JP2016158558A - Methods for isolating dna and rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for easily and efficiently isolating both DNA and RNA from a biological sample containing DNA and RNA.SOLUTION: The method for isolating DNA and RNA from the same biological sample containing DNA and RNA comprises the following steps (A)-(F): (A) a step of mixing the biological sample with a first nucleic acid binding carrier in a liquid phase, (B) a step of separating the first nucleic acid binding carrier and supernatant obtained in the step (A), (C) a step of eluting either DNA or RNA from the first nucleic acid binding carrier separated in the step (B), (D) a step of mixing the supernatant separated in the step (B) with a second nucleic acid binding carrier in a liquid phase, (E) a step of separating the second nucleic acid binding carrier and supernatant obtained in the step (D), and (F) a step of eluting, from the second nucleic acid binding carrier separated in the step (E), nucleic acids of RNA or DNA which has not been eluted in the step (C).SELECTED DRAWING: None

Description

  The present invention relates to a method for isolating DNA and RNA. More particularly, it relates to a method for isolating both DNA and RNA from the same biological sample.

DNA is a bearer of life information, and RNA is a molecule that receives the information and plays an important role in protein synthesis and the like in vivo.
In the fields of biochemistry, genetic engineering, and clinical diagnosis, RNA analysis is performed by methods such as Northern blot analysis and reverse transcription polymerase chain rearrangement. To perform such analysis with high accuracy, high-purity RNA is used. It is necessary to use it.
Therefore, as a method of isolating RNA with high purity, an acidic solution containing lithium salt or chaotropic agent and silica particles are mixed with a biological sample containing RNA, RNA is adsorbed on silica particles, and then RNA is eluted. The method of making it propose is proposed (patent document 1).

  In addition to RNA, DNA is also an important molecule widely used for research and clinical analysis in the field of molecular biology. As a method of obtaining both DNA and RNA from a biological sample, after treating the biological sample with a lysis solution, divide this into two, and after treating one biological sample with a buffer solution of pH 7 or higher, etc. RNA is obtained by, for example, adsorbing RNA to a carrier, and the other biological sample is treated with a buffer solution of pH 7 or higher, and then DNA contained therein is adsorbed to the carrier to obtain DNA. (Patent Document 2).

JP-A-10-75784 Special table 2008-518618 gazette

However, in the method as described in Patent Document 2, RNA is easily mixed into the extracted DNA, and it has been necessary to treat with ribonuclease in order to purify the DNA. In addition, when DNA and RNA are obtained from different biological samples, RNA loss in the DNA extraction process and DNA loss in the RNA extraction process occur, and both the yield of DNA and the yield of RNA are reduced. .
Therefore, the problem to be solved by the present invention is to provide a method for easily and efficiently isolating both DNA and RNA from a biological sample containing DNA and RNA.

  The above problems have been solved by the following means.

<1> A method for isolating DNA and RNA from the same biological sample containing DNA and RNA, the method comprising the following steps (A) to (F):
(A) A step of mixing the biological sample and the first nucleic acid binding carrier in a liquid phase (B) A step of separating the first nucleic acid binding carrier and the supernatant obtained in step (A) (C ) A step of eluting one of DNA and RNA from the first nucleic acid binding carrier separated in step (B); and (D) the supernatant separated in step (B) and the second nucleic acid binding carrier. (E) The step of separating the second nucleic acid-binding carrier obtained from step (D) and the supernatant (F) The second nucleic acid binding separated in step (E) A step of eluting nucleic acid that was not eluted in step (C) from RNA and DNA from the carrier

<2> A method for isolating DNA and RNA from the same biological sample containing DNA and RNA, the method comprising the following steps (A) to (F):
(A) A step of mixing the biological sample and the first nucleic acid-binding carrier in a liquid phase to which a lithium salt is added. (B) Separating the RNA-binding carrier and the supernatant obtained in step (A). (C) The step of eluting RNA from the RNA binding carrier separated in step (B) (D) The supernatant separated in step (B) and the second nucleic acid binding carrier are mixed with a lower alcohol. Step of mixing in the added liquid phase (E) Step of separating the DNA-binding carrier and supernatant obtained in step (D) (F) DNA from the DNA-binding carrier separated in step (E) Step of elution <3> The isolation method of <2>, wherein the step (A) is performed under acidic conditions.
<4> The isolation method of <2> or <3>, wherein the lithium salt is at least one selected from an inorganic salt of lithium and an organic salt of lithium.
<5> The isolation according to any one of <2> to <4>, wherein the lithium salt is at least one selected from lithium chloride, lithium acetate, lithium citrate, lithium carbonate, lithium hydroxide, and lithium borate. Method.

<6> A method for isolating DNA and RNA from the same biological sample containing DNA and RNA, the method comprising the following steps (A) to (F):
(A) The step of mixing the biological sample and the first nucleic acid-binding carrier in a liquid phase to which polyalkylene glycol is added. (B) The DNA-binding carrier and the supernatant obtained in step (A). The step of separating (C) The step of eluting DNA from the DNA binding carrier separated in step (B) (D) The supernatant separated in step (B) and the second nucleic acid binding carrier are mixed with lower alcohol. Or a step of mixing in a liquid phase to which a lithium salt is added (E) a step of separating the RNA-binding carrier and supernatant obtained in step (D) (F) an RNA-binding carrier separated in step (E) The step of eluting RNA <7> The isolation method according to <6>, wherein the lower alcohol or lithium salt is a lower alcohol.
<8> The isolation method according to <6> or <7>, wherein the polyalkylene glycol is a polyalkylene glycol having a weight average molecular weight of 6000 to 10,000.

<9> The isolation method according to any one of <1> to <8>, wherein the first nucleic acid-binding carrier and the second nucleic acid-binding carrier are magnetic particles.
<10> The isolation method according to any one of <1> to <9>, wherein the first nucleic acid-binding carrier and the second nucleic acid-binding carrier are non-silica magnetic particles having a nucleic acid-binding functional group.
<11> The isolation method according to any one of <1> to <10>, wherein the liquid phase in step (A) is a liquid phase to which a chaotropic agent is added.
<12> The isolation method according to <11>, wherein the chaotropic agent is at least one selected from guanidinium salts, urea, iodides, perchlorates, thiocyanates and isothiocyanates.
<13> The isolation method according to <12>, wherein the guanidinium salt is at least one selected from guanidinium hydrochloride, guanidinium acetate, guanidinium phosphate, guanidinium thiocyanate, guanidinium isothiocyanate, guanidinium sulfate, and guanidinium carbonate.
<14> The isolation method according to any one of <1> to <13>, which is a method for isolating DNA and RNA without using a nucleolytic enzyme.

  According to the isolation method of the present invention, both DNA and RNA can be isolated easily and efficiently from a biological sample containing DNA and RNA.

The isolation method of the present invention is a method for isolating DNA and RNA from the same biological sample containing DNA and RNA, and includes the following steps (A) to (F).
(A) A step of mixing the biological sample and the first nucleic acid binding carrier in a liquid phase (B) A step of separating the first nucleic acid binding carrier and the supernatant obtained in step (A) (C ) A step of eluting one of DNA and RNA from the first nucleic acid binding carrier separated in step (B); and (D) the supernatant separated in step (B) and the second nucleic acid binding carrier. (E) The step of separating the second nucleic acid-binding carrier obtained from step (D) and the supernatant (F) The second nucleic acid binding separated in step (E) A step of eluting nucleic acid that was not eluted in step (C) from RNA and DNA from the carrier

[Step (A)]
Step (A) is a step of mixing a biological sample containing DNA and RNA and a first nucleic acid-binding carrier in a liquid phase. By such step (A), either one of DNA and RNA is bound to the first nucleic acid binding carrier to form the first nucleic acid binding carrier.

The biological sample is not particularly limited as long as it contains DNA and RNA. For example, in addition to blood composition components such as whole blood, serum, plasma, blood components, various blood cells, blood clots, and platelets, urine, Examples include feces, semen, tissue fluid, saliva, cells separated from these, cultured cells, and the like, and microorganisms and viruses may be used. The biological sample may be pretreated with a stabilizer or the like.
DNA and RNA may be endogenous or exogenous, and may be enzymatically synthesized in vitro. Examples of DNA include genomic DNA, plasmid DNA, synthetic oligo DNA, PCR products, and the like. Examples of RNA include mRNA, rRNA, tRNA, microRNA, and synthetic RNA.
The concentration of the biological sample in the system is preferably 1 to 100 (w / v)%, and more preferably 10 to 60 (w / v)%.

  The first nucleic acid-binding carrier is not particularly limited as long as it is a carrier that can bind nucleic acid; inorganic substances such as silica, glass, diatomaceous earth, metals, metal oxides; organic substances such as resins; Either may be used, but a non-silica carrier is preferred. Even if the resin is composed of natural polymers such as polysaccharides (agarose, dextran, cellulose, etc.), styrene polymer, styrene-butadiene copolymer, (meth) acrylic ester polymer, (meth) acrylonitrile Although it may be composed of a synthetic polymer such as a polymer, a fluorine polymer, polyethylene, polypropylene, or polyester, a resin having a nucleic acid-binding functional group on its surface is preferred.

The first nucleic acid binding carrier preferably has a nucleic acid binding functional group. The nucleic acid-binding functional group is preferably an acidic group that can bind to a nucleic acid. Specific examples include a carboxy group, a sulfonic acid group, a phosphoric acid group, and a phenolic hydroxyl group. From the viewpoint of the dissociation constant, a carboxy group and a sulfonic acid group are preferable, and a carboxy group is more preferable.
The content of the nucleic acid-binding functional group is preferably 1 to 2000 nmol, more preferably 5 to 1000 nmol, and particularly preferably 10 to 100 nmol per 1 mg of the solid content of the carrier from the viewpoint of isolation efficiency. The content of the nucleic acid binding functional group can be measured by an electric conductivity measurement method or the like. In particular, when the DNA is bound to the first nucleic acid-binding carrier to obtain a DNA-binding carrier, the isolation efficiency is greatly improved by setting the content of the nucleic acid-binding functional group to 10 to 100 nmol.

The form of the first nucleic acid binding carrier is not particularly limited, and any of particles, monoliths, membranes, fibers, chips, powders, containers, plates, etc. may be used. Is preferred. In the present invention, the first nucleic acid-binding carrier is preferably a magnetic particle from the viewpoint of ease of separation. As the magnetic particles, magnetic particles containing a magnetic substance in a resin having a nucleic acid-binding functional group on the surface (hereinafter simply referred to as magnetic particles containing a magnetic substance in the resin) are preferable.
The magnetic material may be ferromagnetic, paramagnetic, or superparamagnetic, but is preferably superparamagnetic from the viewpoint of easy magnetic flux collection and redispersion. Examples of the magnetic material include metals such as ferrite, iron oxide, iron, manganese oxide, manganese, nickel oxide, nickel, cobalt oxide, and cobalt, or alloys.

  Specific examples of the magnetic particles containing a magnetic substance in the resin include the following particles (i) to (iv).

(I) Particles in which magnetic fine particles are dispersed in a continuous phase containing a non-magnetic material such as a resin (ii) Particles having a secondary aggregate of magnetic fine particles as a core and a non-magnetic material such as a resin as a shell (Iii) Core particles having core particles composed of a non-magnetic material such as a resin and a magnetic layer (secondary aggregate layer) containing magnetic fine particles provided on the surface of the core particles are used as a core. Particles in which a non-magnetic layer such as resin is provided as a shell on the outermost layer of the mother particle (iv) A resin or silica in which a non-magnetic layer such as resin is provided as a shell on the outermost layer of the particle Particles in which magnetic fine particles are dispersed in pores of porous particles made of, etc. All the particles (i) to (iv) are known and can be produced according to a conventional method.

  Moreover, the average particle diameter (volume average particle diameter) when the first nucleic acid-binding carrier is a particle is preferably 0.1 to 500 μm, more preferably 0.2 to 100 μm, and particularly preferably 0. .3 to 50 μm. The average particle size can be measured by laser diffraction / scattered particle size distribution measurement or the like.

  Among such first nucleic acid-binding carriers, magnetic particles are preferable, and non-silica magnetic particles having a nucleic acid-binding functional group are more preferable.

  The usage-amount of a 1st nucleic acid binding support | carrier is about 0.01-100 mass parts normally with respect to 100 mass parts of biological samples, Preferably it is 0.1-10 mass parts.

As the liquid phase in the step (A), when an RNA is bound to the first nucleic acid-binding carrier to obtain an RNA-binding carrier, one to which a lithium salt is added is preferable. In addition, when a DNA is bound to the first nucleic acid binding carrier to form a DNA binding carrier, it is preferable to add polyalkylene glycol.
The lithium salt is not particularly limited as long as it generates lithium ions in the liquid phase, and may be an inorganic salt of lithium or an organic salt of lithium. Examples include lithium chloride, lithium acetate, lithium citrate, lithium carbonate, lithium hydroxide, lithium borate and the like, and lithium chloride and lithium acetate are preferred. A lithium salt may be used individually by 1 type, or may be used in combination of 2 or more type.
The concentration of the lithium salt in the system is usually 1 to 10M, preferably 3 to 8M.
Examples of the polyalkylene glycol include ABA block copolymers of polyethylene glycol and polypropylene glycol, such as polyethylene glycol, polypropylene glycol, and pluronic surfactants. You may use combining a seed | species or more. As a weight average molecular weight of polyalkylene glycol, 1000-100,000 are preferable and 6000-10000 are more preferable. Such weight average molecular weight can be measured by NMR or the like.
The concentration of polyalkylene glycol in the system is preferably 5 to 10 (w / v)%.

Moreover, as a liquid phase in a process (A), what added the chaotropic agent is preferable. Examples of chaotropic agents include guanidinium salts, ureas, iodides, perchlorates, thiocyanates, and isothiocyanates, which may be used alone or in combination of two or more. Good. Among these, a guanidinium salt is preferable as the chaotropic agent. Examples of the guanidinium salt include guanidinium hydrochloride, guanidinium acetate, guanidinium phosphate, guanidinium thiocyanate, guanidinium isothiocyanate, guanidinium sulfate, and guanidinium carbonate, and guanidinium hydrochloride and guanidinium acetate are particularly preferable.
The concentration of the chaotropic agent in the system is preferably 3-8M.

In addition, when RNA is bound to the first nucleic acid-binding carrier to obtain an RNA-binding carrier, the mixing in the step (A) is preferably performed under acidic conditions, more preferably at pH 6.0 or lower, It is more preferable to carry out at pH 3.0 to 6.0, and it is particularly preferable to carry out at pH 4.0 to 5.5. On the other hand, when DNA is bound to the first nucleic acid-binding carrier to form a DNA-binding carrier, it is preferably carried out under neutral or alkaline conditions, more preferably at pH 6.0 to 9.0. It is particularly preferable to carry out at pH 6.5 to 7.5. The pH may be adjusted using various buffer solutions.
In the present specification, pH means pH at 25 ° C.

Moreover, the liquid phase in the step (A) may contain water, a surfactant, and the like in addition to the above-described components.
Examples of the surfactant include a nonionic surfactant, an anionic surfactant, and a cationic surfactant, and a nonionic surfactant is preferable. Examples of the nonionic surfactant include polyoxyethylene octylphenyl ether (Triton X-100 and the like) and polyoxyethylene monolaurate sorbitan (Tween 20 and the like). In addition, surfactant may be used individually by 1 type, or may be used in combination of 2 or more type.

  The temperature at the time of mixing in the step (A) is usually 10 to 60 ° C., and the mixing time may be about 1 to 10 minutes.

[Process (B)]
Step (B) is a step of separating the first nucleic acid-binding carrier and supernatant obtained in step (A). Specifically, conventional solid-liquid separation such as centrifugation or separation using a magnetic field may be performed to recover the supernatant or the first nucleic acid binding carrier.
Moreover, when the liquid phase in the step (A) is one to which a lithium salt is added, the liquid is separated into an RNA-binding carrier and a supernatant containing DNA by the step (B). On the other hand, when the liquid phase in the step (A) is one to which polyalkylene glycol is added, it is separated into a DNA-binding carrier and a supernatant containing RNA by the step (B).

The first nucleic acid binding carrier separated in the step (B) is preferably washed prior to the step (C) (hereinafter, this step is referred to as a washing step (α)).
The method of the washing step (α) is usually divided into two types depending on the shape of the carrier. When the carrier is in the form of particles, such as magnetic particles, there is a method in which particles are dispersed in the washing liquid for washing. On the other hand, when the carrier is in the form of a plate, the washing liquid is applied to the surface. There is a method of cleaning by contact.
As the washing step (α) when the carrier is magnetic particles, a method of collecting magnetic particles using a magnetic field to separate the magnetic particles from the liquid phase and then redispersing the magnetic particles in the washing liquid is preferable.
In addition, the frequency | count of the washing | cleaning process ((alpha)) may be 1 time or multiple times.

The cleaning liquid is preferably a cleaning liquid containing one or more selected from lithium salts, surfactants and lower alcohols. In particular, when washing an RNA binding carrier, a washing solution containing one or more selected from a lithium salt and a surfactant and a washing solution containing a lower alcohol are preferred, and when washing a DNA binding carrier, a washing solution containing a lower alcohol. Is preferred.
As said lithium salt and surfactant, the thing similar to what can be used at a process (A) is mentioned. Examples of lower alcohols include methanol, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, and the like. You may use it in combination. Of these, ethanol is preferred.
When the RNA binding carrier is washed, the washing solution is preferably acidic, more preferably pH 6.0 or less, still more preferably pH 3.0 to 6.0, and pH 4.0 to 5 Is particularly preferred.

[Process (C)]
Step (C) is a step of eluting one of DNA and RNA from the first nucleic acid binding carrier separated in step (B). Step (C) may be performed according to conventional nucleic acid elution. Examples of the elution method include a method in which the first nucleic acid binding carrier separated in the step (B) is stirred in an aqueous medium such as various buffer solutions or water. Thereafter, conventional solid-liquid separation such as centrifugation or separation using a magnetic field is performed, and if necessary, one of DNA and RNA can be isolated with high purity by removing the aqueous medium. it can.
In addition, when the liquid phase in the step (A) is a solution to which a lithium salt is added, RNA is eluted from the RNA binding carrier in the step (C). On the other hand, when the liquid phase in the step (A) is one to which polyalkylene glycol is added, the DNA is eluted from the DNA binding carrier in the step (C).

[Process (D)]
Step (D) is a step of mixing the supernatant separated in step (B) and the second nucleic acid-binding carrier in a liquid phase. By such step (D), the nucleic acid that was not eluted in step (C) (the nucleic acid contained in the supernatant separated in step (B)) of RNA and DNA was bound to the second nucleic acid-binding carrier. A second nucleic acid binding carrier is formed.

Examples of the second nucleic acid binding carrier include the same as the first nucleic acid binding carrier. Among these, magnetic particles are preferable, and non-silica magnetic particles having a nucleic acid-binding functional group are more preferable.
The amount of the second nucleic acid binding carrier used may be an amount that can sufficiently bind the nucleic acid contained in the supernatant separated in the step (B). About 0.01 to 100 parts by mass, preferably 0.1 to 10 parts by mass.

  As the liquid phase in the step (D), when a DNA is bound to the second nucleic acid-binding carrier to obtain a DNA-binding carrier, a solution to which a lower alcohol is added is preferable. In addition, when RNA is bound to the second nucleic acid-binding carrier to obtain an RNA-binding carrier, those to which a lower alcohol or a lithium salt is added are preferable, and those to which a lower alcohol is added are more preferable.

As lithium salt, the thing similar to what can be used at a process (A) is mentioned.
When using a lithium salt, the concentration of the lithium salt in the system is preferably 3 to 8M.

Examples of the lower alcohol include methanol, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol and the like, and one kind can be used alone or two or more kinds can be combined. May be used. Among these, ethanol, propanol, and isopropanol are preferable, and isopropanol is particularly preferable.
When using a lower alcohol, the concentration of the lower alcohol in the system is preferably 30 to 60 (w / v)%.

  Moreover, the liquid phase in the step (D) may contain water in addition to the above components.

  The temperature at the time of mixing in the step (D) is usually 10 to 60 ° C., and the mixing time may be about 1 to 10 minutes.

[Process (E)]
Step (E) is a step of separating the second nucleic acid binding carrier and the supernatant obtained in step (D). Specifically, conventional solid-liquid separation such as centrifugation or separation using a magnetic field may be performed to recover the supernatant or the second nucleic acid binding carrier.
Moreover, when the liquid phase in the step (A) is a solution to which a lithium salt is added, the DNA in the supernatant separated in the step (B) is bound to the carrier in the step (D), and the step ( E) separates the DNA binding carrier. On the other hand, when the liquid phase in the step (A) is one to which polyalkylene glycol is added, the RNA in the supernatant separated in the step (B) binds to the carrier in the step (D), and the step (E) separates the RNA binding carrier.

The second nucleic acid binding carrier separated in the step (E) is preferably washed prior to the step (F) (hereinafter, this step is referred to as a washing step (β)).
The technique of the washing step (β) is usually divided into two types depending on the shape of the carrier. When the carrier is in the form of particles, such as magnetic particles, there is a method in which particles are dispersed in the washing liquid for washing. On the other hand, when the carrier is in the form of a plate, the washing liquid is applied to the surface. There is a method of cleaning by contact.
As the washing step (β) when the carrier is magnetic particles, a method of collecting magnetic particles using a magnetic field to separate the magnetic particles from the liquid phase and then redispersing the magnetic particles in the washing liquid is preferable.
In addition, the frequency | count of a washing | cleaning process ((beta)) may be 1 time or multiple times.

The cleaning liquid is preferably a cleaning liquid containing at least one selected from lithium salts, surfactants and lower alcohols, and is preferably a cleaning liquid containing lower alcohols.
As said lithium salt and surfactant, the thing similar to what can be used at a process (A) is mentioned. Moreover, as a lower alcohol, the thing similar to the lower alcohol which can be used by the washing | cleaning process ((alpha)) is mentioned.

[Process (F)]
Step (F) is a step of eluting the nucleic acid that was not eluted in step (C) out of RNA and DNA from the second nucleic acid binding carrier separated in step (E). Step (F) may be performed according to conventional nucleic acid elution. Examples of the elution method include a method in which the second nucleic acid-binding carrier separated in step (E) is stirred in an aqueous medium such as various buffers or water. Thereafter, conventional solid-liquid separation such as centrifugation or separation using a magnetic field is performed, and the aqueous medium is removed as necessary. Thus, the nucleic acid that has not been eluted in step (C) of RNA and DNA is increased. It can be isolated in purity.

And according to the isolation method of this invention including the said process (A)-(F), both DNA and RNA can be isolated easily and efficiently from the biological sample containing DNA and RNA.
Since the DNA isolated by the isolation method of the present invention is less contaminated with RNA, and the DNA isolated into the isolated RNA is also less contaminated, according to the isolation method of the present invention, a nucleolytic enzyme ( Both DNA and RNA can be obtained with high purity without degrading the contaminated nucleic acid by using ribonuclease or deoxyribonuclease).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

[Example 1]
(1) Culture of HT29 cells Human colon adenocarcinoma HT29 cells were cultured at 37 ° C. for 3 days in McCoy's 5A medium (manufactured by Thermo) containing 1 (w / v) fetal bovine serum. . Thereafter, the medium was removed, 1 mL of 2 mM EDTA-containing PBS was added, and the mixture was allowed to stand at 37 ° C. for 10 minutes, and the cells were detached from the petri dish. After the peeled cells were suspended in D-PBS (-) (manufactured by Nissui Pharmaceutical), the number of cells in this suspension was measured using a hemocytometer, and a microtube (referred to as tube A). The sample was diluted with D-PBS (−) so that the concentration of HT29 cells was 1 × 10 ⁶ cells / mL. This cell dilution is designated as cell dilution A.

(2) RNA extraction In tube A, the cell dilution A obtained in step (1) (corresponding to 1 × 10 ⁵ HT29 cells) was added to the lysate (guanidine hydrochloride: 6M, sodium acetate-hydrochloric acid buffer ( 200 μL of pH 4.0): 0.2 M, containing Triton X-100: 0.1 (w / v)%) was added to completely lyse the cells. To this, 100 μL of an aqueous suspension containing 1 mg of magnetic particles (Dynabeads MyOne Calboxyl, Life Technologies) and 200 μL of 8 M lithium chloride solution (solvent: water) were added, mixed, and then allowed to stand at room temperature for 10 minutes. .
Next, the tube A was placed on a magnetic stand, the magnetic particles were collected, and the supernatant (supplied as supernatant A) was transferred to another microtube (referred to as tube B) with a micropipette.
Then, 500 μL of a washing solution (including lithium chloride: 3 M, sodium acetate-hydrochloric acid buffer (pH 4.0): 0.2 M, Triton X-100: 0.1 (w / v)%) is added to tube A, After removing from the magnetic stand and stirring, it was placed on the magnetic stand again to collect the magnetic particles, and the supernatant was removed with a micropipette. After repeating this washing operation once more, washing with 1 mL of 70% ethanol was repeated three times, and then the liquid phase was completely removed. 50 μL of water was added to the tube A, removed from the magnetic stand, and stirred to completely disperse the magnetic particles. Thereafter, the tube A was placed on a magnetic stand, the magnetic particles were collected, and the supernatant (referred to as supernatant B) was collected with a micropipette.

(3) DNA extraction To 500 μL of the supernatant A transferred to the tube B in the procedure (2), 300 μL of 2-propanol and 100 μL of an aqueous suspension containing 1 mg of magnetic particles (Dynabeads MyOne Calboxyl, Life Technologies) After adding and mixing, the mixture was allowed to stand at room temperature for 10 minutes. Next, the tube B was placed on a magnetic stand to collect magnetic particles, and the supernatant was removed with a micropipette.
Then, 1 mL of 70% ethanol was added to tube B, removed from the magnetic stand, stirred, and then placed on the magnetic stand again to collect magnetic particles, and the supernatant was removed with a micropipette. After this washing operation was repeated three times in total, the liquid phase was completely removed. 100 μL of water was added to the tube B, removed from the magnetic stand, and stirred to completely disperse the magnetic particles. Thereafter, the tube B was set on a magnetic stand to collect magnetic particles, and the supernatant (supplied as the supernatant C) was collected with a micropipette.

(4) Synthesis of cDNA by reverse transcription PCR From the RNA contained in the supernatant B obtained in step (2), HT29 cells were synthesized by reverse transcription PCR using a transcripter first strand cDNA synthesis kit (Roche). A constantly expressed cDNA was synthesized. The specific procedure is shown below.
First, reverse transcriptase, RNase inhibitor, deoxynucleotide mix, oligo [dT] ₁₈ primer and transcripter reaction buffer were added to 10 μL of supernatant B so that the concentration was as per the manual attached to the kit, and the volume was 20 μL. After being treated at 55 ° C. for 30 minutes, further treated at 85 ° C. for 5 minutes to synthesize cDNA, and then cooled on ice.

(5) Analysis of extracted RNA β-actin mRNA was amplified and detected by real-time PCR using the cDNA synthesized in procedure (4) as a template. Moreover, using the supernatant B obtained in the procedure (2) as a specimen, β-actin DNA was amplified by real-time PCR, and the presence or absence of β-actin DNA was verified. The specific procedure is shown below.
First, 10 μL of cDNA or 10 μL of supernatant B was mixed with 15 μL of PCR Master Mix having the following composition. Then, real-time PCR was performed under the following conditions using 7500 Real Time PCR System (manufactured by Applied Biosystems), and the Ct value was calculated. The results are shown in Table 1.
PCR Master Mix Composition: PCR buffer containing 10 × MgCl 2.5 μL, PCR grade dNTPs 1 μL, Fw primer 0.3 μL, Rv primer 0.3 μL, Taqman probe 0.075 μL, ROX dye 0.125 μL, FastStart Taq DNA Polymerase 0. 1 μL, 1% BSA 2.5 μL, and PCR grade water 8.1 μL
Real-time PCR conditions: 50 ° C.-2 min → 95 ° C.-10 min → (95 ° C.-15 sec, 60 ° C.-1 min) × 40 cycles

(6) Analysis of extracted DNA Using the DNA contained in the supernatant C obtained in the procedure (3) as a template, β-globin DNA was amplified by real-time PCR and detected. In addition, after the supernatant C obtained in the procedure (3) was treated with DNase, reverse transcription PCR was performed. Using this as a sample, β-globin RNA was amplified by real-time PCR, and the presence or absence of β-globin RNA was verified. . Reverse transcription PCR and real-time PCR were performed in the same manner as in procedures (4) and (5) except that the primers used were changed to primers capable of amplifying the β globin gene.
The results are shown in Table 1.

[Example 2]
A Ct value was calculated in the same manner as in Example 1 except that the magnetic particles used in the procedures (2) and (3) of Example 1 were changed to Magnosphere MS160 Carboxyl (manufactured by JSR Life Sciences). The results are shown in Table 1.

Example 3
The Ct value was calculated in the same manner as in Example 1 except that the magnetic particles used in the procedures (2) and (3) of Example 1 were changed to AMPure XP (manufactured by Beckman Coulter). The results are shown in Table 1.

Example 4
A Ct value was calculated in the same manner as in Example 1 except that the magnetic particles used in the procedures (2) and (3) of Example 1 were changed to SeraMag speedbeads (manufactured by GE healthcare). The results are shown in Table 1.

Example 5
The same procedure as in Example 1 was performed, except that 200 μL of the 8M lithium chloride solution (solvent: water) used in the procedure (2) of Example 1 was changed to 200 μL of the 8M lithium acetate solution (solvent: water). Calculated. The results are shown in Table 1.

Example 6
(1) DNA extraction In a microtube (referred to as tube C), a cell solution A (corresponding to 1 × 10 ⁵ HT29 cells) obtained in the same manner as in the procedure (1) of Example 1 was added to a lysate ( 200 μL of guanidine hydrochloride: 6 M, Tris-HCl buffer (pH 7.0): 0.2 M, containing Triton X-100: 0.1 (w / v)%) was added to completely lyse the cells. To this was added 100 μL of an aqueous suspension containing 1 mg of magnetic particles (Magnosphere MS160 Carboxyl, manufactured by JSR Life Sciences) and 200 μL of a 30% (w / v) solution of polyethylene glycol (Mw = 1000) (solvent: water). After mixing, the mixture was allowed to stand at room temperature for 10 minutes.
Next, the tube C was placed on a magnetic stand to collect magnetic particles, and the supernatant (supplied as supernatant D) was transferred to another microtube (referred to as tube D) with a micropipette.
Then, 1 mL of 70% ethanol was added to tube C, removed from the magnetic stand, stirred, and then placed on the magnetic stand again to collect magnetic particles, and the supernatant was removed with a micropipette. After this washing operation was repeated three times in total, the liquid phase was completely removed. 100 μL of water was added to the tube C, removed from the magnetic stand, and stirred to completely disperse the magnetic particles. Thereafter, the tube C was placed on a magnetic stand to collect magnetic particles, and the supernatant (supplied as supernatant E) was collected with a micropipette.

(2) RNA extraction 100 μL of an aqueous suspension containing 300 μL of 2-propanol and 1 mg of magnetic particles (Magnosphere MS160 Carboxyl, manufactured by JSR Life Sciences) in 500 μL of the supernatant D transferred to the tube D in the procedure (1) After mixing, the mixture was allowed to stand at room temperature for 10 minutes. Next, the tube D was set on a magnetic stand, the magnetic particles were collected, and the supernatant was removed with a micropipette.
Then, 1 mL of 70% ethanol was added to tube D, removed from the magnetic stand, stirred, and then placed on the magnetic stand again to collect magnetic particles, and the supernatant was removed with a micropipette. After this washing operation was repeated three times in total, the liquid phase was completely removed. 100 μL of water was added to the tube D, removed from the magnetic stand, and stirred to completely disperse the magnetic particles. Thereafter, the tube D was placed on a magnetic stand to collect magnetic particles, and the supernatant (supplied as supernatant F) was collected with a micropipette.

(3) Detection of DNA and RNA Using supernatant E obtained in step (1), in the same manner as in step (6) of Example 1, detection of β-globin DNA and verification of the presence or absence of β-globin RNA contamination And went. Further, using the supernatant F obtained in the procedure (2), in the same manner as the procedures (4) and (5) of Example 1, detection of β-actin mRNA and verification of the presence or absence of β-actin DNA contamination were performed. went.
These results are shown in Table 1.

Example 7
The Ct value was calculated in the same manner as in Example 6 except that the magnetic particles used in the procedures (1) and (2) of Example 6 were changed to Dynabeads MyOne Carboxyl (manufactured by Life Technologies). The results are shown in Table 1.

Example 8
A Ct value was calculated in the same manner as in Example 6 except that the magnetic particles used in the procedures (1) and (2) of Example 6 were changed to AMPure XP (manufactured by Beckman Coulter). The results are shown in Table 1.

Example 9
A Ct value was calculated in the same manner as in Example 6 except that the magnetic particles used in the procedures (1) and (2) of Example 6 were changed to SeraMag speedbeads (manufactured by GE healthcare). The results are shown in Table 1.

[Comparative Example 1]
The 8M lithium chloride solution (solvent: water) used in the procedure (1) of Example 1 is 2-propanol, and the 2-propanol used in procedure (2) of Example 1 is the 8M lithium chloride solution (solvent: water). The Ct value was calculated by performing the same operation as in Example 1 except that each was changed. The results are shown in Table 1.

  As shown in Table 1 below, both DNA and RNA could be isolated easily and efficiently by the methods of Examples 1-9.

The characteristics of each magnetic particle used in the examples and comparative examples are shown below.
Dynabeads MyOne Calboxyl (manufactured by Life Technologies): carboxy group-containing non-silica magnetic particles (average particle size: 1.0 μm, surface carboxy group content: 1000 nmol / mg beads)
Magnosphere MS160 Carboxyl (manufactured by JSR Life Sciences): carboxy group-containing non-silica magnetic particles (average particle size: 1.5 μm, magnetic substance content: about 28% by mass, surface carboxy group amount: 30 nmol / mg beads)
AMPure XP (manufactured by Beckman Coulter): carboxy group-containing non-silica magnetic particles (average particle size: 1.0 μm, surface carboxy group content: 1000 nmol / mg beads)
SeraMag speedbeads (manufactured by GE healthcare): carboxy group-containing non-silica magnetic particles (average particle size: 1.0 μm, surface carboxy group content: 1000 nmol / mg beads)

[Comparative Example 2]
(1) Extraction of DNA and RNA DNA and RNA were extracted with reference to the description in JP-T-2008-518618. The specific procedure is shown below.
First, cell dilution A (corresponding to 1 × 10 ⁵ HT29 cells) obtained in the same manner as in the procedure (1) of Example 1 was added to a lysis solution (guanidine hydrochloride: 6 M, Tris-HCl buffer (pH 7. 0): 0.2 M, containing Triton X-100: 0.1 (w / v)%) was added in an amount of 200 μL to completely lyse the cells. The cell lysate was divided into 100 μL separate microtubes (referred to as tubes E and F).

In tube E containing 100 μL of cell lysate, 100 μL of aqueous suspension containing 1 mg of magnetic particles (Dynabeads MyOne Calboxyl, Life Technologies) and 200 μL of 8M lithium chloride solution (solvent: water) were added and mixed. And allowed to stand at room temperature for 10 minutes.
Then, 500 μL of a cleaning solution (including lithium chloride: 3 M, sodium acetate-hydrochloric acid buffer (pH 4.0): 0.2 M, Triton X-100: 0.1 (w / v)%) is added to tube E, After removing from the magnetic stand and stirring, it was placed on the magnetic stand again to collect the magnetic particles, and the supernatant was removed with a micropipette. After repeating this washing operation once more, washing with 1 mL of 70% ethanol was repeated three times, and then the liquid phase was completely removed. 50 μL of water was added to the tube E, removed from the magnetic stand, and stirred to completely disperse the magnetic particles. Thereafter, the tube E was set on a magnetic stand to collect magnetic particles, and the supernatant (supplied as the supernatant G) was collected with a micropipette.

In tube F containing 100 μL of cell lysate, 100 μL of an aqueous suspension containing 1 mg of magnetic particles (Dynabeads MyOne Calboxyl, Life Technologies) and 200 μL of 8M lithium chloride solution (solvent: water) are added and mixed. And allowed to stand at room temperature for 10 minutes.
Next, the tube F was placed on a magnetic stand, the magnetic particles were collected, and the supernatant (supplied as supernatant H) was transferred to another microtube (referred to as tube G) using a micropipette.
To 500 μL of the supernatant H transferred to the tube G, 300 μL of 2-propanol and 100 μL of an aqueous suspension containing 1 mg of magnetic particles (Dynabeads MyOne Calboxyl, Life Technologies) were added and mixed, and then allowed to stand at room temperature for 10 minutes. I put it. Next, the tube G was placed on a magnetic stand to collect magnetic particles, and the supernatant was removed with a micropipette.
Then, 1 mL of 70% ethanol was added to tube G, removed from the magnetic stand, stirred, and then placed on the magnetic stand again to collect magnetic particles, and the supernatant was removed with a micropipette. After this washing operation was repeated three times in total, the liquid phase was completely removed. 100 μL of water was added to the tube G, removed from the magnetic stand, and stirred to completely disperse the magnetic particles. Thereafter, the tube G was placed on a magnetic stand to collect magnetic particles, and the supernatant (supplied as Supernatant I) was collected with a micropipette.

(2) Detection of DNA and RNA Using supernatant G obtained in procedure (1), detection of β-actin mRNA and contamination of β-actin DNA in the same manner as procedures (4) and (5) of Example 1 And the presence or absence of was verified. Further, using the supernatant I obtained in the procedure (1), in the same manner as in the procedure (6) of Example 1, detection of β-globin DNA and verification of the presence or absence of contamination with β-globin RNA were performed.
These results are shown in Table 2. In addition, the extraction amount of both DNA and RNA was less than half compared with the case where it isolated by the method of Examples 1-9.

[Reference Example 1]
(1) Extraction of DNA and RNA In cell dilution A (corresponding to 1 × 10 ⁵ HT29 cells) obtained in the same manner as in procedure (1) of Example 1, lysate (guanidine hydrochloride: 6M, Tris- 200 μL of hydrochloric acid buffer (pH 7.0): 0.2 M, containing Triton X-100: 0.1 (w / v)%) was added to completely lyse the cells. This cell lysate was divided into 100 μL separate microtubes (referred to as tubes H and I).
From 100 μL of the cell lysate contained in tube H, DNA was extracted (referred to as extract A) using Blood & Cell culture DNA Mini Kit (manufactured by QIAGEN). In addition, RNA was extracted (referred to as extract B) from 100 μL of the cell lysate contained in tube I using RNeasy Mini Kit (manufactured by QIAGEN). In addition, Blood & Cell culture DNA Mini Kit is equipped with RNase.
(2) Detection of DNA and RNA Detection of β-actin mRNA and contamination of β-actin DNA using extract B obtained in procedure (1) in the same manner as procedures (4) and (5) of Example 1 And the presence or absence of was verified. Further, using the extract A obtained in the procedure (1), in the same manner as in the procedure (6) of Example 1, detection of β-globin DNA and verification of the presence or absence of β-globin RNA were performed.
These results are shown in Table 2.

  As shown in Table 2, in the method of Comparative Example 2, a large amount of RNA was mixed in the extracted DNA, and in order to isolate DNA by such a method, the use of RNase as used in Reference Example 1 was used. Is necessary and the number of processes increases. Moreover, since it was necessary to divide the sample in half, the amount of the extracted nucleic acid was very small.

Claims (14)

A method for isolating DNA and RNA from the same biological sample containing DNA and RNA, the method comprising the following steps (A) to (F):
(A) A step of mixing the biological sample and the first nucleic acid binding carrier in a liquid phase (B) A step of separating the first nucleic acid binding carrier and the supernatant obtained in step (A) (C ) A step of eluting one of DNA and RNA from the first nucleic acid binding carrier separated in step (B); and (D) the supernatant separated in step (B) and the second nucleic acid binding carrier. (E) The step of separating the second nucleic acid-binding carrier obtained from step (D) and the supernatant (F) The second nucleic acid binding separated in step (E) A step of eluting nucleic acid that was not eluted in step (C) from RNA and DNA from the carrier A method for isolating DNA and RNA from the same biological sample containing DNA and RNA, the method comprising the following steps (A) to (F):
(A) A step of mixing the biological sample and the first nucleic acid-binding carrier in a liquid phase to which a lithium salt is added. (B) Separating the RNA-binding carrier and the supernatant obtained in step (A). (C) The step of eluting RNA from the RNA binding carrier separated in step (B) (D) The supernatant separated in step (B) and the second nucleic acid binding carrier are mixed with a lower alcohol. Step of mixing in the added liquid phase (E) Step of separating the DNA-binding carrier and supernatant obtained in step (D) (F) DNA from the DNA-binding carrier separated in step (E) Elution process   The isolation method according to claim 2, wherein step (A) is carried out under acidic conditions.   The isolation method according to claim 2 or 3, wherein the lithium salt is at least one selected from an inorganic salt of lithium and an organic salt of lithium.   The isolation method according to any one of claims 2 to 4, wherein the lithium salt is at least one selected from lithium chloride, lithium acetate, lithium citrate, lithium carbonate, lithium hydroxide, and lithium borate. . A method for isolating DNA and RNA from the same biological sample containing DNA and RNA, the method comprising the following steps (A) to (F):
(A) The step of mixing the biological sample and the first nucleic acid-binding carrier in a liquid phase to which polyalkylene glycol is added. (B) The DNA-binding carrier and the supernatant obtained in step (A). The step of separating (C) The step of eluting DNA from the DNA binding carrier separated in step (B) (D) The supernatant separated in step (B) and the second nucleic acid binding carrier are mixed with lower alcohol. Or a step of mixing in a liquid phase to which a lithium salt is added (E) a step of separating the RNA-binding carrier and supernatant obtained in step (D) (F) an RNA-binding carrier separated in step (E) Eluting RNA from   The isolation method according to claim 6, wherein the lower alcohol or lithium salt is a lower alcohol.   The isolation method according to claim 6 or 7, wherein the polyalkylene glycol is a polyalkylene glycol having a weight average molecular weight of 6000 to 10,000.   The isolation method according to any one of claims 1 to 8, wherein the first nucleic acid-binding carrier and the second nucleic acid-binding carrier are magnetic particles.   The isolation method according to any one of claims 1 to 9, wherein the first nucleic acid-binding carrier and the second nucleic acid-binding carrier are non-silica magnetic particles having a nucleic acid-binding functional group.   The isolation method according to any one of claims 1 to 10, wherein the liquid phase in the step (A) is a liquid phase to which a chaotropic agent is added.   The isolation method according to claim 11, wherein the chaotropic agent is at least one selected from guanidinium salts, urea, iodides, perchlorates, thiocyanates and isothiocyanates.   The isolation method according to claim 12, wherein the guanidinium salt is at least one selected from guanidinium hydrochloride, guanidinium acetate, guanidinium phosphate, guanidinium thiocyanate, guanidinium isothiocyanate, guanidinium sulfate, and guanidinium carbonate.   The isolation method according to any one of claims 1 to 13, which is a method for isolating DNA and RNA without using a nucleolytic enzyme.