beads. did. Next, 450 μL of washing solution II (5 mM in the same manner)
The particles were washed with (Tris-HCl buffer). Finally, 50 μL of eluate (sterilized water) is added to the collected particles, the particles are suspended, heated to the temperature shown in Table 1 for 5 minutes, the microtube is placed on a magnetic stand, and the supernatant is removed. It was collected. The amount of DNA in the supernatant was measured by absorbance. The result is shown in FIG.

As a comparative example (conventional method), it was washed with 70% ethanol instead of the washing solution II, and elution was performed by stirring with a vortex mixer at room temperature for 5 minutes.

Table 1 shows the ratio of the amount of DNA extracted by the method of the present invention to the amount of DNA extracted in the comparative example (control) at each temperature as the recovery rate.

Thus, even if the silica to which DNA is bound is washed with a low salt concentration aqueous solution, the DNA remains firmly bound to the silica, and by heating to a temperature higher than 70 ° C., preferably 80 ° C. or higher, DNA can be recovered.

Claims (8)

Mixing a sample containing deoxyribonucleic acid (DNA) with a lysate containing a chaotropic substance and a nucleic acid binding solid phase carrier, and adsorbing the DNA to the carrier;
Washing the carrier on which the DNA has been adsorbed with a washing solution containing no organic solvent;
Eluting the DNA adsorbed on the carrier into an eluate at a temperature higher than 70 ° C .;
A method for extracting DNA comprising The extraction method according to claim 1, wherein the cleaning liquid is water or a low salt concentration aqueous solution. The extraction method according to claim 2, wherein the salt concentration of the low salt concentration aqueous solution is 100 mM or less. The extraction method according to claim 1, wherein the eluate is water or a low salt concentration aqueous solution. The extraction method according to claim 4, wherein the salt concentration of the low salt concentration aqueous solution is 100 mM or less. The DNA adsorbed on the carrier is eluted at less than 100 ° C.
6. The method according to any one of 5 above. The solution is 3-7M guanidine salt, 0-5% nonionic surfactant, 0-0.
The method according to claim 1, which is a neutral lysate containing 2 mM EDTA and 0 to 0.2 M reducing agent. The method according to claim 1, wherein the carrier is magnetic particles.