CN1241933C - Plasmid DNA purifying method by phase separation method - Google Patents
Plasmid DNA purifying method by phase separation method Download PDFInfo
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- CN1241933C CN1241933C CN 03141590 CN03141590A CN1241933C CN 1241933 C CN1241933 C CN 1241933C CN 03141590 CN03141590 CN 03141590 CN 03141590 A CN03141590 A CN 03141590A CN 1241933 C CN1241933 C CN 1241933C
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Abstract
The present invention relates to a new dual phase method for separating DNA and other biomacromolecules, which is characterized in that the method comprises the following steps: (1) adding liquid to be suspended, (2) adding alkali to be cracked, (3) neutralization and agglutination, (4) phase formation and separation and (5) filtration and recovery. Plasmid DNA is separated and purified by phase separation technology. The present invention has the advantages of high separation purity and high speed (about 10 to 20 minutes), can simultaneously separate different bacterium components and provides convenient conditions for separating and purifying the bacterium components.
Description
Technical field
The present invention relates to a kind ofly adopt novel two phase process that DNA and other biological macromole are separated.And can be separated to different cell components simultaneously, the condition of providing convenience for the separation and purification of these cell components.
Background technology
Traditional plasmid DNA purification process is with reagent cracking bacteriums such as SDS (sodium laurylsulfonate), TX-100, and plasmid DNA is discharged in the supernatant, removes protein through phenol, chloroform extracting again, and the plasmid DNA in the supernatant reclaims through ethanol or isopropanol precipitating.But still retain materials such as amounts of protein, lipopolysaccharides, polysaccharide in the plasmid DNA of purifying like this, these materials have seriously disturbed many important biological experiment results, especially biological studies such as cell transfecting, gene therapy, dna vaccination immunity are higher to the purity requirement of plasmid DNA, this purification process can not satisfy to the requirement on the plasmid DNA purity, needs to adopt the purification process of other form to come the separation and purification plasmid DNA.
Other has bibliographical information to adopt DEAD-silica resin (resin) to come plasmid DNA purification.Have the phosphate radical gene on the dna molecular, make dna molecular electronegative.In DEAE groups density on the silica far above the DEAE density on traditional polyose resin, DNA more is firmly bonded on this resin that contains high-density DEAE, and need DNA to be eluted with very high salt concn, but other impurity such as protein is just eluted when lower salt concn, and DNA reaches fully with other impurity and separates.But this situation only just occurs during not in conjunction with negatively charged ion such as SDS on the biomacromolecule surface.But, in containing the supernatant of plasmid DNA, in fact retained a large amount of and SDS bonded denatured protein, LPS, polysaccharide.The SDS molecule have sulphate groups because of, electronegative, can with protein equimolecular mortise, cause these molecular surfaces to have a large amount of negative charges, changed original chromatography behavior greatly, make the elution requirement of protein, LPS and polysaccharide that fundamental change take place, can't be fully impurity such as DNA and protein, LPS be separated up hill and dale.
Chinese patent literature CN1378592A discloses " using the method that tangential flow filtration comes plasmid DNA purification under the condition of no RNA enzyme and organic solvent ", and this method comprises the steps: that (a) digests described cell; (b) with about 4 to 24 hours of cell cultures carrying out cracking and dissolving, but RNA is not carried out enzymatic digestion; (c) remove lysate impurity from cell so that a kind of plasmid DNA solution to be provided; (d) this solution stream is filtered through tangential flow filtration device, to obtain to contain the phegma of this plasmid DNA; (e) reclaim this phegma.This method velocity of separation and effect are not ideal enough.
Summary of the invention
The technical issues that need to address of the present invention are, overcome disadvantages of background technology, provide a kind of DNA of making and other biological macromole to separate, and velocity of separation and effect be DNA purification process preferably all.
Phase Partition Purified Plasmid DNA Method of the present invention is characterized in that by following operation steps:
1) liquid feeding suspends: after containing that the bacterium liquid of plasmid is centrifugal and abandoning supernatant, add bacterium suspension and make thorough suspension;
2) alkaline splitting: add the gentle mixing of bacterium lysate, make the abundant cracking of thalline, form bright solution;
3) in and aggegation: add the gentle mixing of neutralization buffer, to forming the aggegation piece;
4) become to be separated: the gentle mixing of bonus point phase solution, form emulsion, centrifugal, abandon phase, will change the residual INTERPHASE CARBIDE PRECIPITATION of microstrainer elimination down mutually over to;
5) filtered and recycled: in filtrate, add the DNA binding soln and change DNA preparation pipe over to, make solution filter pellosil, DNA is attached on the pellosil and obtains concentrating and reclaiming with negative pressure or centrifuging.
Above-mentioned steps 1), 2) and 5) be conventional steps.3) to 4) be novel phase-separated system.In this individual system, at first must contain at ethanol/undissolved salt of Virahol height, and also protein, LPS (bacteria lipopolysaccharide), RNA and lipid are separated out sex change in this salt or are not dissolved, and opposite DNA must dissolve fully.Ammonium sulfate, cesium chloride possess such characteristic.Because ammonium sulfate is cheap, and is again protein LPS, effective denaturing agent of RNA.Bacterium with the solution neutralization of sulfur acid ammonium, makes the protein distortion and forms insoluble closely mixture with genomic dna after the SDS/NaOH dissolving and discharging plasmid DNA in this individual system.But at this moment not enough so that other impurity of ammonium sulfate concentrations all effectively precipitate in the solution system.Adding organic solution ethanol: behind the Virahol (v/v=2～10: 1), moisture content has been arrived in the organic phase (going up phase) by extracting, makes inorganic ammonium sulfate concentrations in mutually almost reach state of saturation, is the dewatering state of height.Except plasmid DNA still is dissolved state is positioned at down mutually, the bacterium fat-soluble component by extracting to last phase, every other bacterium composition since with saturated ammonium sulphate and ethanol: Virahol is all incompatible and be precipitated out by sex change, RNA is because density is big, at the pipe end, other sex change compositions are deposited in alternate by centrifugation.
The key principle of purifying be exactly in the ammonium sulfate of state of saturation plasmid DNA still keep dissolved state completely, and other impurity are by sex change fully and be precipitated out.Add ethanol: isopropyl alcohol mixed solvent makes the moisture content in the sulfur acid ammonium solution be walked by extracting, makes down the ammoniumsulphate soln that reaches nearly state of saturation mutually.The key that forms this two-phase system is the solution that (1) contains finite concentration ammonium sulfate; (2) certain volume and a certain proportion of ethanol: isopropanol solvent.If ammonium sulfate concentrations is low excessively, be difficult for forming two-phase, and ammonium sulfate can be separated out.Concentration is too high, and ammonium sulfate also can be separated out.Only when adding the suitable proportion volume of ethanol: could form biphasic system during isopropyl alcohol mixed solvent, and the concentration of the ammonium sulfate in making down mutually reaches nearly saturated state.Ethanol when adding: the isopropyl alcohol mixed solvent volume is too small, can not become phase, causes ammonium sulfate precipitation on the contrary.Volume is excessive, also forms precipitation, although DNA does not separate out, is unfavorable for follow-up operation.Ethanol also can form two-phase system with the solution of ammonium sulfate, but required ethanol volume is excessive, and ratio has deviation slightly, can cause or can not become phase, perhaps causes ammonium sulfate precipitation to separate out.Add a certain amount of Virahol, ammoniumsulphate soln and ethanol: both proportional range of Virahol organic solvent is widened greatly.
Described neutralization buffer is ammonium sulfate (or cesium chloride) 380～480 grams, Guanidinium hydrochloride 28～98 grams, NaH
2PO
42H
2O 16～18 grams add water to 1 liter; Described phase-splitting solution is ethanol: Virahol (v/v)=2～10: 1.Wherein, Guanidinium hydrochloride can impel the free property of plasmid DNA, makes DNA effectively enter down phase, and is unlikely to form the copolymerization aggegation with protein.
Be added with in the described neutralization buffer and impel ammonium acetate 36～45 gram, the then better effects if of saltouing.
Conventional DNA binding soln: 5～7M Guanidinium hydrochloride also has 20～100mM NaH
2PO
4, 40～200mM NaAc, pH5.0～6.0.
In actually operating, when volume is 1 volume when finishing with liquid feeding suspension step, 3), 4) add neutralization buffer in the step: phase-splitting solution (v/v)=1.6～2.4: 2.6～3.2, just can reach required effect.
Institute's bonus point phase solution is carried out precooling, can make the phase-splitting better effects if, precooling is not higher than 10 ℃ usually.
Only being separated of uniqueness of the present invention needs a step be about to different cell components to separate simultaneously, be the innovative point of the technology of the present invention.In this phase-separated system, RNA is deposited in the pipe end 4 in centrifugal process, genomic dna and denatured protein form insoluble mixture and are positioned at alternate 2, polysaccharide, lipopolysaccharides, lipid, opsonigenous substances and pigment by extracting to last mutually in 1, and plasmid DNA still is dissolved state and is positioned at down mutually 3, sees Fig. 1.Down mutually in highly purified plasmid DNA be attached under the specific high salt condition silica film/on, the purpose that reaches quick recovery and be further purified.Through washings, demineralised liquid (100mM NH
4Ac, 55～70% ethanol, 10mM Tris-HCl, pH7.0) after the impurity and high salt concentration ion that remains on the silica film is removed in washing, the plasmid DNA that is adsorbed onto on the silica film elutes through minor amount of water or tris buffer, and can be used for various molecular biology experiments immediately.
With the plasmid DNA separation and purification, has the separation purity height with phase detachment technique of the present invention, the advantage of speed fast (finishing in about 10-20 minute), and can be separated to different cell components simultaneously, the condition of providing convenience for the separation and purification of these cell components.
Description of drawings
Fig. 1 is the result schematic diagram that is separated.
Embodiment
Below in conjunction with embodiment, the present invention done describing in further detail.In an embodiment,
Bacterial suspension: contain the 25mM Tris-HCl damping fluid of RNase A1, pH8.0;
Bacterial lysate: 1%SDS/0.18～0.2M NaOH (SDS: sodium laurylsulfonate);
Neutralization buffer: ammonium sulfate or cesium chloride 380～480 grams, Guanidinium hydrochloride 28～98 grams, NaH
2PO
42H
2O 16～18 grams add water to 1 liter;
Phase-splitting solution: ethanol: Virahol (v/v)=2～10: 1;
The DNA binding soln: 5～7M Guanidinium hydrochloride also has 20～100mM NaH
2PO
4, 40～200mM NaAc, PH5.0～6.0.
General employing, bacterial suspension: bacterial lysate: neutralization buffer: phase-splitting solution: DNA binding soln (v/v)=1: 1: 1.6～2.4: 2.6～3.2: 2.
Demineralised liquid: 100mM NH
4Ac, 55～70% ethanol, 10mM Tris-HCl, pH7.0;
Washings: 2.5-5M Guanidinium hydrochloride, 20mM Tris-HCl, pH6.5-7.0;
RNase A1 is the RNA enzyme component A1 of 100 μ g/ml;
Tris is a Tutofusin tris;
Embodiment 1---and ultrapure plasmid DNA is purifying in a small amount
One, experiment is prepared
1. when using for the first time, will all add with the RNase A1 (the RNA enzyme components of 100 μ g/ml) that test kit carries bacterial suspension (the 25mM Tris-HCl damping fluid that contains RNase A1, pH8.0) in, mix 4 ℃ of airtight storages.
2. when using for the first time, press demineralised liquid (100mM NH
4Ac final concentration, 56% ethanol, pH value 7.0) specified volume adds dehydrated alcohol on the reagent bottle, mix.The airtight storage of room temperature.
3. notice before using observing whether the adularescent precipitation occurs in the bacterial lysate,,, and be cooled to room temperature and re-use please in 37 ℃ of incubations dissolvings if any precipitation.
4.4 ℃ precooling neutralization buffer and phase-splitting solution.
Two, operation steps
1, liquid feeding suspends: collect the plasmid bacterium liquid of overnight incubation in 1～4ml LB substratum, centrifugal 30 seconds of 12000 * g abandons most supernatant.The bacterial suspension that has added RNase A1 with the 250 μ l bacterial precipitation that fully suspends.
Attention: bacterium excessive can influence cracking, in and efficient.If use rich medium, bacteria liquid is long-pending should to reduce by half or still less.
Attention: confirm to have added RNase A1 in the bacterial suspension; Suspending needs evenly should not leave little bacterium piece, otherwise can influence the cracking of thalline.
2, alkaline splitting: (1%SDS/0.2M NaOH, gentle but spin upside down fully and mix 4～6 times, this step should not be above 5 minutes to add 250 μ l bacterial lysates.
Attention: avoid acutely rocking, otherwise will cause the pollution of genomic dna; Bacterial lysate covers tight bottle cap after using immediately, in order to avoid airborne CO
2In and the NaOH in the bacterial lysate, reduce bacteriolyze efficient.
3, in and aggegation: add the neutralization buffer (NaH2PO42H2O 16 grams, ammonium acetate 36 grams add water to 1 liter for ammonium sulfate 420 grams, Guanidinium hydrochloride 47 grams) of 4 ℃ of precoolings of 450 μ l, leniently spin upside down immediately 8～10 times, thorough mixing is even.
Attention: avoid acutely rocking, otherwise will cause the pollution of genomic dna.
4, become to be separated: the liquid that the is separated (ethanol: Virahol (v/v)=3: 1), leniently spin upside down 10 times, slightly firmly mix making solution form muddy emulsion, centrifugal 1 minute of 12000 * g for several times again that adds 4 ℃ of precoolings of 650 μ l.The blue phase that goes up is abandoned in suction, will colourless following phase transition to microstrainer (placing the 1.5ml centrifuge tube), centrifugal 30 seconds of 12000 * g.
Attention: on need not mutually to abandon fully to the greatest extent, shift the colourless idol of phase time down have sneak on phase, can phase-splitting rapidly in the Tip head, be easy to abandon (see figure 1).As in transfer process, sucking a small amount of interphase precipitate, needn't remove, can when filtering, remove.
5, filtered and recycled: abandon microstrainer, add 450 μ l DNA binding solns in filtrate, mix.
Step 6.1 can be selected negative pressure method or centrifuging operation.
6.1A. negative pressure method
6.1A.1. DNA is prepared on the socket that pipe is inserted into negative pressure device.Mixed solution in the step 5 is added DNA prepare in the pipe, open and also regulate negative pressure, slowly exhaust solution in the pipe with the flow velocity of 1 of about per second.
6.1A.2. negative pressure is transferred to maximum, add 500 μ l washingss, negative pressure exhausts solution in the pipe.
6.1A.3. the maintenance negative pressure adds the demineralised liquid that 700 μ l have added dehydrated alcohol around tube wall, negative pressure exhausts solution in the pipe; With same method more once with the washing of 700 μ l demineralised liquids.
Attention: confirm in demineralised liquid, to have pressed specified volume adding dehydrated alcohol on the reagent bottle; Adding demineralised liquid around the tube wall helps cleaning down to fall to stick in salt on the tube wall.
6.1B. centrifuging
Place the 2-ml Eppendorf tube 6.1B.1. DNA is prepared pipe, the mixed solution in the step 5 is added DNA prepare in the pipe centrifugal 30 seconds of 12000 * g.
6.1B.2. abandon filtrate, DNA is prepared pipe put and get back in the 2-ml Eppendorf tube, add 500 μ l washingss, centrifugal 30 seconds of 12000 * g.
6.1B.3. abandon filtrate, DNA is prepared pipe put and get back in the 2-ml Eppendorf tube, add the demineralised liquid that 700 μ l have added dehydrated alcohol, centrifugal 30 seconds of 12000 * g, with same method more once with the washing of 700 μ l demineralised liquids.
Attention: confirm in demineralised liquid, to have pressed specified volume adding dehydrated alcohol on the reagent bottle.
6.2. being prepared pipe, DNA places the 1.5ml centrifuge tube, centrifugal 1 minute of 12000 * g.
Place another clean 1.5ml centrifuge tube 6.3. DNA is prepared pipe, add 60 μ l Eluent or deionized waters in silica film central authorities, room temperature left standstill 1 minute.Centrifugal 1 minute eluted dna of 12000 * g.
Operation steps
1, liquid feeding suspends: collect the high copy number plasmid bacterium liquid of overnight incubation in the 40ml LB substratum, or the low copy plasmid of incubated overnight in the 100ml LB substratum.The centrifugal 8min of 〉=3000 * g abandons supernatant.Centrifuge tube was inverted on the paper handkerchief several minutes, eliminates supernatant.The bacterial suspension that has added RNase A1 with the 4.5ml bacterium that fully suspends.
Attention: bacterium excessive can influence cracking, in and the yield of efficient and plasmid DNA.If use rich medium, bacteria liquid is long-pending should to reduce by half or still less.
Attention: confirm to have added RNase A1 in the bacterial suspension.Suspending needs evenly should not leave little bacterium piece.Otherwise can influence the cracking of thalline.
2, alkaline splitting: add the 4.5ml bacterial lysate, gentle but spin upside down fully and mix 4～6 times, this step should not be above 5 minutes.
Attention: cover tight bottle cap immediately after bacterial lysate uses, in order to avoid among the airborne CO2 and the NaOH in the bacterial lysate, reduce bacteriolyze efficient;
Avoid acutely rocking, otherwise will cause the pollution of genomic dna.
3, in and aggegation: add the neutralization buffer of 4 ℃ of precoolings of 8ml, gentle immediately and spin upside down fully and mix, form the aggegation piece of consolidation until precipitation.
Attention: after adding neutralization buffer, should mix immediately, to avoid forming partial coagulated lumps; Avoid acutely rocking, otherwise will cause the pollution of genomic dna.
4, become to be separated: add the liquid 1 that is separated of 4 ℃ of precoolings of 12ml, leniently spin upside down 10 times, slightly firmly mix making solution form muddy emulsion, 4 ℃, centrifugal 8 minutes of 〉=10000 * g for several times more up and down.The blue phase that goes up is abandoned in suction, will descend to change over to mutually in the middle amount filter, with push injection solution is filled in 50ml centrifuge tube or other containers.
Attention: on need not mutually to abandon fully to the greatest extent, shift the colourless idol of phase time down have sneak on phase, can phase-splitting rapidly in moving the drop head, be easy to abandon.As in transfer process, sucking a small amount of interphase precipitate, needn't remove, can when filtering, remove.
5. filtered and recycled: firmly dandle is equipped with the reagent bottle of DNA binding soln-R, and wherein the silica gel particle of fully suspending adds 8ml DNA binding soln-R in filtrate, mixes.
Attention: silica gel particle is the DNA adsorption medium, needs fully to suspend evenly.
Step 6.1 can be selected negative pressure method or push injection operation.
6.1A. negative pressure method
6.1A.1. correctly connect VITAGENE (Chinese) negative pressure device, middle amount prepared on the socket that pipe is inserted into negative pressure device.The mixed solution of drawing in the step 5 is transferred in the middle amount preparation pipe, opens and also regulates negative pressure, and the flow velocity about 2 with per second slowly exhausts solution in the pipe.
6.1A.2. negative pressure is transferred to maximum, add 9ml Buffer W1 (being washings), negative pressure exhausts solution in the pipe.
6.1A.3. the maintenance negative pressure, Xiang Zhongliang prepares and adds the demineralised liquid that 9ml has added dehydrated alcohol in the pipe, and negative pressure exhausts solution in the pipe.
Attention: confirm in demineralised liquid solution, to have pressed specified volume adding dehydrated alcohol on the reagent bottle.
6.1B. push injection
6.1B.1. the mixed solution of drawing in the step 5 is transferred in the middle amount preparation pipe, inserts piston, slowly injects vertically downward, drains solution in the pipe with about 2 flow velocity of per second.
6.1B.2. contain silica gel particulate purification column on the amount preparation pipe during rotation is taken off, withdraw from piston, again purification column is reinstalled on the syringe, add 9ml Buffer W1, insert piston, inject vertically downward, drain liquid.
Attention: silica gel particle is a white particle, as does not see that white particle is deposited in the post, and whether check step 7 fully suspends silica gel particle evenly.
6.1B.3. in kind, added amount preparation pipe in the demineralised liquid washing of dehydrated alcohol with 9ml.
Attention: confirm in demineralised liquid, to have pressed specified volume adding dehydrated alcohol on the reagent bottle.
6.2. rotation is taken off and is contained silica gel particulate purification column, places the 1.5ml centrifuge tube, centrifugal 2 minutes of top speed.
6.3. purification column is placed clean 1.5ml centrifuge tube, on silica gel particle, adds 500 μ l water.Stir silica gel particle gently with the Tip head it is soaked into fully by elutriant, room temperature left standstill 1 minute.Centrifugal 1 minute of 12000 * g.
6.4. abandon purification column, in the plasmid DNA of wash-out, add 400 μ l Virahols, mix centrifugal 10 minutes of 12000 * g.
Abandon supernatant 6.5. carefully be inverted centrifuge tube.70% ethanol that adds 500 μ l-20 ℃ precoolings, centrifugal 2 minutes of 12000 * g.
Abandon supernatant 6.6. carefully be inverted centrifuge tube, drying at room temperature 10 minutes.
6.7. precipitate with the suitable quantity of water dissolving DNA.
Claims (5)
1, a kind of Phase Partition Purified Plasmid DNA Method is characterized in that by following operation steps:
1) liquid feeding suspends: after containing that the bacterium liquid of plasmid is centrifugal and abandoning supernatant, add bacterium suspension and make thorough suspension;
2) alkaline splitting: add the gentle mixing of bacterium lysate, make the abundant cracking of thalline, form bright solution;
3) in and aggegation: add the gentle mixing of neutralization buffer, to forming the aggegation piece; Described neutralization buffer is ammonium sulfate or cesium chloride 380～480 grams, Guanidinium hydrochloride 28～98 grams, and SODIUM PHOSPHATE, MONOBASIC 16～18 grams add water to 1 liter;
4) become to be separated: the gentle mixing of bonus point phase solution, form emulsion, centrifugal, abandon phase, will change the residual INTERPHASE CARBIDE PRECIPITATION of microstrainer elimination down mutually over to; Described phase-splitting solution is ethanol: Virahol (v/v)=2～10: 1.
5) filtered and recycled: in filtrate, add the DNA binding soln and change DNA preparation pipe over to, make solution filter pellosil, DNA is attached on the pellosil and obtains concentrating and reclaiming with negative pressure or centrifuging.
2, Phase Partition Purified Plasmid DNA Method according to claim 1 is characterized in that: be added with ammonium acetate 36～45 grams in the described neutralization buffer.
3, Phase Partition Purified Plasmid DNA Method according to claim 1 and 2 is characterized in that: with 1) volume was 1 volume when step finished, 3), 4) add neutralization buffer in the step: phase-splitting solution (v/v)=1.6～2.4: 2.6～3.2.
4, Phase Partition Purified Plasmid DNA Method according to claim 1 and 2 is characterized in that: the precooling of described phase-splitting solution.
5, Phase Partition Purified Plasmid DNA Method according to claim 3 is characterized in that: the precooling of described phase-splitting solution.
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|---|---|---|---|
| CN 03141590 CN1241933C (en) | 2003-07-10 | 2003-07-10 | Plasmid DNA purifying method by phase separation method |
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| CN1241933C true CN1241933C (en) | 2006-02-15 |
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| CN101016330B (en) * | 2006-02-10 | 2011-07-20 | 上海主健生物工程有限公司 | Application of silica gel solution in extraction of human oral cavity cell DNA |
| CN115404154A (en) * | 2021-05-10 | 2022-11-29 | 艾棣维欣(苏州)生物制品有限公司 | Device for extracting plasmid DNA from bacteria |
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Assignee: Aisjin Biotechnology (Hangzhou) Co.,Ltd. Assignor: Yu Weiming Contract fulfillment period: 2004.11.10 to 2023.7.9 Contract record no.: 2008330001913 Denomination of invention: Plasmid DNA purifying method by phase separation method Granted publication date: 20060215 License type: Exclusive license Record date: 20081111 |
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Free format text: EXCLUSIVE LICENSE; TIME LIMIT OF IMPLEMENTING CONTACT: 2004.11.10 TO 2023.7.9; CHANGE OF CONTRACT Name of requester: AISIJIN BIOTECHNOLOGY ( HANGZHOU ) CO., LTD. Effective date: 20081111 |
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Effective date of registration: 20151203 Address after: 310052, room 2, building 256, 102-106 Hung Hong Road, Hangzhou, Zhejiang, Binjiang District Patentee after: GENE ERA BIOTECH CO.,LTD. Address before: Stone town of Hangzhou city in Zhejiang province 310022 AFP Village City Industrial Park building B2-1 (Weitejie) Patentee before: Yu Weiming |
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