CN101120089A - Method for separating and purifying nucleic acid - Google Patents

Abstract

A method for separating and purifying nucleic acid, the method comprising: (1) a step of contacting a sample solution containing nucleic acid with a solid phase to adsorb the nucleic acid on the solid phase; (2) a step of contacting a washing solution with the solid phase to wash the solid phase in a state that the nucleic acid is adsorbed on the solid phase; and (3) a step of contacting a recovering solution with the solid phase to desorb the nucleic acid from the solid phase, wherein the sample solution is prepared by including a step of removing a precipitate component, and adding a surfactant and a water-soluble organic solvent to a supernatant solution of the precipitate.

Description

The method of separation and purification of nucleic acid

Technical field

The present invention relates to extensively to be used in being used in the recombinant nucleic acid technical field separates method with plasmid DNA purification.

Background technology

In many cases, only can obtain the nucleic acid of denier, and in addition, the operation more complicated of separation and purifying, and require a great deal of time.The time-consuming complex operations of this common needs tends to cause the loss of nucleic acid.Conventional method comprises with required DNA introducing e. coli plasmid vector and in carrier it is cultivated that therefore preparing a large amount of required DNA is a kind of operations of often carrying out in the recombinant nucleic acid technical field.At this stage, implement from E.coli recovery by the following method: after carrying out cracking operation to plasmid DNA, remove genomic dna, protein etc. by alkalescence-SDS method, thereby obtain the mixture of RNA and plasmid DNA, and use (for example) post partition method (making spent ion exchange resin) or cesium chloride density gradient ultracentrifuge method from the mixture of RNA and plasmid DNA, to obtain purified plasmid DNA usually.Yet in making the post partition method of spent ion exchange resin, required plasmid DNA is through a large amount of elutriant dilutions.As a result, need the plasmid DNA behind the wash-out be concentrated, and this need carry out complicated operations.In addition, in cesium chloride density gradient ultracentrifuge method, centrifugal treating is carried out for a long time with the large-scale plant high speed rotating.As a result, should careful noting, and should handle also complicatedly, and is uneconomic.

On the other hand, as separating simply, efficiently and the method for purification of nucleic acid, report has a kind of like this separate nucleic acid purification process: be used for making nucleic acid to be adsorbed onto solution and the solution that is used for making nucleic acid desorption on the solid phase on the solid phase, nucleic acid is adsorbed onto on the solid phase, and make its desorption, wherein, described solid phase contains hydroxyl organic polymer (JP-A-2003-128691) in its surface.

Summary of the invention

Yet the technology that proposes in JP-A-2003-128691 can not be removed genomic dna fully and be derived from lower and yield plasmid of the purity of the RNA of bacteria RNA, the plasmid that reclaimed in the process of plasmid purification from E.coli also be not enough.Therefore, must make improvement.

Given this, implementing from the solution that contains RNA and plasmid DNA, to separate and during the method for plasmid DNA purification,, all needing the high yield high purity ground required plasmid DNA of recovery fast for many samples.

The objective of the invention is to provide the method for separation and purification of nucleic acid, this method comprises is adsorbed onto on the solid phase surface sample solution that contains RNA and plasmid DNA, and by the washing etc. with nucleic acid from the solid phase desorption, thereby obtain highly purified plasmid DNA fast with high yield.

As the result of the further investigation of carrying out for the problem that overcomes in the routine techniques, the inventor finds: by will by comprise remove precipitate component and the sample solution that tensio-active agent and water-miscible organic solvent add the method preparation of the step in the sedimentary supernatant liquor is adsorbed onto on the solid phase can high yield, high purity ground reclaims required nucleic acid (plasmid DNA) fast.Particularly, the present invention is verified: the solid phase that includes organic polymer by use can improve the yield and the purity of required nucleic acid significantly, wherein, described organic polymer is that the mixture saponification of the cellulose acetate by making cellulose acetate or acetylation value inequality makes.Finish the present invention based on above-mentioned discovery.

According to the present invention, the method of a kind of like this separation and the required nucleic acid of purifying (plasmid DNA) is provided, this method is used respectively and is used for the solution that the solution and being used to of required nucleic acid that absorption contains the sample solution of required nucleic acid (plasmid DNA) makes nucleic acid desorption on the solid phase, wherein, described sample solution is that preparation method by above sample solution makes.

The present invention realizes above-mentioned purpose by following content.

1. the method for separation and purification of nucleic acid, described method comprises:

(1) sample solution that will contain nucleic acid contacts with solid phase, thereby makes described nucleic acid be adsorbed onto step on the solid phase;

(2) washings is contacted with described solid phase, thereby be attracted to the step of washing solid phase under the state on the described solid phase at described nucleic acid; With

(3) will reclaim liquid and contact, thereby make the step of described nucleic acid desorption on the solid phase with described solid phase;

Wherein, described sample solution is by comprising the method preparation of removing precipitate component and tensio-active agent and water-miscible organic solvent being added the step in the sedimentary supernatant liquor.

2. as the method for above 1 described separation and purification of nucleic acid,

Wherein, described glass or plastic containers.

3. as the method for above 1 or 2 described separation and purification of nucleic acid,

Wherein, described tensio-active agent is a polyoxygenated ethylidene class tensio-active agent.

4. the method for any described separation and purification of nucleic acid as in above 1 to 3,

Wherein, described tensio-active agent is a polyoxygenated ethylidene anhydrosorbitol tensio-active agent.

5. the method for any described separation and purification of nucleic acid as in above 1 to 4,

Wherein, described sample solution is to make by add pretreatment fluid in the sample that contains nucleic acid, and described pretreatment fluid contains and is selected from least a in chaotropic salt, defoamer, nucleic acid stability agent, buffer reagent, sour agent, alkaline agent and the enzyme.

6. the method for any described separation and purification of nucleic acid as in above 1 to 5,

Wherein said solid phase is membranaceous solid phase.

7. the method for any described separation and purification of nucleic acid as in above 1 to 6,

Wherein, described water-miscible organic solvent comprises and is selected from least a in methyl alcohol, ethanol, propyl alcohol and isomer thereof and butanols and the isomer thereof.

8. the method for any described separation and purification of nucleic acid as in above 1 to 7,

Wherein, described solid phase comprises silicon-dioxide or derivatives thereof, diatomite or aluminum oxide.

9. the method for any described separation and purification of nucleic acid as in above 1 to 8,

Wherein said solid phase includes organic polymer.

10. as the method for above 9 described separation and purification of nucleic acid,

Wherein, described solid phase comprises and is selected from least a in Teflon (registered trademark), polyester, polyethersulfone, polycarbonate, acrylate copolymer, urethane, polybenzimidazole, polyolefine, polyvinyl chloride and the polyvinylidene difluoride (PVDF).

11. as the method for above 9 described separation and purification of nucleic acid,

Wherein, described solid phase comprises the nylon of positively charged or negative charge.

12. as the method for above 9 described separation and purification of nucleic acid,

Wherein, described organic polymer has polysaccharide structures.

13. as the method for above 9 described separation and purification of nucleic acid,

Wherein, described organic polymer comprises and is selected from least a in Mierocrystalline cellulose, cellulose mixed esters, nitrocellulose, cellulose acetate and the soluble cotton.

14. a device, it is used for implementing as the step in the method for above 1 to 13 any described separation and purification of nucleic acid automatically.

15. a kit utility, it is used for implementing the method as above 1 to 13 any described separation and purification of nucleic acid, and described kit utility comprises:

(i) separate nucleic acid purification column;

(ii) tensio-active agent;

(iii) pretreatment fluid, it contains and is selected from least a in chaotropic salt, defoamer, nucleic acid stability agent, buffer reagent, sour agent, alkaline agent and the enzyme;

(iv) washings; And

(v) reclaim the reagent of liquid.

Description of drawings

Fig. 1 separates and the nucleic acid of purifying and molecular weight marker carry out agarose gel electrophoresis and the electrophoresis photo that obtains the method according to this invention; And

Fig. 2 separates with the nucleic acid of purifying, separates as a comparison case and the nucleic acid of purifying and molecular weight marker carry out agarose gel electrophoresis and the electrophoresis photo that obtains the method according to this invention,

Wherein M represents molecular weight marker, the 1kb dna ladder band of Invitrogen company; 1 expression does not contain tensio-active agent (lysate A); 2 expression lysates (B); 3 expression lysates (C); 4 expression lysates (D); 5 expression lysates (E); 6 expression lysates (F); 7 expressions contain 0% alcoholic acid lysate (lysate G) (kit utility that QIAGEN company produces); 8 expressions contain 17% alcoholic acid lysate (lysate H) (kit utility that QIAGEN company produces); 9 expressions contain 34% alcoholic acid lysate (lysate I) (kit utility that QIAGEN company produces); 10 expressions contain 0% alcoholic acid lysate (lysate G) (dispersion liquid, alkali solution and the neutralizer that use among the embodiment 1); 11 expressions contain 17% alcoholic acid lysate (lysate H) (dispersion liquid, alkali solution and the neutralizer that use among the embodiment 1); And 12 expression contain 34% alcoholic acid lysate (lysate I) (among the embodiment 1 use dispersion liquid, alkali solution and neutralizer).

Preferred forms of the present invention

Below will describe in detail according to separate nucleic acid of the present invention and purification process.The preparation of sample solution

The sample that contains nucleic acid that uses among the present invention comprises bacterium or cell.

The bacterium or the cell that can use is not particularly limited, as long as it is to contain those of plasmid DNA.

In the present invention, what contained nucleic acid can be in annular and the straight chain in the sample is any, can be in strand and the two strands any, and can be among DNA and the RNA any.Molecular weight to used nucleic acid does not have any qualification.In addition, can be double-stranded (ds) plasmid DNA or strand (ss) phage DNA as the plasmid DNA of required nucleic acid, and to its molecular weight without any qualification.

Sample used herein is meant the optional sample that contains nucleic acid.The kind of sample amplifying nucleic acid can be one or more (two or more).Length to single nucleic acid is not particularly limited.For example, can use and have optional length the nucleic acid of (for example, from several bp to several Mbp).From processing property, the length of nucleic acid is generally several bp to hundreds of kbp.

In the method for the invention, the sample solution that contains nucleic acid by bacterium or cell preparation is contacted with solid phase, thereby the nucleic acid in the sample solution is adsorbed onto on the solid phase, make the nucleic acid that is adsorbed onto on solid phase desorption on the solid phase then.

As mentioned above, in the method for the invention, remove precipitate component and the method for the step in the sedimentary supernatant liquor that adds gained prepares sample solution with tensio-active agent and water-miscible organic solvent by comprising, and it is adsorbed on the solid phase as sample solution, separates thus and the required nucleic acid of purifying (plasmid DNA).

Preferably, prepare described sample solution by adding pretreatment fluid, wherein, described pretreatment fluid contains and is selected from least a in chaotropic salt, tensio-active agent, defoamer, nucleic acid stability agent, buffer reagent, sour agent, alkaline agent and the enzyme.

More preferably, prepare described sample solution by following method:

Use dispersion liquid disperse bacterial or cell;

Add alkali solution with dissolution of bacteria or cell;

Add neutralizer;

Remove precipitate component; And

Tensio-active agent and water-miscible organic solvent (lysate) are added in the sedimentary supernatant liquor.

Described dispersion liquid can contain and is selected from least a in nucleic acid stability agent and the buffer reagent.

Described alkali solution contains alkaline agent and is selected from least a in tensio-active agent, defoamer, buffer reagent and the enzyme.The adding alkali solution can make bacterium or cell and the nucleic acid dissolving in the dispersion liquid institute dispersive solution.As a result, the structure that constitutes cell is dissolved, and nucleic acid can be dispersed in the sample solution.Used alkali solution comprises that hydroxide ion concentration is the aqueous solution of the basic metal analogue of 0.1 to 5 mol.On the other hand, can utilize the protein poor heat resistance and nucleic acid (as, DNA) the stronger characteristic of thermotolerance, uses the heat modification method to replace the alkali solution method.Under the situation of using the heat modification method, heating condition preferably: Heating temperature is 80 ℃ to 100 ℃, and be 5 to 20 minutes heat-up time.Adding under the situation of alkali solution, can use the heat modification method separately or two kinds of methods are used in combination.

Neutralizer contains acid and is selected from least a in chaotropic salt, tensio-active agent, defoamer, nucleic acid stability agent, buffer reagent and the basic metal analogue.Neutralizer preferably plays adding the alkaline bleach liquor cleavage thing acidifying effect of gained behind the alkali solution by adding mineral acid and inorganic salt.Described acid is preferably acetate.For the purpose of the present invention, Suan concentration is unimportant and can change.Can use any inorganic salt to acid, as long as it dissolves in water.Preferred inorganic salt are its negatively charged ion and the sour identical salt of negatively charged ion.For example, under the situation of using acetate, inorganic salt are preferably alkali metal acetate or alkaline-earth metal acetate, are preferably potassium acetate especially.Use acid fully pH to be reduced to and be preferably 4.0 to 6.0, more preferably 4.5 to 5.5 acid range.The variation of the concentration of salt also can be similar with the concentration of acid, but owing to following reason, the concentration of preferably salt is higher.Have at gained solution under the situation of high ion concentration, this solution helps chromosomal DNA and other contamination precipitation, and this makes separate easily plasmid DNA and these impurity.The concentration of salt most preferably is 1.0 mol to 10 mol (based on an alkali valency salt).

In the present invention, described lysate is the solution that contains aforesaid tensio-active agent and water-miscible organic solvent, and it can also contain the compound that is selected from chaotropic salt, defoamer, nucleic acid stability agent, buffer reagent and the basic metal analogue.When described lysate contains tensio-active agent, can improve the yield of required nucleic acid.

Not it is desirable in and keep not dispersive cell by the solution that disperses gained with dispersion liquid.Preferably, when disperse bacterial or cell, use methods such as vortex, the mixing of rapping, overturn with its homodisperse.

Sample solution can contain enzyme.In addition, enzyme can be added in above-mentioned any solution.

RNA degrading enzyme solution can be joined by in the solution that adds the lysate preparation, thereby degrade unwanted RNA in advance.

By specific dna degradation enzyme solution being joined the unwanted DNA that to degrade in the solution by lysate preparation (as, chromogene group DNA).In addition, by specific dna degradation enzyme solution being joined the unwanted DNA that to degrade in the solution that contains required nucleic acid to be recycled (as, chromogene group DNA).

Tensio-active agent

The example of the tensio-active agent that uses among the present invention comprises nonionogenic tenside, anion surfactant, cats product and amphoterics.

Among these, can preferably use anion surfactant and nonionogenic tenside.

Examples of anionic surfactants comprises sulfuric acid ester tensio-active agent, sulfonic acid class tensio-active agent, carboxylic-acid tensio-active agent and phosphoric acid based surfactant.Preferred use alkyl sulfuric ester salt, and more preferably use sodium lauryl sulphate.Preferably, tensio-active agent can be included in the alkali solution, so that promote the dissolving of bacterium or cell.

The example of nonionogenic tenside comprises polyoxygenated ethylidene class tensio-active agent and fatty acid alkyl amide, among these, preferably uses polyoxygenated ethylidene class tensio-active agent.The example of polyoxygenated ethylidene class tensio-active agent comprises polyoxygenated ethylidene alkyl phenyl ethers tensio-active agent and polyoxygenated ethylidene alkyl ethers tensio-active agent.At above-mentioned polyoxygenated ethylidene (after this for easy, sometimes being also referred to as " POE ") in the alkyl ethers tensio-active agent, preferred example comprises POE ether in the last of the ten Heavenly stems, POE lauryl ether, POE tridecyl ether, POE alkylidene group ether in the last of the ten Heavenly stems, POE Span 20, POE dehydrating sorbitol monooleate, POE anhydrosorbitol monostearate, four oleic acid polyoxygenated ethylidene Sorbitol Powders, POE alkylamine and POE alkyne diol.Specifically, the POE anhydrosorbitol class tensio-active agent such as POE Span 20, POE dehydrating sorbitol monooleate, POE anhydrosorbitol monostearate and four oleic acid polyoxygenated ethylidene sorbyl alcohols is preferred.

These tensio-active agents can use separately or use in the mode of its two or more mixture.Surfactant concentrations in alkali solution, neutralizer and the lysate is preferably 0.1 quality % to 30 quality % (in this manual, quality % is equivalent to weight %).

Buffer reagent

The example of the buffer reagent that uses among the present invention comprises the conventional pH buffer reagent that uses.Preferred pH buffer reagent is the pH buffer reagent that biological chemistry is used.The example of the pH buffer reagent that biological chemistry is used comprises the buffer reagent that contains Citrate trianion, phosphoric acid salt or acetate, Tris-HCl, TE (Tris-HCl/EDTA), TBE (Tris-borate/EDTA), TAE (Tris-acetate/EDTA) and Good ' s buffer reagent.The example of Good ' s buffer reagent comprises MES (2-morpholino ethyl sulfonic acid), Bis-Tris (two (2-hydroxyethyl) imino-s three (methylol) methane), HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl] ethyl sulfonic acid), PIPES (piperazinyl-1,4-two (2-ethanesulfonic acid)), ACES (N-(2-kharophen)-2-aminoethyl sulfonic acid), CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) and TES (N-three (methylol) methyl-2-aminoethyl sulfonic acid).

The concentration of these buffer reagents in dispersion liquid, alkali solution, neutralizer, lysate, washings and the recovery liquid be preferably 1 to 300 mmole/liter.

The nucleic acid stability agent

The example of the nucleic acid stability agent of using among the present invention comprises the compound with the effect that makes the nuclease inactivation.For example, the difference of kind per sample may contain the nuclease of the nucleic acid of degrading in sample.(for example) nucleic acid by the situation of homogenize under, nuclease is had an effect to nucleic acid, the result can make the yield of nucleic acid reduce significantly.The nucleic acid stability agent can make the nucleic acid stability ground in the sample exist, and this is preferred.

As nucleic acid stability agent, can use the compound that is often used as reductive agent with the effect that makes the nuclease inactivation.The example of used reductive agent comprises: hydrogen; Hydride is as hydrogen iodide, hydrogen sulfide, lithium aluminum hydride or sodium borohydride; High electropositive metal, as basic metal, magnesium, aluminium or zinc, or their mercury alloys; Organic oxygen compound is as aldehydes, carbohydrate, formic acid or oxalic acid; And sulfhydryl compound.In the middle of these materials, preferably use sulfhydryl compound.The example of sulfhydryl compound comprises: N-acetylcystein, mercaptoethanol and alkyl sulfhydryl.Particularly, preferably use beta-mercaptoethanol.Sulfhydryl compound can use separately or use with two or more mixture.

The concentration of spendable nucleic acid stability agent is preferably 0.1 quality % to 20 quality % in treatment solution, and more preferably 0.3 quality % to 15 quality %.

Can also be with sequestrant as nucleic acid stability agent with the effect that makes the nuclease inactivation.The example of spendable sequestrant comprises EDTA, NTA and EGTA.Sequestrant can use separately or use with the mixture of two kinds or more of sequestrants.For example, can according to 1 to 300 mmole/liter activity use EDTA.Preferably, sequestrant can be contained in the dispersion liquid, thereby play the effect that makes endogenous nuclease inactivation.

The basic metal analogue

The preferred example of used basic metal analogue comprises muriate and acetylate among the present invention.More preferably sodium salt, sylvite and lithium salts.Can use the concentration of the basic metal analogue in dispersion liquid, alkali solution, neutralizer, lysate, washings and recovery liquid to be preferably 0.01 mol or higher, and more preferably 0.01 to 5 mol.

Enzyme

The example of used enzyme comprises protein degrading enzyme, nucleolysis enzyme and muramidase among the present invention.Preferably, use a kind of enzyme at least.In addition, preferably, the mixture of described enzyme with two kinds of enzymes or plurality of enzymes can be used.

Protein degrading enzyme is not particularly limited, and for example, can preferably uses Sumizyme MP.

The nucleolysis enzyme is not particularly limited, and for example, can preferably uses the RNA degrading enzyme.

The RNA degrading enzyme is not particularly limited, and for example, can preferably uses RNaseA or RNase T1.

The example of the dna degradation enzyme that can preferably use comprises the specific nucleic acid excision enzyme of APT dependency exonuclease (trade(brand)name: Plasmid-Safe is positioned at the product of the Epicenter technologies company in Wisconsin, USA Madison city) and strand.These enzyme spcificity ground enzymes are cut linear DNA (for example genomic dna), but can not cut superhelix shape plasmid DNA by enzyme.

Muramidase is not particularly limited, and for example, can preferably uses N,O-Diacetylmuramidase.

The concentration of enzyme is preferably and adds fashionable per 1 ml sample overall solution volume 0.001 to 10IU in the sample solution, more preferably 0.1 arrives 1IU.Can be to be 0.05 to 20mg/mL concentration use sample solution according to activity for another mode of selecting for use.

In order stably to keep the action effect of enzyme, buffer reagent can be joined in the sample solution.In this case, the amount of (for example) Tris-HCl that can add be 1 to 200 mmole/liter.

Can also more preferably use the protein degrading enzyme or the muramidase that do not contain the nucleolysis enzyme.

In addition, can preferably use the enzyme that contains the proteolytic degradation enzyme stabilizers.The stablizer that can preferably use is a metal ion.The example of described metal ion comprises magnesium ion and calcium ion.These ions can be respectively add with the form of (for example) magnesium chloride and calcium acetate.Contain the protein degradation enzyme stabilizers and can greatly reduce the amount that reclaims the required proteolytic degradation enzyme of nucleic acid, thus, can reduce the required expense of nucleic acid that reclaims.The solution of protein degrading enzyme can contain buffer reagent or polyvalent alcohol.For example, can according to 0.1 to 200 mmole/liter amount add Tris-HCl as buffer reagent, perhaps can add glycerine according to 1% to 70% amount as polyvalent alcohol.These buffer reagents and polyvalent alcohol can use separately separately or use in the mode of its two or more mixture.

Defoamer

The example of used defoamer comprises among the present invention: silicone based defoamer (as, silicone oil, dimethyl polysiloxane, organic silicon emulsion, modified polyorganosiloxane or organic silicon composite) and alcohol defoaming agent (as, alkyne diol, enanthol, ethylhexanol, higher alcohol or polyoxyalkylene glycol), the ethers defoamer (for example, heptyl cellosolve or nonyl cellosolve-3-heptyl Sorbitol Powder), oils and fats defoamer (animal oil and vegetables oil), the metallic soap defoamer (for example, aluminum stearate or calcium stearate), the fatty acid ester defoamer (as, natural wax or tributyl phosphate), the phosphoric acid ester defoamer (for example, the octyl group sodium phosphate), the amine defoamer (for example, diamyl amine), the amides defoamer (for example, stearic amide) and other defoamer (for example, ferric sulfate and alumina).As particularly preferred defoamer, two kinds of compositions in silicone based defoamer and the alcohol defoaming agent can be used in combination.In addition, as alcohol defoaming agent, preferably use the acetylenic diols tensio-active agent.The concentration of the defoamer that uses in alkali solution, neutralizer, lysate and the sample solution is preferably 0 to 10 quality %, and more preferably 0.01 quality % to 5 quality %.

Chaotropic salt

The example of used chaotropic salt comprises guanidinesalt, sodium isocyanate, sodium iodide, potassiumiodide among the present invention.In these chaotropic salts, preferably use guanidinesalt.The example of guanidinesalt comprises Guanidinium hydrochloride, guanidinium isothiocyanate and guanidine thiocyanate.In these, preferably use Guanidinium hydrochloride.These chaotropic salts can use separately, or use in the mode of two or more chaotropic salt mixtures.The concentration of the chaotropic salt that uses in neutralizer, lysate or the sample solution is preferably 0.5 mol, 0.5 to 4 mol more preferably, and 1 to 3 mol more preferably.

Urea can be used as from the liquid material, to replace chaotropic salt.

Water-miscible organic solvent

As mentioned above, lysate contains tensio-active agent and water-miscible organic solvent.Sample solution by comprising the method preparation that adds this step of lysate is contacted with solid phase.By this operation, the nucleic acid in the sample solution is adsorbed onto on the solid phase.In order to adsorb, water-miscible organic solvent to be mixed with dissolved nucleic acid blended liquid phase, and need to have salt in the gained sample solution by aforesaid operations dissolved nucleic acid.

In other words, the hydration structure that is present in the water molecules around the nucleic acid by destruction makes nucleic acid be dissolved as unsteady state.When the nucleic acid that is in this state contacts with solid phase, can think that interact between the polar group on lip-deep polar group of nucleic acid and the solid phase surface (solid phase surface preferably as described below), thus, nucleic acid is adsorbed onto on the solid phase surface.The method according to this invention by water-miscible organic solvent being mixed with dissolved nucleic acid blended liquid phase and making the step that has salt in the gained sample solution, plays pendulum nucleic acid.

The example of water-miscible organic solvent comprises alcohols, acetone, acetonitrile and dimethyl formamide.In these water-miscible organic solvents, preferably use alcohols.Alcohols can be any one in primary alconol, secondary alcohol and the tertiary alcohol.In these alcohols, preferably use methyl alcohol, ethanol, propyl alcohol and isomer thereof and butanols and isomer thereof.

The ultimate density that contains the water-miscible organic solvent in the sample solution of nucleic acid is preferably 5 quality % to 90 quality %, and more preferably 20 quality % to 60 quality %.Particularly preferably be, the concentration of the water-miscible organic solvent of adding is high as much as possible in not producing the accumulative scope.

The preferred example of the salt that exists in the gained sample solution comprises various from liquid material (as guanidinesalt, sodium iodide or sodium perchlorate), sodium-chlor, Repone K, ammonium chloride, Sodium Bromide, Potassium Bromide, Calcium Bromide, brometo de amonio, sodium acetate, potassium acetate and ammonium acetate.

The pH of sample solution is preferably 3 to 10, and more preferably 4 to 9, and most preferably be 5 to 8.

The surface tension of gained sample solution is preferably 0.05J/cm ²Perhaps lower, viscosity is preferably 1 to 10,000mPa, and proportion is preferably 0.8 to 1.2.The solution that preparation has this physicals has the following advantages.In next procedure, after the sample solution that contains nucleic acid passes solid phase and nucleic acid and is adsorbed on the solid phase, can remove residual solution at an easy rate.

Solid phase

The solid phase that solid phase is preferably adsorbed nucleic acid by the interaction that does not relate to ionic linkage substantially.This means that under working conditions " ionization " do not take place the solid phase side, and can infer: because polar changes, nucleic acid and solid phase attract each other on every side.Thus, can separate and purification of nucleic acid with the separation performance and the good washing efficient of excellence.Can infer: solid phase is the solid phase of possess hydrophilic property group, and because polar change on every side, the hydrophilic radical of nucleic acid and the hydrophilic radical of solid phase attract each other.

Hydrophilic radical used herein is meant and can interactional polar group (atomic group) takes place with water, participates in the group (atomic group) of absorption nucleic acid corresponding to all.Preferably, hydrophilic radical be with glassware for drinking water have the medium tenacity level interactional hydrophilic radical (referring to by altogether upright publish described in " the chemical dictionary " that Co., Ltd. publishes about " group that wetting ability is not too strong " in " hydrophilic radical ").The example of this hydrophilic radical comprises hydroxyl, carboxyl, cyano group and oxyethylene group.In these hydrophilic radicals, preferably use hydroxyl.

The solid phase of possess hydrophilic property group used herein is meant such solid phase: form the possess hydrophilic property group of material of solid phase own, or refer to such solid phase: by handling or applying hydrophilic radical is introduced in the material that forms solid phase.Under the situation that the material that forms solid phase is handled or applied, form any that the material of solid phase can be in organic materials and the inorganic materials.The example of spendable solid phase comprises: wherein form this solid phase as the organic materials of possess hydrophilic property group of material; Solid phase to the organic materials that do not contain hydrophilic radical is handled, thereby to the solid phase of wherein introducing hydrophilic radical; By applying with the solid phase of the material with hydrophilic radical to the organic materials that do not contain hydrophilic radical, thereby to the solid phase of wherein introducing hydrophilic radical; Wherein form this solid phase of material as the inorganic materials of possess hydrophilic property group; Solid phase to the inorganic materials that do not contain hydrophilic radical is handled, thereby to the solid phase of wherein introducing hydrophilic radical; With apply with the solid phase of the material with hydrophilic radical the inorganic materials that do not contain hydrophilic radical, thereby to the solid phase of wherein introducing hydrophilic radical.Consider from the angle that operation is easy to carry out, preferably use organic materials (as organic polymer) as the material that forms solid phase.

The solid phase of the material of possess hydrophilic property group can comprise the solid phase of the organic materials with hydroxyl.The example of the solid phase of this organic materials with hydroxyl comprises: poly-hydroxyethyl vinylformic acid, poly-hydroxyethyl methacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, polymethyl acrylic acid, polyoxyethylene, the polymeric amide (nylon of acryl coating for example; this nylon can be positively charged, also can be electronegative), polypropylene and have the organic polymer of polysaccharide structures.

The example of the organic polymer with polysaccharide structures that can preferably use comprises Mierocrystalline cellulose, hemicellulose, dextran, agarose, dextrin, amylose starch, starch extract, starch, glycogen, amylopectin, mannosans, glucomannan, moss starch, isolichenin, laminariose, carrageenin, xylan, Polylevulosan, alginic acid, hyaluronic acid, chrondroitin, chitin and chitosan.But organic polymer is not limited to above-mentioned materials, as long as it has polysaccharide structures and polysaccharide structures derivative.Also can preferably use the ester derivative of any above-mentioned polysaccharide structures.In addition, can preferably use the saponification resultant of the ester derivative of any above-mentioned polysaccharide structures.

Preferably, the ester of the ester derivative of any above-mentioned polysaccharide structures is selected from least a in carboxylicesters, nitric ether, sulfuric ester, sulphonate, phosphoric acid ester, phosphonic acid ester and the pyrophosphate.In addition, can preferably use the saponification resultant of carboxylicesters, nitric ether, sulfuric ester, sulphonate, phosphoric acid ester, phosphonic acid ester and the pyrophosphate of any above-mentioned polysaccharide structures.

Preferably, the carboxylicesters of any above-mentioned polysaccharide structures is selected from least a in alkyl-carbonyl ester, alkenyl carbonyl ester, aromatic base carbonyl ester and the aromatic alkyl carbonyl ester.In addition, can preferably use the saponification resultant of alkyl-carbonyl ester, alkenyl carbonyl ester, aromatic base carbonyl ester and the aromatic alkyl carbonyl ester of any above-mentioned polysaccharide structures.

Preferably, the ester group of the alkyl-carbonyl ester of any above-mentioned polysaccharide structures is selected from least a in ethanoyl, propionyl, butyryl radicals, pentanoyl, oenanthyl, capryloyl, decanoyl, lauroyl, tridecanoyl, hexadecanoyl and the octadecanoyl.In addition, can preferably use saponification resultant with any above-mentioned polysaccharide structures that is selected from least a ester group in ethanoyl, propionyl, butyryl radicals, pentanoyl, oenanthyl, capryloyl, decanoyl, lauroyl, tridecanoyl, hexadecanoyl and the octadecanoyl.

Preferably, the ester group of the alkenyl carbonyl ester of any above-mentioned polysaccharide structures is selected from least a in acryl and the methacryloyl.In addition, can preferably use saponification resultant with any above-mentioned polysaccharide structures that is selected from least a ester group in acryl and the methacryloyl.

Preferably, the ester group of the aromatic base carbonyl ester of any above-mentioned polysaccharide structures is selected from least a in benzoyl group and the naphthoyl base (naphthaloyl).In addition, can preferably use saponification resultant with any above-mentioned polysaccharide structures that is selected from least a ester group in benzoyl group and the naphthoyl base.

The example of the nitric ether of any above-mentioned polysaccharide structures that can preferably use comprises nitrocellulose, the nitric acid hemicellulose, the nitric acid dextran, the nitric acid agarose, the nitric acid dextrin, the nitric acid amylose starch, the starch nitrate essence, the nitric acid glycogen, the nitric acid amylopectin, the nitric acid mannosans, the nitric acid glucomannan, the nitric acid moss starch, the nitric acid isolichenin, the nitric acid laminariose, the nitric acid carrageenin, nitric acid xylan (nitroxylam), the nitric acid Polylevulosan, the nitric acid alginic acid, the nitric acid hyaluronic acid, the nitric acid chrondroitin, nitric acid chitin and nitric acid chitosan.

In addition, can preferably use the saponification resultant of above-mentioned nitrocellulose, nitric acid hemicellulose, nitric acid dextran, nitric acid agarose, nitric acid dextrin, nitric acid amylose starch, starch nitrate essence, nitric acid glycogen, nitric acid amylopectin, nitric acid mannosans, nitric acid glucomannan, nitric acid moss starch, nitric acid isolichenin, nitric acid laminariose, nitric acid carrageenin, nitric acid xylan, nitric acid Polylevulosan, nitric acid alginic acid, nitric acid hyaluronic acid, nitric acid chrondroitin, nitric acid chitin and nitric acid chitosan.

The example of the sulfuric ester with any above-mentioned polysaccharide structures that can preferably use comprises sulfate cellulose, the sulfuric acid hemicellulose, T 500, the sulfuric acid agarose, Dextrin sulfate, the sulfuric acid amylose starch, the starch sulfate essence, the sulfuric acid glycogen, Amylopectin Sultate, the sulfuric acid mannosans, the sulfuric acid glucomannan, the sulfuric acid moss starch, the sulfuric acid isolichenin, the sulfuric acid laminariose, the sulfuric acid carrageenin, the sulfuric acid xylan, the sulfuric acid Polylevulosan, the sulfuric acid alginic acid, sulfated hyaluronic acid, chondroitin sulfate, sulfuric acid chitin and sulfuric acid chitosan.In addition, can preferably use the saponification resultant of above-mentioned sulfate cellulose, sulfuric acid hemicellulose, T 500, sulfuric acid agarose, Dextrin sulfate, sulfuric acid amylose starch, starch sulfate essence, sulfuric acid glycogen, Amylopectin Sultate, sulfuric acid mannosans, sulfuric acid glucomannan, sulfuric acid moss starch, sulfuric acid isolichenin, sulfuric acid laminariose, sulfuric acid carrageenin, sulfuric acid xylan, sulfuric acid Polylevulosan, sulfuric acid alginic acid, sulfated hyaluronic acid, chondroitin sulfate, sulfuric acid chitin and sulfuric acid chitosan.

Preferably, the sulphonate with any above-mentioned polysaccharide structures is selected from least a in alkyl sulfonic ester, olefin sulfonic acid ester, aromatic base sulphonate and the sweet-smelling alkyl sulfonic acid ester.In addition, can preferably use the saponification resultant of abovementioned alkyl sulphonate, olefin sulfonic acid ester, aromatic base sulphonate and sweet-smelling alkyl sulfonic acid ester.

The example of the phosphoric acid ester with any above-mentioned polysaccharide structures that can preferably use comprises phosphorylated cotton, the phosphoric acid hemicellulose, phosphorylated glucan, the phosphoric acid agarose, the phosphoric acid dextrin, the phosphoric acid amylose starch, the starch phosphates essence, the phosphoric acid glycogen, the phosphoric acid amylopectin, phosphomannan, the phosphoric acid glucomannan, the phosphoric acid moss starch, the phosphoric acid isolichenin, the phosphoric acid laminariose, the phosphoric acid carrageenin, the phosphoric acid xylan, the phosphoric acid Polylevulosan, the phosphoric acid alginic acid, the phosphoric acid hyaluronic acid, the phosphoric acid chrondroitin, phosphoric acid chitin and calcium phosphate-chitosan.In addition, can preferably use the saponification resultant of above-mentioned phosphorylated cotton, phosphoric acid hemicellulose, phosphorylated glucan, phosphoric acid agarose, phosphoric acid dextrin, starch phosphates enzyme, starch phosphates essence, phosphoric acid glycogen, phosphoric acid amylopectin, phosphomannan, phosphoric acid glucomannan, phosphoric acid moss starch, phosphoric acid isolichenin, phosphoric acid laminariose, phosphoric acid carrageenin, phosphoric acid xylan, phosphoric acid Polylevulosan, phosphoric acid alginic acid, phosphoric acid hyaluronic acid, phosphoric acid chrondroitin, phosphoric acid chitin and calcium phosphate-chitosan.

The example of the phosphonic acid ester with any above-mentioned polysaccharide structures that can preferably use comprises the phosphonic acids Mierocrystalline cellulose, the phosphonic acids hemicellulose, the phosphonic acids dextran, the phosphonic acids agarose, the phosphonic acids dextrin, the phosphonic acids amylose starch, the phosphonic acids starch extract, the phosphonic acids glycogen, the phosphonic acids amylopectin, the phosphonic acids mannosans, the phosphonic acids glucomannan, the phosphonic acids moss starch, the phosphonic acids isolichenin, the phosphonic acids laminariose, the phosphonic acids carrageenin, the phosphonic acids xylan, the phosphonic acids Polylevulosan, the phosphonic acids alginic acid, the phosphonic acids hyaluronic acid, the phosphonic acids chrondroitin, phosphonic acids chitin and phosphonic acids chitosan.In addition, can preferably use the saponification resultant of above-mentioned phosphonic acids Mierocrystalline cellulose, phosphonic acids hemicellulose, phosphonic acids dextran, phosphonic acids agarose, phosphonic acids dextrin, phosphonic acids amylose starch, phosphonic acids starch extract, phosphonic acids glycogen, phosphonic acids amylopectin, phosphonic acids mannosans, phosphonic acids glucomannan, phosphonic acids moss starch, phosphonic acids isolichenin, phosphonic acids laminariose, phosphonic acids carrageenin, phosphonic acids xylan, phosphonic acids Polylevulosan, phosphonic acids alginic acid, phosphonic acids hyaluronic acid, phosphonic acids chrondroitin, phosphonic acids chitin and phosphonic acids chitosan.

The example of the pyrophosphate with any above-mentioned polysaccharide structures that can preferably use comprises the tetra-sodium Mierocrystalline cellulose, the tetra-sodium hemicellulose, the tetra-sodium dextran, the tetra-sodium agarose, the tetra-sodium dextrin, the tetra-sodium amylose starch, the tetra-sodium starch extract, the tetra-sodium glycogen, the tetra-sodium amylopectin, the tetra-sodium mannosans, the tetra-sodium glucomannan, the tetra-sodium moss starch, the tetra-sodium isolichenin, the tetra-sodium laminariose, the tetra-sodium carrageenin, the tetra-sodium xylan, the tetra-sodium Polylevulosan, the tetra-sodium alginic acid, the tetra-sodium hyaluronic acid, the tetra-sodium chrondroitin, tetra-sodium chitin and tetra-sodium chitosan.In addition, can preferably use above-mentioned tetra-sodium Mierocrystalline cellulose, the tetra-sodium hemicellulose, the tetra-sodium dextran, the tetra-sodium agarose, the tetra-sodium dextrin, the tetra-sodium amylose starch, the tetra-sodium starch extract, the tetra-sodium glycogen, the tetra-sodium amylopectin, the tetra-sodium mannosans, the tetra-sodium glucomannan, the tetra-sodium moss starch, the tetra-sodium isolichenin, the tetra-sodium laminariose, the tetra-sodium carrageenin, the tetra-sodium xylan, the tetra-sodium Polylevulosan, the tetra-sodium alginic acid, the tetra-sodium hyaluronic acid, the tetra-sodium chrondroitin, the saponification resultant of tetra-sodium chitin and tetra-sodium chitosan.

Spendable example with ether derivative of above-mentioned polysaccharide structures comprises methylcellulose gum, ethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, propyloic-carbamyl ethyl cellulose, Walocel MT 20.000PV, Natvosol, hydroxypropylcellulose, Vltra tears, hydroxyethylmethyl-cellulose, cyanoethyl cellulose and carbamyl ethyl cellulose.But ether derivative is not limited to above-mentioned materials.In these ether derivatives, preferably use Walocel MT 20.000PV and Natvosol.

Preferably the hydroxyl in the wherein any above-mentioned polysaccharide structures of use is by the compound that replaces with any degree halogenation.

The solid phase that contains the organic polymer with polysaccharide structures that can preferably use is a cellulose acetate.In addition, also can use the organic polymer solid phase that contains the mutually different cellulose acetate mixture of acetylation value.The example of the mutually different cellulose acetate mixture of the acetylation value that can preferably use comprises: the mixture of cellulosetri-acetate and Cellulose diacetate; The mixture of cellulosetri-acetate and single cellulose acetate; The mixture of cellulosetri-acetate, Cellulose diacetate and single cellulose acetate; Mixture with Cellulose diacetate and single cellulose acetate.In these cellulose acetate mixtures, can especially preferably use the mixture of cellulosetri-acetate and Cellulose diacetate.The ratio of mixture of cellulosetri-acetate and Cellulose diacetate (mass ratio) is preferably 99: 1 to 1: 99 and more preferably 90: 10 to 50: 50.

The particularly preferred solid phase that contains cellulose acetate is the surperficial saponification resultant of the cellulose acetate described in patent documentation JP-A-2003-128691.The surperficial saponification resultant of cellulose acetate is the material by the mixture saponification gained that makes the mutually different cellulose acetate of acetylation value.Also can preferably use the saponification resultant of the mixture of cellulosetri-acetate and Cellulose diacetate, the saponification resultant of the mixture of cellulosetri-acetate and single cellulose acetate, the saponification resultant of the mixture of the saponification resultant of the mixture of cellulosetri-acetate, Cellulose diacetate and single cellulose acetate and Cellulose diacetate and single cellulose acetate.More preferably use the saponification resultant of the mixture of cellulosetri-acetate and Cellulose diacetate.The ratio of mixture of cellulosetri-acetate and Cellulose diacetate (mass ratio) is preferably 99: 1 to 1: 99, and more preferably 90: 10 to 50: 50.In this case, just can control hydroxy radical content (density) on the solid phase surface according to the degree (saponification deg) that saponification is handled.In order to improve the separation efficiency of nucleic acid, the hydroxyl of preferred high level (density).The saponification degree (surperficial saponification degree) of the solid phase that obtains by saponification is preferably 5% to 100%, and more preferably 10% to 100%.In addition, in order to increase the surface-area of solid phase, preferably, make the saponification of cellulose acetate solid phase.

Saponification used herein is to instigate cellulose acetate and saponification liquor (for example, aqueous sodium hydroxide solution) contact.By this processing, the cellulose acetate that contacts with saponification liquor partly is converted into regenerated cellulose, thereby introduces hydroxyl.So the regenerated cellulose of preparation is different with the original fibers element at aspects such as crystal forms.The particularly preferred regenerated cellulose solid phase that is to use is as solid phase among the present invention.

In order to change saponification degree, can carry out saponification by changing concentration sodium hydroxide.Can easily measure saponification degree (for example, can measure) by NMR by the minimizing degree of carbonyl peak value.

Method in the organic materials solid phase do not contain hydrophilic radical that hydrophilic radical introduced is that the grafted polymer with possess hydrophilic property group on main polymer chain or the side chain is bonded on the solid phase.

Two kinds of methods are arranged as the method on the solid phase that grafted polymer is bonded to organic materials.A kind of method is the method that makes solid phase and grafted polymer chemical bonding, and another kind of method is to use solid phase as starting point, makes the compound polymerization with polymerizable double bond, thereby forms the method for grafted polymer.

In the method that makes solid phase and grafted polymer chemical bonding, use the polymkeric substance that on the end of polymkeric substance or side chain, has the functional group that can react with solid phase.Between the functional group of this functional group and solid phase chemical reaction taking place and carries out grafting.To being not particularly limited with the functional group that solid phase reacts, if it can with the functional group reactions of solid phase.The example of the functional group that reacts with solid phase comprises: silane coupled base (as, organoalkoxysilane), isocyanate group, amino, hydroxyl, carboxyl, sulfonic group, phosphate, epoxy group(ing), allyl group, methacryloyl and acryl.

The example that can be used as the compound of the polymkeric substance that has reactive functional groups on polymer ends or side chain especially comprises: its end have trialkoxysilyl polymkeric substance, its end have amino polymkeric substance, its end have carboxyl polymkeric substance, have the polymkeric substance of epoxy group(ing) and the polymkeric substance that has isocyanate group at its end at its end.The polymkeric substance that uses in this embodiment is not particularly limited, as long as it has the hydrophilic radical that participates in absorption nucleic acid.The example of this polymkeric substance comprises: poly-hydroxyethyl vinylformic acid, poly-hydroxyethyl methacrylic acid and their salt; Polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, polymethyl acrylic acid and salt thereof; And polyoxyethylene.

With solid phase as starting point, make compound carry out polymerization and the method that forms grafted polymer is commonly called the surface grafting polymerization method with polymerizable double bond.The surface grafting polymerization method is meant a kind of like this method: by plasma radiation, optical radiation or heating etc., make substrate surface produce reactive site, thus will have polymerizable double bond and the compound that is set to contact with solid phase by polyreaction and solid phase bonding.

Can be used for forming these two prerequisites of hydrophilic radical that compound with the grafted polymer of base material bonding need have polymerisable double bonds simultaneously and participate in absorption nucleic acid.Any compound in the monomer of the oligopolymer of the polymkeric substance of possess hydrophilic property group, possess hydrophilic property group and possess hydrophilic property group all can be used as this compounds, as long as have two keys in its molecule.Useful especially compound is the monomer of possess hydrophilic property group.

The useful especially monomeric example of possess hydrophilic property group is monomer for example vinylformic acid 2-hydroxyl ethyl ester, methacrylic acid 2-hydroxyl ethyl ester and the monomethyl vinylformic acid glyceryl ester that contains hydroxyl.In addition, can preferably use the monomer that contains carboxyl, for example acrylic or methacrylic acid or its an alkali metal salt or amine salt.

The method that other hydrophilic radical is incorporated in the solid phase of the organic materials of possess hydrophilic property group not is with the material coating solid phase with hydrophilic radical.The material that is used to apply is not particularly limited, as long as it has the hydrophilic radical that participates in nucleic acid absorption.But on the angle of carrying out from processing ease, the preferred polymkeric substance that comprises organic materials that uses is as the material that is used to apply.The example of this polymkeric substance comprises: poly-hydroxyethyl vinylformic acid, poly-hydroxyethyl methacrylic acid or their salt; Polyvinyl alcohol, polyvinylpyrrolidone and their salt; The mixture of the cellulose acetate that polyoxyethylene, cellulose acetate and acetylation value have nothing in common with each other.The preferred polymkeric substance that uses with polysaccharide structures.

Spendable a kind of method is: the mixture of cellulose acetate or the mutually different cellulose acetate of acetylation value is coated on the organic materials solid phase of possess hydrophilic property group not, and the coating of the mixture of the cellulose acetate that has nothing in common with each other of Dichlorodiphenyl Acetate Mierocrystalline cellulose or acetylation value is carried out saponification.In the method, saponification degree is preferably 5% to 100%, and more preferably 10% to 100%.

The example of the inorganic materials solid phase of possess hydrophilic property group comprises the solid phase that contains silicon-dioxide or derivatives thereof, diatomite or alumina cpd.The solid phase that contains silicon dioxide compound can be the glass filter membrane.The example of solid phase can also comprise as Japanese Patent No.3, the silicon dioxide porous membrane described in 058,342.This silicon dioxide porous membrane can prepare by the following method: the developping solution with the cationically ampholytic material that forms the bilayer capsule ability is spread on the substrate, remove the liquid membrane from substrate then and desolvate, thereby make the plurality of layers of double molecular film of amphiprotic substance, the plurality of layers of double molecular film is contacted with the solution that contains silicon dioxide compound, and extraction plurality of layers of double molecular film is also removed.

Two kinds of methods are arranged as hydrophilic radical is introduced the not method of the inorganic materials solid phase of possess hydrophilic property group.A kind of method is the method with the grafted polymer chemical bonding of solid phase and possess hydrophilic property group; Another kind method is to use the method for grafted polymer, wherein uses the monomer of intramolecularly possess hydrophilic property group and two keys, and with solid phase as starting point, polymerization becomes grafted polymer.

Under the situation with the grafted polymer chemical bonding of solid phase and possess hydrophilic property group, the functional group that will react with the functional end-group of grafted polymer introduces in the inorganic materials, and graftomer is bonded on it.In addition, at the monomer that uses intramolecularly possess hydrophilic property group and two keys, and as starting point, polymerization becomes under the situation of grafted polymer with solid phase, will the functional group as starting point be incorporated in the inorganic materials when the compound with two keys carries out polymerization.

The monomer of the graftomer of possess hydrophilic property group and intramolecularly possess hydrophilic property group and two keys can preferably use the graftomer of the possess hydrophilic property group described in the method in the above-mentioned organic materials solid phase that hydrophilic radical is incorporated into possess hydrophilic property group not and the monomer of intramolecularly possess hydrophilic property group and two keys respectively.

Method in the another kind of inorganic materials solid phase of hydrophilic radical being introduced possess hydrophilic property group not is to apply with the material with hydrophilic radical.Be not particularly limited applying used material, as long as it has the hydrophilic radical that participates in nucleic acid absorption.On the angle of carrying out from processing ease, the preferred polymkeric substance that comprises organic materials that uses is as the material that is used to apply.The example of this polymkeric substance comprises: poly-hydroxyethyl vinylformic acid, poly-hydroxyethyl methacrylic acid and their salt; Polyvinyl alcohol, polyvinylpyrrolidone and their salt; The mixture of polyoxyethylene, cellulose acetate and the mutually different cellulose acetate of acetylation value.

Spendable a kind of method is: the mixture with cellulose acetate or the mutually different cellulose acetate of acetylation value applies the not organic materials solid phase of possess hydrophilic property group, and the coating of the mixture of the cellulose acetate that has nothing in common with each other of Dichlorodiphenyl Acetate Mierocrystalline cellulose or acetylation value is carried out saponification.In the method, saponification degree is preferably 5% to 100%, and more preferably 10% to 100%.

Containing not, the example of the solid phase of the inorganic materials of possess hydrophilic property group comprises: metal (as aluminium), glass, cement, pottery (as porcelain and pottery) and the solid phase that processing such as new ceramics, silicon or gac are made.

The preferred solid phase of using strainer or form membrane, its reason is that solution can pass through from its inside.In this case, the thickness of solid phase is preferably 10 to 500 μ m, and 50 to 250 μ m more preferably.Consider the easy degree of washing, the solid phase that preferred thickness is thin as far as possible.

The mean pore size of the solid phase that solution can therefrom pass through is preferably 0.1 to 10 μ m, and 1 to 5 μ m more preferably.Because solid phase has the mean pore size in this scope, thus can obtain being used to adsorb enough surface-area of nucleic acid, and the hole is not easy to be blocked.Can use bubble point method (according to ASTM 316-86 and JIS 3832) to determine the mean pore size of the solid phase that solution can therefrom pass.

The solid phase that solution can therefrom pass through can be the porous-film that its front surface and rear surface are mutually symmetrical, but can preferably use front surface and rear surface porous-film asymmetrical relative to one another." front surface and rear surface are asymmetrical relative to one another " of Shi Yonging is meant one side from porous-film to its another side in this article, and the physics or the chemical property of film change.

An example of film physical properties is a mean pore size, and an example of chemical property is a saponification degree.

When using its front surface and rear surface porous-film asymmetrical relative to one another in the present invention, preferably make mean pore size, become " less mean pore size " from " big mean pore size " along the direction of solution by film.In addition, preferably using the ratio of maximum diameter of hole and minimum-value aperture is 2 or higher porous-film.More preferably, the maximum diameter of hole is 5 or higher with the ratio of minimum-value aperture.Because have this aperture ratio, thus can obtain being used to adsorb enough surface-area of nucleic acid, and the hole is not easy to be blocked.

The porosity of the solid phase that solution can therefrom pass through is preferably 50% to 95%, and more preferably 65% to 80%.In addition, bubble point is preferably 0.1 to 10kgf/cm ², and more preferably 0.2 arrive 4kgf/cm.

The pressure-losses of the solid phase that solution can therefrom pass through is preferably 0.1 to 100kPa.Owing to have this pressure-losses, so can obtain uniform pressure when exerting pressure.More preferably, the pressure-losses is 0.5 to 50kPa." pressure-losses " used herein is meant that water whenever passes through the needed minimum pressure of film of 100 μ m thickness.

When water at 25 ℃, 1kg/cm ²Pressure during down by solid phase, the permeable amount of the solid phase that solution can therefrom pass through is preferably the every 1cm of per minute ²1 to 5000 milliliter of film, the more preferably every 1cm of per minute ²5 to 1000 milliliters of films.

The nucleic acid adsorptive capacity of the solid phase that solution can therefrom pass through is preferably every milligram of porous-film and adsorbs 0.1 μ g or higher nucleic acid, and more preferably every milligram of porous-film adsorbs 0.9 μ g or higher nucleic acid.

In order to make solution and porous-film reach suitable duration of contact, when the sample solution that contains nucleic acid passed through solid phase, its flow velocity was preferably per unit solid phase area (cm ²) 2 to 1,500 μ L/ seconds.

If the duration of contact of solution and porous-film is too short, then can not reach sufficient separation and purification effect.On the other hand, from the angle of processing property, be disadvantageous oversize duration of contact.In addition, flow velocity per unit solid phase area (cm more preferably ²) 5 to 700 μ L/ seconds.

When solutions employed can be passed through the inside of solid phase, this solution can be a kind of solution, but also can use multiple solution.Multiple solid phase can contain identical or different material.

Washing

After being adsorbed onto nucleic acid on the solid phase, the washing solid phase improves the yield and the purity of nucleic acid thus and makes the amount of the sample that contains required nucleic acid reduce to a small amount of.In addition, by washing automatically and reclaimer operation, can implement this operation quickly and easily.For quick complete operation can only carry out the once washing step.More importantly under the situation, preferably repeat washing operation several times in purity.

Washings is preferably the solution that contains water-miscible organic solvent.If desired and necessary, washings can also contain water-soluble salt, buffer reagent and tensio-active agent.Require washing operation to play such effect: to be adsorbed onto impurity on the solid phase with nucleic acid in its flush away sample solution.In order to reach this requirement, washings need have such composition, and it makes nucleic acid can not make impurity desorb on the solid phase from desorption on the solid phase.The reason of doing like this is because nucleic acid is insoluble in the water-miscible organic solvent (as alcohols), and therefore, this composition is suitable for making other composition desorption except nucleic acid under the condition that keeps nucleic acid.In addition, add the adsorption effect that water-soluble salt can increase nucleic acid, thus, the effect of optionally removing impurity and unnecessary composition is strengthened.

The example of contained water-miscible organic solvent comprises alcohols and acetone in the washings.The preferred alcohols that uses.The example of alcohols comprises described methyl alcohol, ethanol, propyl alcohol and butanols.Propyl alcohol can be any in Virahol and the n-propyl alcohol, and butanols can be any in straight chain butanols and the side chain butanols.Can use two or more mixture of these alcohols.In these alcohols, preferably use ethanol.The amount of contained water-miscible organic solvent is preferably 20 quality % to 100 quality % in the washings, and more preferably 40 quality % to 100 quality %.

When containing water-soluble salt in the washings, preferred halogen.In these halogens, more preferably muriate.Water-soluble salt is preferably univalent cation or divalent cation.Preferred as alkali salt and alkaline earth salt.In these an alkali metal salts and alkaline earth salt, more preferably sodium salt and sylvite, and sodium salt most preferably.

When containing water-soluble salt in the washings, the concentration of water-soluble salt be preferably 10 mmoles/liter or higher.Upper limit of concentration is not particularly limited, as long as this upper limit is not in reducing the deliquescent scope of impurity.Yet this upper limit is preferably 1 mol, and 0.1 mol more preferably.Preferred embodiment is that water-soluble salt is a sodium-chlor, and its concentration is 20 Bo mol or higher.

Washings can not contain from the liquid material.This situation can reduce behind the washing step from the liquid material sneaks into possibility in the recycling step.Washings contains under the situation of liquid material in recycling step, should can suppress enzyme reaction such as PCR reaction usually from the liquid material.Therefore, the enzyme reaction after considering etc., ideal situation is not contain from the liquid material in the washings.In addition, has corrodibility and toxicity from the fluidity material.Therefore, from this respect, if can not use from the liquid material, then this operational safety to the experimenter is very favourable.Used herein is urea, guanidinesalt, sodium isocyanate, sodium iodide, potassiumiodide etc. from the liquid material.

Conventionally, during the washing step of separate nucleic acid purification process, washings has the wettability of height to the container such as post, so washings usually is retained in the container, causes washings to be sneaked in the washing step recycling step afterwards.This is the reason that (for example) nucleic acid purity drop or the reactivity in later step reduce.Owing to this reason, when using container such as post to carry out the absorption of nucleic acid and desorption, importantly, residual washings should not be retained in the post, makes that solutions employed (particularly washings) can not influence next step in absorption and washing.

Therefore, for the recovery liquid that uses in the washings that prevents to use in the washing step and the later step mixes, and make the washings residual quantity in the post reach minimum, the surface tension of washings is preferably less than 0.035J/m ²When the surface tension of washings hour, can improve the wettability between washings and the pillar, thus, can suppress the amount of residual solution.

In order to improve detersive efficiency, can increase the ratio of water in the washings.Yet in this case, the surface tension of washings increases, and the residual quantity of solution increases.Surface tension at washings is 0.035J/m ²Or under the bigger situation, can suppress the amount of residual solution by the repellency that strengthens post.By strengthening the repellency of post, and form drop, and drop flows downward, thus, can suppress the amount of residual solution.For example, be used to strengthen the method for the method of repellency, perhaps when making the post moulding, introduce the method for water-resisting agent (as silicone resin), but be not limited thereto for coating water-resisting agent on the post surface.

The amount of washings is preferably 2 μ l/mm in the washing step ²Or it is higher.When the amount of washings is big, can improve washing effect.Yet, when the amount of washings is 200 μ l/mm ²Or when lower, can keep processing property, and can suppress the outflow of sample, this is preferred.

In washing step, by under the situation of solid phase, flow velocity is preferably: per unit membrane area (cm at washings ²) 2 μ L/ seconds to 1,500 μ L/ seconds, more preferably: per unit membrane area (cm ²) 5 μ L/ seconds are to 700 μ L/ seconds.Reducing through-rate, and spended time can wash fully than under the long situation.But, can carry out the separation and the purification process of nucleic acid apace by flow velocity being set in the above scope, and can not reduce detersive efficiency, and this is preferred.

In washing step, the temperature of washings is preferably 4 to 70 ℃, and room temperature more preferably.In addition, in washing step, when carrying out washing step, can apply mechanical vibration stirring or ultrasonic stirring to the separate nucleic acid purification column.Can also wash by implementing centrifugally operated.

Before washing step or in the washing step, when the required nucleic acid that will reclaim is DNA, can contact degradation of rna in advance with solid phase by making RNA degrading enzyme solution.When required nucleic acid is RNA, can contact degradation of dna in advance by making dna degradation enzyme solution and solid phase.In either case, subsequently by using washings washing solid phase from solid phase, to remove the RNA degrading enzyme or the dna degradation enzyme all is very important.

Washing solid phase is afterwards contacted with the solution that can make the nucleic acid that is adsorbed onto on the solid phase carry out desorption.Solution after the contact contains required nucleic acid.Therefore, reclaim the solution after this contact, and be used for the operation such as the nucleic acid amplification that is undertaken by PCR (polymerase chain reaction) subsequently.

The volume-adjustment that the reclaims liquid volume for the sample solution that contains nucleic acid made by sample can be able to be carried out the desorption of nucleic acid then.Contain to some extent and to separate and the amount of the recovery liquid of the nucleic acid of purifying depends on the amount of used sample.Usually the amount of used recovery liquid is that tens microlitres are to several hectolambdas.But when the amount of sample is minimum when maybe needing to separate with a large amount of nucleic acid of purifying, the volume that reclaims liquid can change in the scope of L to tens milliliter of 1 μ.

Reclaim liquid and can preferably use purified distilled water and Tris/EDTA buffer reagent etc.The pH that reclaims liquid is preferably 2 to 11, and more preferably 5 to 9.Specifically, ionic strength and salt concn can produce favourable influence to the wash-out that is adsorbed nucleic acid.The ionic strength that reclaims liquid be preferably 290 mmoles/liter or lower, and salt concn be 90 mmoles/liter or lower.In this case, salt can be an alkali metal salt.Be set to have above-mentioned character by reclaiming liquid, can improve the recovery of nucleic acid, thus, can reclaim a large amount of nucleic acid.

When reducing to reclaim the volume of liquid, can obtain containing the recovery liquid of spissated nucleic acid with reference to the volume that contains the initial sample solution of nucleic acid.(reclaiming the volume of liquid) is preferably 1: 100 to 99: 100 with the ratio of (volume of sample solution), and more preferably 1: 10 to 9: 10.By the ratio in this scope, can be according to easy mode condensed nucleic acid, and need not to implement concentration operation in the step after the separate nucleic acid purifying.Thus, can provide a kind of method that is used to make nucleic acid solution, wherein, by the height in the concentration ratio sample of the spissated nucleic acid of aforesaid method.

Unqualified to reclaiming the number of times that liquid injects, inject number of times and can be once or twice or repeatedly.Usually, separating quickly and easily and during purification of nucleic acid, can undertaken by the mode of a reclaimer operation.On the other hand, in the time that a large amount of nucleic acid will be reclaimed, can repeatedly inject recovery liquid in batches.

In recycling step,, can reclaim to nucleic acid and add stablizer in the liquid for the degraded of the nucleic acid that suppresses to reclaim.The example of the stablizer that can add comprises antibacterial agent, anti-mycotic agent and nucleolysis inhibitor.The nucleolysis inhibitor is the inhibitor of nucleolysis enzyme, and EDTA especially.As other recovery embodiment, can in returnable, add stablizer in advance.In addition, by in the nucleic acid solution that reclaims, adding the specific dna degradation enzyme unwanted DNA (as chromogene group DNA) that can degrade.

The method according to this invention can be used for separating in the situation with plasmid DNA purification, and can be preferred for separating in a similar fashion in the situation with purifying phasmid DNA.

In the present invention, preferably use the separate nucleic acid purification unit, this separate nucleic acid purification unit has: (a) solid phase; (b) have the container of at least two openings, this container is used to hold solid phase; (c) the pressure difference generation device that links to each other with an opening of described container.

Described separate nucleic acid purification unit below will be described.

The material of container is not particularly limited, as long as this container can hold solid phase, and at least two openings can be set get final product.Go up from being easy to make, preferably use plastics.For example preferably use transparent or opaque resin as plastics, described resin such as polystyrene, polymethacrylate, polyethylene, polypropylene, polyester, nylon or polycarbonate.

Container is provided with the solid phase holding portion, and solid phase can be contained in this holding portion.In suction with when discharging sample solution etc., solid phase can not break away from holding portion, and pressure difference generation device (as syringe) can be connected on the opening.For this container, preferably, this container is divided into two portions at first, and after holding solid phase, these two portions can be engaged.In addition, break away from holding portion, the net of being made by the material of contaminated nucleic acid not can be put into the upside and the downside of solid phase for fear of solid phase.

The shape that is contained in the solid phase in the container is not particularly limited, and solid phase can be circle, square, rectangle, ellipse, cylindrical (under the situation of film), coiling shape (under for the situation of film), pearl shape (its surface scribbles the organic polymer of hydroxyl) etc.On the angle that is fit to make, the highly symmetric shape of preferred use is as circular, square, cylindrical or coiling shape and pearl shape.

Usually hold under the situation that body and lid be separated in solid phase and make container, and solid phase is held body and lid respectively is provided with at least one opening.Opening is used as the entrance and exit of sample solution, washings that contains nucleic acid and the solution that can make the nucleic acid desorption that is adsorbed on the solid phase (purpose for easy hereinafter all is called " sample solution etc. ").Opening is connected on the pressure difference generation device that can make internal tank form decompression state or pressurized state.The shape that solid phase is held body is not particularly limited.But make for convenience, and sample solution etc. is diffused on the whole surface of solid phase easily, preferred solid phase is held the cross section of body for circular.In order to prevent to produce the solid phase smear metal, tetragonal cross section also is preferred.

Lid need be connected to solid phase and hold on the body, so that make internal tank be in decompression state or pressurized state by the pressure difference generation device.But, can select any method of attachment, as long as can reach this state.The example of method of attachment comprise use tackiness agent to connect, bolt connects, be connected, make and be screwed and use the connection of ultrasonic wave heating and melting.

The internal volume of container is only by the decision of the amount of pending sample solution, but normally by holding solid phase volume indicate.Specifically, internal volume is preferably such volume: this volume can hold about 1 to 6 solid phase, and the thickness of each solid phase is about 1mm or lower (50 to 500 μ m according to appointment), and its diameter is about 2 to 20mm.

Preferably, the edge section of the solid phase in the container and the inner-wall surface of container closely touch such degree, and this degree makes that sample solution etc. can be by the space between solid phase and the inwall.

Bottom towards the solid phase of the opening of the inlet that is used as sample solution etc. is built as such mode, and it is not tight to make that the inwall of solid phase and container contacts, thereby the space is provided, so that sample solution etc. are diffused on the whole surface of solid phase as far as possible equably.

Preferably, the top in the solid phase of facing the opening that is connected with the pressure difference generation device is provided with it has perforation (hole) near the center element.This element has the function of the solid phase of pressing down, and has the effect of efficient discharge sample solution etc.This element be preferably shape with inclined-plane (as, doline or bowl-type) so that solution is concentrated on central hole.By the amount of considering pending sample solution etc. and the size of holding the container of solid phase, the thickness of the size in hole, the angle on inclined-plane and element can suitably be determined by those skilled in the art.Preferably, be provided with the space between described element and opening, this space is used to store sample solution that overflows etc., thereby prevents that it is inhaled in the pressure difference generation device.The big I of this spatial is suitably determined by those skilled in the art.In order to collect nucleic acid effectively, preferably suck the sample solution that contains nucleic acid with so at least amount, this solution amount makes that whole solid phases are enough to immerse wherein.

For prevent sample solution etc. only concentrate on the opening that carries out suction operation under, thereby make sample solution etc. relatively equably by solid phase inside, also preferably between solid phase and described element, be provided with the space.In order to reach this structure, be provided with a plurality of thrusts towards solid phase by element.The size of thrust and number can be determined by those skilled in the art.Yet, preferably keeping spatial simultaneously, keep the port area of solid phase big as much as possible.

Under the situation that is provided with at least three openings on the container, need not explanation, need the unnecessary opening of temporary sealing, so that can suck and discharge solution by decompression operation and pressurized operation.

The pressure difference generation device at first has by reducing the function that pressure in the container that accommodates solid phase in it sucks the sample solution that contains nucleic acid.The pressure difference generation device has the pump that can carry out suction operation and pressurized operation, as syringe, transfer pipet and peristaltic pump.Among these, syringe is suitable for manual operations, and peristaltic pump is suitable for automatic operation.The advantage of transfer pipet is that it can easily use one-handed performance.Preferably, the pressure difference generation device is connected on the opening of container in removable mode.

The following describes the separate nucleic acid and the purification process that use above-mentioned separate nucleic acid purification unit.

Preferably, in separate nucleic acid according to the present invention and purification process, use the separate nucleic acid purification column can implement the absorption and the desorption of nucleic acid, wherein, described separate nucleic acid purification column has been received solid phase in the container contents with at least two openings.

More preferably, use the separate nucleic acid purification column can implement the absorption and the desorption of nucleic acid, wherein, described separate nucleic acid purification column has: (a) solid phase, (b) the pressure difference generation device that has at least two openings, hold the container of solid phase and (c) link to each other with an opening of container.

In this case, first embodiment according to separate nucleic acid of the present invention and purification process can comprise the steps.

(a) step of adding dispersion liquid in sample (bacterium or cell);

(b) add alkali solution in the solution that in above-mentioned steps (a), obtains, thus the step of dissolving sample wherein;

(c) add neutralizer in the solution that in above-mentioned steps (b), obtains, thereby make the step of removing required epinucleic unwanted precipitation of material;

(d) to above-mentioned steps (c) thus in add the lysate preparation sample solution step of (being used to make nucleic acid to be adsorbed onto solution on the solid phase) in the sedimentary supernatant liquor that obtains;

(e) make first opening of separate nucleic acid purification unit insert step in the sample solution;

(f) make nucleic acid be adsorbed onto solution on the solid phase by using pressure in the pressure difference generation device reduction container link to each other with other opening of separate nucleic acid purification unit to suck to be used to, thus the step that this solution is contacted with solid phase;

(g) make being used to of being inhaled into make nucleic acid be adsorbed onto the step that the solution on the solid phase is discharged in the container by using the pressure difference generation device link to each other with other opening of separate nucleic acid purification unit to increase pressure in the container;

(h) with the step in first opening insertion washings of separate nucleic acid purification unit;

(i) by using the pressure in the pressure difference generation device reduction container link to each other with other opening of separate nucleic acid purification unit to suck washings, thus the step that this washings is contacted with solid phase;

(j) by using the pressure difference generation device link to each other with other opening of separate nucleic acid purification unit to increase the step that pressure in the container is discharged the washings that is inhaled in the container;

(k) first opening of separate nucleic acid purification unit is inserted step in the solution (recovery liquid) that can make the nucleic acid desorption that is adsorbed on the solid phase;

(l) by using the pressure in the pressure difference generation device reduction container link to each other with other opening of separate nucleic acid purification unit to suck the solution that can make the nucleic acid desorption that is adsorbed on the solid phase, thus the step that this solution is contacted with solid phase; And

(m) by using the pressure difference generation device link to each other with other opening of separate nucleic acid purification unit to increase the step that solution that pressure in the container allows to be adsorbed to the nucleic acid desorption on the solid phase is discharged in the container.

In step (f), (i) with (l), preferably, suck solution with the amount that contacts with whole solid phases basically.Yet under solution was inhaled into situation in the pressure difference generation device, this device can be by this contaminated aqueous solution.Therefore, the soakage with solution controls to appropriate vol.After sucking an amount of solution, with the pressure difference generation device internal tank is pressurizeed, thereby discharge the solution that is inhaled into.Before carrying out this operation, do not need to have the timed interval, and can be immediately with its discharge after sucking solution.

Second embodiment according to separate nucleic acid of the present invention and purification process can may further comprise the steps.

(a) step of adding dispersion liquid in sample (bacterium or cell);

(b) add alkali solution in the solution that in above-mentioned steps (a), obtains, thus the step of dissolving sample wherein;

(c) add neutralizer in the solution that in above-mentioned steps (b), obtains, thereby make the step of removing required epinucleic unwanted precipitation of material;

(d) to above-mentioned steps (c) thus in add the lysate preparation sample solution step of (being used to make nucleic acid to be adsorbed onto solution on the solid phase) in the sedimentary supernatant liquor that obtains;

(e) sample solution is injected into step in first opening of separate nucleic acid purification unit;

(f) make being used to of being injected into make nucleic acid be adsorbed onto the step that the solution on the solid phase is discharged from other opening by the pressure that uses the pressure difference generation device that links to each other with first opening of separate nucleic acid purification unit to increase in the container;

(g) washings is injected into step in first opening of separate nucleic acid purification unit;

(h) make the washings that is injected into step by using the pressure difference generation device link to each other with first opening of separate nucleic acid purification unit to increase pressure in the container from other opening discharge;

(i) can make the solution (recovery liquid) of the nucleic acid desorption that is adsorbed on the solid phase be injected into step in first opening of separate nucleic acid and purification unit; And

(j) make the solution that can make the nucleic acid desorption that is adsorbed on the solid phase that is injected into from other opening discharge by the pressure that uses the pressure difference generation device link to each other with first opening of separate nucleic acid purification unit to increase in the container, the step that makes the nucleic acid desorption that is adsorbed to solid phase thus and nucleic acid is discharged in the container.

In above-mentioned steps, to the injection mode of sample solution in the container without limits, preferably use experiment with utensil (as transfer pipet or syringe).More preferably, these utensils are that do not contain nuclease or not hydrogenous.

Blending means to sample and various solution is not particularly limited.For example, preferably, use whipping appts, implement to mix 1 second to 3 minute under the condition of 000rpm 30 to 3.By this mixing, can improve yield through the nucleic acid of separation and purifying.Can be preferably to mix and mix for 5 to 30 times for another mode of selecting for use by upset.In addition, also can mix for 10 to 50 times by carrying out pipetting.

Can make and use a kind of kit utility, this kit utility comprises: (i) separate nucleic acid purification column; (ii) tensio-active agent; (iii) pretreatment fluid, it contains at least a in chaotropic salt, defoamer, nucleic acid stability agent, buffer reagent, sour agent, alkaline agent and the enzyme; (iv) washings; And (v) reclaim liquid reagent.

The example of automatic gear is described below, the enforcement automatically from the sample that contains nucleic acid of this automatic gear use separate nucleic acid purification column and pressure difference generation device separates the step with purification of nucleic acid, wherein said separate nucleic acid purification column has been received solid phase in the container contents with at least two openings, but automatic gear is not limited thereto.

Automatic gear be carry out following separation and purification process automatically be used to separate device with purification of nucleic acid.Use the separate nucleic acid purification column that holds solid phase in it, wherein, solution can be by the inside of pillar.The solution (sample solution) that will be used to nucleic acid is adsorbed onto on the solid phase is injected in the separate nucleic acid purification column, pressurization then, thus the nucleic acid in the sample solution is adsorbed onto on the solid phase.Washings is injected in the separate nucleic acid purification column, pressurize, thereby remove impurity.To reclaim liquid and inject in the separate nucleic acid purification column, thereby make the nucleic acid desorption that is adsorbed on the solid phase, and nucleic acid after the desorption and recovery liquid will be reclaimed simultaneously.Thus, this automatic gear has: supporting device, its supporting separate nucleic acid purification column, reception sample solution and the waste fluid container of washings and the returnable of the recovery liquid that reception contains nucleic acid; Compressed air-feed mechanism, its pressurized air is introduced in the separate nucleic acid purification column; And dispensing mechanism, it injects the separate nucleic acid purification column to washings and recovery liquid respectively.

Preferably, supporting device has: be installed on the stand on the apparatus main body; By the column holder moving up and down of stand carrying, its supporting separate nucleic acid purification column; And pan straddle, its supporting waste fluid container and returnable, their position can exchange with respect to the separate nucleic acid purification column below pillar.

Preferably, compressed air-feed mechanism has: from the valve of its underpart jet compression air; Pressure head is used to carry valve and valve is moved up and down with respect to the separate nucleic acid purification column by the column holder supporting; And being located at locating device on the pressure head, this locating device is used for determining being positioned at the position of separate nucleic acid purification column of the stand of supporting device.

Preferably, dispensing mechanism has: the washings coupler that injects washings; Inject the recovery liquid coupler that reclaims liquid; The coupler movable stand of supporting washings coupler and recovery liquid coupler, this movable stand can move on by the separate nucleic acid purification column of supporting device supporting successively; In it, accommodate suction washings the Washing liquid bottle of washings and washings is supplied with the washings supply pump of washings coupler; And in it, accommodate the recovery liquid bottle that reclaims liquid suction and reclaim liquid and supply with the recovery fluid supply pump that reclaims the liquid coupler reclaiming liquid.

According to aforesaid automatic gear, it is provided with: the supporting device of supporting separate nucleic acid purification column, waste fluid container and returnable; The compressed air-feed mechanism in the pressurized air introducing separate nucleic acid purification column; And respectively the injecting mechanism of washings and recovery liquid injection separate nucleic acid purification column.In addition, this device automatically carries out each step in following separate nucleic acid and the purification process: be injected into the separate nucleic acid purification column that is equipped with the solid phase parts adding the solution of depressing being used for nucleic acid is adsorbed onto on the solid phase, thereby nucleic acid is adsorbed onto on the solid phase parts; Inject washings, thereby wash and discharge impurity off; And inject and reclaim liquid, thereby separation and recovery are adsorbed to the nucleic acid on the solid phase parts.Thus, can compactly designed a kind of like this mechanism, its can at short notice efficiently, automatically implement to separate and purification of samples solution in the step of nucleic acid.

When supporting device has stand, column holder moving up and down (it is supporting the separate nucleic acid purification column) and supports the pan straddle of waste fluid container and returnable in tradable mode, just can easily implement each or one group exchange in separate nucleic acid purification column and waste fluid container and the returnable.

When compressed air-feed mechanism has valve, moves up and down the pressure head of valve and is used for determining the locating device of position of nucleic acid separation and purification post, just can supply with pressurized air safely with simple mechanism.

When dispensing mechanism comprises the washings coupler, reclaims the liquid coupler, the coupler movable stand that can on the separate nucleic acid purification column, move successively, from Washing liquid bottle the suction washings and washings supplied with the washings supply pump of washings coupler and from reclaim the liquid bottle suction reclaim liquid and when reclaiming liquid and supply with the recovery fluid supply pump that reclaims the liquid coupler, just can inject washings and recovery liquid successively with simple mechanism.

Below by embodiment the present invention is described in more detail, but be to be understood that the present invention is not limited in these embodiment.

Example

[embodiment 1]

(1) preparation separate nucleic acid purification column

The separate nucleic acid purification column is made by high-impact polystyrene, and its internal diameter is 7mm, and has the part of holding as the porous-film of solid phase.

(2) will be by the porous-film (aperture: 2.5 μ m that the porous-film saponification that comprises cellulosetri-acetate is obtained, diameter: 7mm, thickness: 100 μ m, saponification degree: 95%) be used as porous-film, and it is contained in the solid phase holding portion of the separate nucleic acid purification column of preparation in above-mentioned (1).

(3) preparation dispersion liquid, alkali solution, neutralizer, lysate, washings and recovery liquid.

What preparation had following prescription separately is used to separate dispersion liquid, alkali solution, neutralizer, lysate, washings and recovery liquid with plasmid DNA purification.

Dispersion liquid (being used for separating and plasmid DNA purification)

1 mol Tris-hydrochloric acid (product of the limited 26g of Wako Pure Chemical Industries company)

0.5 mol EDTA (product of the limited public 11g of Wako Pure Chemical Industries department)

Distilled water 465g

Alkali solution (being used for separating and plasmid DNA purification)

1 mol NaOH (product of the Wako Pure Chemical Industries 104g of company limited)

10 weight %SDS (the 50g product of Wako Pure Chemical Industries company limited)

Distilled water 350g

Neutralizer (being used for separating and plasmid DNA purification)

Potassium acetate (product of Wako Pure Chemical Industries company limited) 147g

Acetate (product of Wako Pure Chemical Industries company limited) 68g

Distilled water 356g

Lysate (being used for separating and plasmid DNA purification)

Lysate is according to the formulation shown in the following table 1.

Table 1

	A	B	C	D	E	F
							Polysorbas20 (g)	0	33	45	56	78	100
Bis-Tris(g)	3.4	3.4	3.4	3.4	3.4	3.4
							Ethanol (mL)	344	344	344	344	344	344
Distilled water (mL)	143	110	98	87	65	43

Washings (being used for separating and plasmid DNA purification)

1 mol Tris-hydrochloric acid (Wako Pure Chemical Industries has the product of 5.6g limit company)

Ethanol (99.5%) (product of the limited public 400mL of Wako Pure Chemical Industries department)

Distilled water 94g

Reclaim liquid (being used for separating and plasmid DNA purification)

1 mole/L Tris-hydrochloric acid (product of the limited 5.2g of Wako Pure Chemical Industries company)

Distilled water 494g

(4) from E.coli pBluescript IISK (-)/DH5 α, extract plasmid DNA

(i) preparation E.coli pBluescript IISK (-)/DH5 α

To be inoculated into 100mL with the E.coli DH5 α transformant that plasmid pBluescript IISK (-) (product of Stratagene company) transforms and contain the Luria-Bertani nutrient solution (sodium-chlor (pH:7.5) that the yeast extract that the tryptone that 10g/ rises, 5g/ rise, 5g/ rise) of the Ampicillin Trihydrate of 100 μ g/mL, and be 220 minutes in 37 ℃ of heated culture temperatures, jolting speed ^-1Condition under incubation 15 hours.Behind the incubation, nutrient solution is divided in the nuclease free of injecting 1.5mL, the no hydrogen microtubule (platinotron, the product of BM Equipment company) with the amount of 1.0mL.Use the micro-refrigerated centrifuge of high speed (trade(brand)name: MX-300, the product of Tomy Seiko company) with nutrient solution under the condition of 6000 * g centrifugal 15 minutes.Remove supernatant liquor, thereby obtain biomass.With these biomass as the material that is used to extract.

(ii) separate and plasmid DNA purification

With 3 μ L concentration is that the RNase A solution (product of Wako Pure ChemicalIndustries) of 10mg/mL and RNase T1 solution (product of Sigma company) that 1 μ L concentration is 1mg/mL add respectively in the dispersion liquid of preparation in the 100 μ L above-mentioned steps (3), this dispersion liquid contains in steps the biomass of preparation in (i), with gained solution vortex stirring at room temperature 15 seconds, to guarantee that biomass are disperseed.In this solution, add the alkali solution of preparation in the 100 μ L above-mentioned steps (3) then, and overturn and mix 5 times, so that biomass are carried out the bacteriolyze cracking.In this solution, add the neutralizer of preparation in the 140 μ L above-mentioned steps (3) then, and overturn and mix 5 times, with in and sample solution.Use the micro-refrigerated centrifuge of high speed (commodity are called MX-300, the product of Tomy Seiko company) will precipitate residue under the condition of 18000 * g centrifugal 10 minutes, thereby reclaim 330 μ L supernatant liquors.In new 1.5mL nuclease free, no hydrogen microtubule, add the lysate for preparing in the 320 μ L above-mentioned steps (3) in (platinotron, the product of BM Equipment company) in advance, and in this container, add the supernatant liquor of above gained.Carry out vortex and stirred 30 seconds, thereby obtain sample solution.

Each solution is injected first opening of separate nucleic acid purification column (it accommodates above (2) prepared porous-film as solid phase) respectively, and pressure difference generation device (tubing sucker-rod pump) is connected on this opening.Make the inside of separate nucleic acid purification column be in pressurized state (80kPa), and make the solution of injection pass through the porous-film solid phase, thereby this solution is contacted with solid phase.Solution is discharged from other opening of separate nucleic acid purification column.Washings prepared in above (3) is injected first opening of separate nucleic acid purification column, and tubing sucker-rod pump is connected on this first opening.Make the inside of separate nucleic acid purification column be in pressurized state (80kPa), and make the washings that is injected, discharge from other opening then by the porous-film solid phase.Recovery liquid prepared in above (3) is injected first opening of separate nucleic acid purification column, and tubing sucker-rod pump is connected on first opening of this separate nucleic acid purification column.Make the inside of separate nucleic acid purification column be in pressurized state (80kPa), and make the recovery liquid that is injected, discharge from other opening then by the porous-film solid phase.Reclaim this solution then.Carried out separate nucleic acid and purification process (sample solution that contains nucleic acid from injection to its recovery) needs 6 minutes consuming time.

(5) to the quantitative assay of nucleic acid yield

Respectively reclaim liquid about what in above-mentioned example, reclaim, the electrophoresis result of DNA is shown among Fig. 1.

Absorbancy at the 260nm place is as shown in table 2 below.

Table 2

Lysate	A	B	C	D	E	F
							Absorbancy	2.2	4.2	4.0	4.3	3.9	3.5
	Comparative Examples	The present invention	The present invention	The present invention	The present invention	The present invention

Can find out significantly by electrophoretogram shown in Figure 1 and the result shown in the table 2: in embodiments of the invention (the 2nd swimming lane to the 6 swimming lanes), can prepare DNA efficiently.That is, method of the present invention shows excellent separation performance and good washing efficient, and the result can obtain plasmid DNA in high yield, high purity ground fast in the above-mentioned time period.

[embodiment 2]

According to preparing separate nucleic acid purification column and biomass to (3) identical mode with embodiment 1 (1).About dispersion liquid, alkali solution and neutralizer, the solution that in embodiment 1, uses, also use QIAprep Miniprepkit P1, P2 and N3 solution (product of QIAGEN company).About sample solution, according to embodiment 1 (4) in (i) identical mode prepare sedimentary supernatant liquor.

With in the alkali and the sedimentary supernatant liquor that obtained, and reclaim 330 μ L according to described embodiment through the neutral supernatant liquor.In each new 1.5mL nuclease free, no hydrogen microtubule (product of BMEquipment company), add in advance respectively in the lysate (G is to I) of 320 μ L according to preparation shown in the following table 3, and in this container, add the supernatant liquor of above gained, by vortex with contents stirred 30 seconds, thereby prepare each sample solution.

Table 3

Lysate (being used for separating and plasmid DNA purification)

	G	H	I
				Polysorbas20 (g)	39	39	39
Bis-Tris(g)	3.4	3.4	3.4
				Ethanol (mL)	0	172	344
Distilled water (mL)	444	276	104

Each solution is injected first opening of separate nucleic acid purification column (it accommodates above (2) prepared porous-film as solid phase) respectively, and pressure difference generation device (tubing sucker-rod pump) is connected on this opening.Make the inside of separate nucleic acid purification column be in pressurized state (80kPa), and make the solution of injection pass through the porous-film solid phase, thereby this solution is contacted with solid phase.Solution is discharged from other opening of separate nucleic acid purification column.Washings prepared in above (3) is injected first opening of separate nucleic acid purification column, and tubing sucker-rod pump is connected on this first opening.Make the inside of separate nucleic acid purification column be in pressurized state (80kPa), and make the washings that is injected, discharge from other opening then by the porous-film solid phase.Recovery liquid prepared in above (3) is injected first opening of separate nucleic acid purification column, and tubing sucker-rod pump is connected on first opening of this separate nucleic acid purification column.Make the inside of separate nucleic acid purification column be in pressurized state (80kPa), and make the recovery liquid that is injected, discharge from other opening then by the porous-film solid phase.Reclaim this solution then.Carried out separate nucleic acid and purification process (sample solution that contains nucleic acid from injection to its recovery) needs 6 minutes consuming time.

(6) to the quantitative assay of nucleic acid yield

Respectively reclaim liquid about what in above-mentioned example, reclaim, the electrophoresis result of DNA is shown among Fig. 2.

Absorbancy at the 260nm place is as shown in table 4 below.

Table 4

Dispersion liquid/alkali solution/neutralizer	QIAGEN *	QIAGEN	QIAGEN	Embodiment	1 **	Embodiment 1	Embodiment 1
								Lysate	G	H	I	G	H	I
Absorbancy	0.140	0.830	2.320	0.150	0.680	6.130
								Comparative Examples	The present invention	The present invention	Comparative Examples	The present invention	The present invention

QIAGEN ^*: use QIAprep Miniperpkit P1, P2 and N3 solution (producing) by QIAGEN company.

Embodiment 1 ^*: use dispersion liquid, alkali solution and the neutralizer identical with embodiment 1.

Can find out significantly by electrophoretogram shown in Figure 2 and the result shown in the table 4: plasmid DNA purification efficiently in the embodiment of the invention (the 8th, 9,11 and 12 swimming lane).That is, method of the present invention shows excellent separation performance and good washing efficient, and the result can obtain plasmid DNA in high yield, high purity ground fast in the above-mentioned time period.

Industrial usability

Method of the present invention can be isolated highly purified plasmid DNA efficiently from the sample solution that contains nucleic acid that is made by bacterium or cell.

In this application, required whole disclosures of each foreign patent application of foreign priority to incorporate this paper by reference into, as all listing.

Claims (15)

1. the method for separation and purification of nucleic acid, this method comprises:

(1) sample solution that will contain nucleic acid contacts with solid phase, thereby makes described nucleic acid be adsorbed onto step on the described solid phase;

(2) washings is contacted with described solid phase, thereby be attracted to the step of the described solid phase of washing under the state on the described solid phase at described nucleic acid; With

(3) will reclaim liquid and contact, thereby make the step of described nucleic acid desorption on the described solid phase with described solid phase;

Wherein, described sample solution is by comprising the method preparation of removing precipitate component and tensio-active agent and water-miscible organic solvent being added the step in this sedimentary supernatant liquor.

2. the method for separation according to claim 1 and purification of nucleic acid,

Wherein, described glass or plastic containers.

3. the method for separation according to claim 1 and 2 and purification of nucleic acid,

Wherein, described tensio-active agent is a polyoxygenated ethylidene class tensio-active agent.

4. according to the method for any described separation and purification of nucleic acid in the claim 1 to 3,

Wherein, described tensio-active agent is a polyoxygenated ethylidene anhydrosorbitol tensio-active agent.

5. according to the method for any described separation and purification of nucleic acid in the claim 1 to 4,

Wherein, described sample solution is to make by add pretreatment fluid in the sample that contains nucleic acid, and described pretreatment fluid contains and is selected from least a in chaotropic salt, defoamer, nucleic acid stability agent, buffer reagent, sour agent, alkaline agent and the enzyme.

6. according to the method for any described separation and purification of nucleic acid in the claim 1 to 5,

Wherein said solid phase is membranaceous solid phase.

7. according to the method for any described separation and purification of nucleic acid in the claim 1 to 6,

Wherein, described water-miscible organic solvent comprises and is selected from least a in methyl alcohol, ethanol, propyl alcohol and isomer thereof and butanols and the isomer thereof.

8. according to the method for any described separation and purification of nucleic acid in the claim 1 to 7,

Wherein, described solid phase comprises silicon-dioxide or derivatives thereof, diatomite or aluminum oxide.

9. according to the method for any described separation and purification of nucleic acid in the claim 1 to 8,

Wherein said solid phase includes organic polymer.

10. the method for separation according to claim 9 and purification of nucleic acid,

Wherein, described solid phase comprises and is selected from least a in Teflon (registered trademark), polyester, polyethersulfone, polycarbonate, acrylate copolymer, urethane, polybenzimidazole, polyolefine, polyvinyl chloride and the polyvinylidene difluoride (PVDF).

11. the method for separation according to claim 9 and purification of nucleic acid,

Wherein, described solid phase comprises the nylon of positively charged or negative charge.

12. the method for separation according to claim 9 and purification of nucleic acid,

Wherein, described organic polymer has polysaccharide structures.

13. the method for separation according to claim 9 and purification of nucleic acid,

Wherein, described organic polymer comprises and is selected from least a in Mierocrystalline cellulose, cellulose mixed esters, nitrocellulose, cellulose acetate and the soluble cotton.

14. a device, it is used for implementing according to the step in the method for any described separation of claim 1 to 13 and purification of nucleic acid automatically.

15. a kit utility, it is used for implementing the method according to any described separation of claim 1 to 13 and purification of nucleic acid, and this kit utility comprises:

(i) separate nucleic acid purification column;

(ii) tensio-active agent;

(iii) pretreatment fluid, it contains and is selected from least a in chaotropic salt, defoamer, nucleic acid stability agent, buffer reagent, sour agent, alkaline agent and the enzyme;

(iv) washings; And

(v) reclaim the reagent of liquid.

Images