CN1313478C - New method for rapid purifying DNA plasmid in large quantities - Google Patents

Abstract

The present invention relates to a new method for rapid purifying plasmid DNA in a large-scale mode, which belongs to the field of the research of molecular biology. In the method, nine kinds of reagents and a centrifuge tube with the capacity of 50 to 100 milliliters, wherein the reagents comprise thallus cleaning solution, thallus heavy-suspension buffer solution, thallus cracking solution, cracking neutralization solution, isopropyl alcohol, bergmeal suspension, protein washing solution, ethanol with the concentration of 80%, and DNA diluting solution. Compared with the existing purification method, the present invention has easy operation, stable result, high yield, high purity and no pollution of protein and RNA, and one extraction only needs ninety minutes. Compared with the same plasmid DNA extracted by a Qiagen stud type purification Kit and a home-made SBS resin type kit, the DNA extracted by the method has likewise clear electrophoresis strips and does not have RNA. the present invention has low price and does not need poisonous reagents such as phenol, chloroform, etc. The DNA extracted by the method can be used for preparing DNA vaccines after amplified.

Description

A kind of novel method of rapid purifying DNA plasmid in large quantities

[technical field] the present invention relates to the molecular biology research field, is a kind of novel method of rapid purifying DNA plasmid in large quantities.

[background technology] plasmid DNA generally is meant the small molecules double-stranded DNA that is independent of karyomit(e) covalently closed circle in addition in the tenuigenin, and it has independently replication, and can be assigned in the daughter cell with karyomit(e) in cell fission.Plasmid can shift in of the same race, generic bacterium, also can shift between bacterium not of the same race.Some plasmid can also be incorporated on the chromosomal DNA, so the transfer of plasmid is again the transfer of gene simultaneously.Because plasmid molecule is little, be convenient to separate and extract, after transforming, can become the important carrier of clone gene, at present, be applied to engineered main plasmid and pBR322 arranged and the plasmid of deriving, expressive plasmid and fabric shuttle-type plasmid, be plasmid vector after artificial reconstructed (referring to Sun Shuhan. Principles of Gene Engineering and method. Beijing: People's Medical Officer Press, 2001,43-44) use plasmid as gene cloning carrier, most important prerequisite is the plasmid DNA molecule that will obtain large-scale purification, thus efficiently apace from bacterial cell plasmid purification significant.

The present existing conventional method of extracting plasmid is an alkaline lysis.But the purification process for plasmid has a lot: sucrose or cesium chloride-ethidium bromide density gradient centrifugation is the standard molecular biology method of plasmid purification, but not only consuming time being difficult to of this method amplifies, also use toxicity and mutagenicity reagent, therefore be not suitable for the plasmid DNA preparation.Chromatography is the topological constraints of utilizing the molecular size of nucleic acid to be separated and chemical property, causing with part bonded ability with because of the molecule superhelix, by the interaction of nucleic acid and stationary phase, and separation and purify DNA molecule from impurity selectively.At present the main mode of chromatogram purification has anion-exchange chromatography, Size Exclusion Chromatograph SEC, hydrophobic chromatography, triple strand dna-affinity chromatography, reverse chromatograms etc. (referring to people such as PrazeresDM, Chromatography A, 1998 (806): 31; People such as Horn NA, Human gene therapy, 1995 (6): 565; People such as Diogo MM, Bioseparation, 2002 (10): 211; People such as Wils P, Gene Therapy, 1997,4 (4): 323; People such as Green AP, Biopharm, 1997,5:52).Anion-exchange chromatography can be caught plasmid DNA well because of its very strong specificity.Size Exclusion Chromatograph SEC can be used for the exchange of damping fluid and separablely goes out some micromolecular pollutents.Hydrophobic chromatography also is used to the mass preparation super spirial plasmid in recent years, though and reverse chromatograms and affinity chromatography also are used to the purifying of plasmid DNA, mainly only be applied to laboratory level.In recent years, nanotechnology has become the focus of biophysics and biochemical research.Schluep etc. use triple-helix structure as intermediary, plasmid DNA purification from bacterial lysate.They will be connected with the magnetic nanoparticle of Streptavidin and the reaction of oligonucleotide probe that synthetic is connected with vitamin H, hatch with the bacterial lysate for preparing again, make plasmid DNA and oligonucleotide probe fully form triple-helix structure, wash under strong magnetic field action, plasmid DNA elutes from triple-helix structure.Levison then with the agarose microbeads that is surrounded by the superparamagnetism composition from bacterial lysate, successfully isolate highly purified plasmid DNA (referring to Sun Minli. open whitely, the biotechnology journal, 2001,17 (6): 601) to have made up with the Mn-Zn ferro-magnetic be magnetic nuclear for Lei Han etc., the styrene-propene acid copolymer is the polymer shell, the magnetic macromolecular microsphere of surface band carboxyl, the polyglycol solution of high density and certain salt concn pure condition under, DNA can optionally be attached on the band carboxyl microballoon of surface, reaction through the short period of time, this DNA-magnetic microsphere mixture can thoroughly be cleaned by 70% cold ethanol, with elutriant DNA is eluted afterwards, it is not destroyed (referring to people such as Lei Han to obtain highly purified DNA and superhelix conformation like this, the hi-tech communication, 2002,8:75) we can say, the magneticsubstance isolated plasmid dna has safety, low consumption, fast, characteristics such as efficient, but whether can be applied to mass preparation, still there is not relevant report at present.Lander etc. are to using hexadecyl brometo de amonio (CTAB) selective precipitation plasmid DNA, structure is successfully separated plasmid DNA (referring to LanderRJ from other host's pollutent, Biotechnol Bioeng, 2002,79 (7), 776) CTAB is a kind of cationic detergent, has the characteristic of precipitate nucleic acids and acidic polysaccharide from the solution of low ionic strength.With this understanding, protein and neutral saccharan are still stayed in the solution.In the solution of high ionic strength, the poly beyond CTAB and protein and the acidic polysaccharide forms mixture, but can not precipitate nucleic acids; In addition, the affinity of adjacent hydrophobic tail has strengthened it regularly with the effect of other molecular separation in the CTAB molecule, therefore CYAB can be used for preparing DNA from the part bacterial strain of some Gram-negative bacteria, and other alternative methods are to come the isolated plasmid dna molecule also to obtain success with ammonium sulfate, spermidine and spermine etc. by selective precipitation.Other has research to combine with the cellulose nitrate membrane filtration with ion-exchange, and the liquid behind the ion-exchange wash-out is passed through 0.4 μ m nitrocellulose membrane.Most plasmid DNA macromole passes film and can reclaim from filtrate, and the chromosomal DNA of sex change is by membrane retention.Under the effect of 1.5mol/LNaCl, the RNA molecule is also by membrane retention.The sample that utilizes the Southern hybridization analysis to collect finds that the RNA pollution level 27% drops to below 1% from surpassing (referring to people such as Levy MS, Biotechnol, 2000,76 (2-3): 197).Giovannini etc. then use high performance membrane chromatographic separation super spirial plasmid DNA, the result is under the condition of gradient elution of synchronous wash-out or its beginning liquid of high ionic strength, linear plasmid, open loop plasmid and super spirial plasmid are eluted successively (referring to people such as Giovannini R, AnalChem, 1998,70 (16): 1971).Poeder etc. (1998) have reported the method for preparing super spirial plasmid DNA by the preparation scale electrophoresis, earlier by after the alkaline lysis method, supernatant liquor is packed in the multicell electrophoresis apparatus, when strength of electric field is 8cm, when cell concn is 30g, can reach 75% ± 2% through the rate of recovery behind the electrophoresis of 60min.

In a word, the improvement of on traditional method basis, being done, its plasmid DNA of obtaining must be measured low, of low quality, and leaching process length consuming time, and phenol and chloroform toxic substance are arranged again, and both contaminate environment endangered the test operation personnel health again, was not suitable for extensive purification.Though the novel method of development in recent years has overcome the shortcoming of traditional method, simplified purification process greatly, its material therefor price height, the operative technique difficulty is big etc., and characteristics have determined them generally not use.Therefore, use aforesaid method to be difficult to satisfy the high quality plasmid DNA of needed a large amount of purifications in the clinical immunity test, thereby need find out a kind of New Method of Purification of plasmid DNA that can be quick, a large amount of, simple and easy, efficient, high-quality, inexpensive and harmless.

[summary of the invention] inventor had done comparative studies work widely, find that as a result of it itself is a kind of good adsorbent of adsorbing nucleic acid that diatomite has, but the length of its absorption nucleic acid also has certain restriction, usually between 500bp～5kb, and general plasmid commonly used is all in this scope; The chromosomal DNA of bacterium even be adsorbed, is the characteristics that also can be washed down in washing owing to generally all greater than 10kb, therefore be difficult for being adsorbed when absorption.

Based on above characteristics, the present invention has found through the diatomite of special adsorption function can be arranged plasmid DNA after certain processing.Utilize the plasmid DNA of conventional alkaline lysis preparation, process is mixed with the diatomite of special processing, special absorption plasmid DNA, thus significantly reduced bacterial chromosome and endotoxic pollution.By added RNase in extracting solution R1, the RNA of bacterium is most of digestion when extracting, and the pollution of its RNA has also been dropped to lower level, and this has just significantly reduced ordinary method subsequent disposal institute's time spent.Therefore can clearly see the pollution that does not have bacterial chromosome and RNA with the plasmid electrophoresis of Purification of Diatomite; Whole leaching process shortened in one and a half hours, simplified leaching process, improved working efficiency, reduced experimental cost, and extract the plasmid obtain can be directly used in comprise that enzyme is cut, the various molecular biology operations and the test of clinical animal immune of determined dna sequence etc., whole purification process need not used specific apparatus, and testing crew also need not carry out special training on operation.The inventor has done further research based on the purpose that it is applied to put into practice production, has found the method for utilizing the quick purifying DNA plasmid of diatomite really.

So, the purpose of this invention is to provide a kind of New Method of Purification of quick, a large amount of, simple and easy, efficient, high-quality, inexpensive and harmless plasmid DNA.

In the plasmid DNA extractive technique, obtain the plasmid DNA that quality is good, purity is high, gordian technique is the elimination of cellular lysate, protein and RNA.Core of the present invention is the salt that agents useful for same adopts different pH and different concns, under high salt state, single-minded ground of the diatomite energy adsorption of DNA of handling, and under less salt or aqueous solution state, DNA is eluted, the plasmid DNA purity of being extracted can satisfy the relevant test of molecular biology, comprises that the clone identifies that enzyme is cut, connection, sequencing and transfection etc., and can satisfy clinical zooperal requirement.

The present invention includes 9 kinds of reagent, i.e. thalline scavenging solution, the resuspended damping fluid of thalline (R1); Cellular lysate liquid (R2); Cracking neutralizer (R3); Virahol (R4), diatomite suspension (R5); Albumen washings (R6); 80% ethanol (R7); DNA diluent (R8).Their composition and proportioning are respectively:

Reagent thalline scavenging solution

Component: 0.1mmol/L NaCl

10mmol/L Tris-HCl(pH?8.0)

1mmol/L EDTA(pH?8.0)

Proportioning: preparation 100ml solution

Get 2.5mol/L NaCl 4ml

1mol/L?Tris-HCl 1ml

5mol/L?EDTA 1ml

Distilled water 80ml

Be settled to 100ml with the sterilization distilled water, sterilized 4 ℃ of preservations 20 minutes for 115 ℃.

Reagent R1:

Component: 50mmol/L glucose

50mmol/L Tris-HCl(pH?8.0)

10mmol/L EDTA(pH?8.0)

150μg/ml RNaseA

Proportioning: preparation 100ml R1

Get glucose (C ₆H ₁₂O ₆) 0.991 gram

Distilled water 80ml

0.5mol?EDTA(pH?8.0) 2ml

1mol?Tris-HCl(pH?8.0)?2.5ml

Be settled to 100ml with the sterilization distilled water, 115 ℃ the sterilization 20 minutes, during use by adding 150mgRnaseA in every 100ml solution.4 ℃ of preservations.

Reagent R2:

Component: 0.2molNaOH

1％ SDS

Proportioning: preparation 100ml R2

Get 10mol NaOH 2ml

Distilled water 80ml

10％SDS 10ml

Be settled to 100ml with the sterilization distilled water, room temperature preservation.

Reagent R3:

Component: 4mmol/L KAc, pH4.8

Get Potassium ethanoate 23.55 grams

Distilled water 60ml

After the stirring and dissolving, add the 28.5ml Glacial acetic acid, transferring pH with about 1.5ml concentrated hydrochloric acid is 4.8, mends the sterilization distilled water to 100ml.Room temperature preservation.

Reagent R4: Virahol (analytical pure) is a commercialization reagent.

Reagent R5:

Component: Guanidinium hydrochloride 69.9 grams

Distilled water 60ml

Diatomite 5 grams

Get of the sterilization distilled water dissolving of 68.9 gram Guanidinium hydrochlorides, be settled to 100ml again with 40ml.

Diatomaceous processing: the concentrated nitric acid of getting a certain amount of diatomite adding certain volume was handled about 30 minutes.Discard nitric acid, after cleaning with distilled water, centrifugal, oven dry gets final product.

Reagent R6:

Component: 50% ethanol

0.1mol/L NaCl

0.01mol/L Tris-HCl?pH7.5，

0.05mol/L EDTA

Proportioning: preparation 100ml R5

Get dehydrated alcohol 50ml

2.5mol/L?NaCl 4ml

1mol/L?Tris-HCl 10ml

0.05mol/L?EDTA(pH8.0) 10ml

Steam water with disinfectant water and be settled to the 100ml. room temperature preservation.

Reagent R7:

Component: dehydrated alcohol 80ml

Distilled water 20ml

After mixing, room temperature preservation.

Reagent R8:

Component: TE damping fluid (pH7.6) 100ml

Proportioning: configuration 100ml TE damping fluid

Get Tris salt (analytical pure) 1.58 grams

EDTA (analytical pure) 0.372 gram

Distilled water 80ml

After the stirring and dissolving, transfer to pH7.6, be settled to 100ml with the sterilization distilled water.4 ℃ of preservations.

Comparison shows that with existing reported method the main advantage of the present invention is: easy and simple to handle, the result is stable; Weak point consuming time, 90 minutes with the interior all processes of finishing; Must measure height, to a high copy number plasmid, the conventional cultivation 18 hours, when bacterial growth density OD600 was about 3.5,1.5ml bacterium liquid can obtain 10-15 μ g DNA; Purity is good, and no protein and RNA pollute, and the plasmid DNA of extraction can supply endonuclease digestion, connection, sequencing, eukaryotic cell transfection equimolecular biological test usefulness, and through amplifying the preparation that can be used for dna vaccination; Inexpensive, compare with similar products at home and abroad, price reduces by 50%; Since need be not anti-with phenol and chlorine, the worry of environmental pollution both eliminated, harmless to test operation personnel health again; Simple to operate, need not testing crew is carried out special training on operation; It is simple to test used instrument, need not buy the instrument of special expensive.

Using present method extraction plasmid DNA can operate by following concrete grammar:

(1) shakes bottle a large amount of culturing bacterium amplification plasmids (seeing " molecular cloning test guide " for details) routinely;

(2) the conventional thalline of cultivating 18-20 hour of learning from else's experience is got 500ml bacterium liquid, and room temperature centrifugal 10 minutes with 5000rpm is abandoned supernatant;

(3) with the resuspended thalline of STE of 40ml precooling, be transferred in 2 50ml centrifuge tubes, room temperature is abandoned supernatant with 6000rpm 5 minutes;

(4) add the R1 of 5ml precooling in the precipitation, mixing fully vibrates;

(5) add the freshly prepared R2 of 10ml, put upside down mixing gently 5 times, about 10 minutes of ice bath;

(6) R3 of adding 7.5ml precooling puts upside down mixing 5 times, about 5 minutes of ice bath gently;

(7) 4 ℃, with 12000rpm centrifugal 10 minutes, get supernatant to other 50ml centrifuge tube;

(8) add the R4 of 0.6 times of volume, put upside down mixing, room temperature was placed 10 minutes, and 4 ℃, with 12000rpm centrifugal 10 minutes, abandon supernatant, 70% washing with alcohol precipitation, 37 ℃ to be evaporated to the precipitation edge transparent;

(9) add 5ml R8 (TE) and make it dissolving;

(10) add 5ml R5, shake up when taking, room temperature absorption 5 minutes, centrifugal 5 minutes of 12000rpm discards centrifugate;

(11) add 5ml R6, centrifugal 5 minutes of room temperature 12000rpm discards centrifugate;

(12) add 5ml R7, centrifugal 5 minutes of room temperature 12000rpm discards centrifugate, and room temperature was placed 10 minutes, makes it dry;

(13) with 1ml R8 dissolving, room temperature is placed 8 minutes (or 37 ℃ placed 5 minutes), desorption, and centrifugal 5 minutes of room temperature 12000rpm,--20 ℃ of preservations are standby;

The gained plasmid DNA is got 2 μ L electrophoresis (0.8% agarose, 80V, 30 minutes) and is detected its purity and quantitative with ultraviolet spectrophotometer.

[Brief Description Of Drawings] Fig. 1 uses present method to the thalline that contains same plasmid DNA and uses Qiagen pillar purification kit and the plasmid DNA electrophoretogram of homemade match Parkson resin type test kit extraction.Among the figure, Mark is DL15000 molecular weight marker 1A, the 2A plasmid DNA for using present method to extract; 3A, 4A corresponding plasmid DNA for using Qiagen pillar purification kit to be extracted; 5A, 6A are the corresponding plasmid DNA that use match Parkson resin type test kit is extracted.Fig. 1 result shows that the plasmid DNA of using the present invention to extract compares with domestic and international plasmid purification test kit, is not having obvious difference aspect purity and the output.

Claims (4)

1. the novel method of a rapid large-scale purifying DNA plasmid, its method feature is to operate as follows:

(1) shakes bottle a large amount of culturing bacterium amplification plasmids (seeing " molecular cloning test guide " for details) routinely;

(2) the conventional thalline of cultivating 18-20 hour of learning from else's experience is got 500ml bacterium liquid, and room temperature centrifugal 10 minutes with 5000rpm is abandoned supernatant;

(3) with the resuspended thalline of STE of 40ml precooling, be transferred in 2 50ml centrifuge tubes, room temperature is abandoned supernatant with 6000rpm 5 minutes;

(4) add the R1 of 5ml precooling in the precipitation, mixing fully vibrates;

(5) add the freshly prepared R2 of 10ml, put upside down mixing gently 5 times, about 10 minutes of ice bath;

(6) R3 of adding 7.5ml precooling puts upside down mixing 5 times, about 5 minutes of ice bath gently;

(7) 4 ℃, with 12000rpm centrifugal 10 minutes, get supernatant to other 50ml centrifuge tube;

(8) add the Virahol R4 of 0.6 times of volume, put upside down mixing, room temperature was placed 10 minutes, and 4 ℃, with 12000rpm centrifugal 10 minutes, abandon supernatant, 70% washing with alcohol precipitation, 37 ℃ to be evaporated to the precipitation edge transparent;

(9) add 5ml R8 (TE) and make it dissolving;

(10) add 5ml R5, shake up when taking, room temperature absorption 5 minutes, centrifugal 5 minutes of 12000rpm discards centrifugate;

(11) add 5ml R6, centrifugal 5 minutes of room temperature 12000rpm discards centrifugate;

(12) add 5ml R7, centrifugal 5 minutes of room temperature 12000rpm discards centrifugate, and room temperature was placed 10 minutes, makes it dry;

(13) with 1ml R8 dissolving, room temperature is placed 8 minutes (or 37 ℃ placed 5 minutes), desorption, and centrifugal 5 minutes of room temperature 12000rpm,--20 ℃ of preservations are standby;

2. the novel method of the described purifying DNA plasmid in large quantities of claim 1, its material characteristics is to comprise:

(1) thalline scavenging solution, component are 0.1mmol/L NaCl, 10mmol/L Tris-HCl (pH 8.0), 1mmol/L EDTA (pH 8.0);

(2) the resuspended damping fluid of thalline (R1), the component of R1 are 50mmol/L glucose, 25mmol/L Tris-Cl (pH8.0), 10mmol/L EDTA (pH 8.0), 200 μ g Rnase A;

(3) cellular lysate liquid (R2), the component of R2 is 200mmol/L NaOH, 1%SDS;

(4) cracking neutralizer (R3), the component of R3 is 4mmol/L KAc, pH4.8;

(5) Virahol (R4), the component of R4 is the business analysis pure reagent;

(6) diatomite suspension (R5), the component of R5 are that 5 gram diatomite mix with 50ml 7mol/ Guanidinium hydrochloride, shake up when taking;

(7) albumen washings (R6), the component of R6 is 50% ethanol, 100mmol/L NaCl, 10mmol/L Tris-HCl (pH7.5), 5mmol/L EDTA;

(8) 80% ethanol (R7), the component of R7 are 80% analytically pure ethanol;

(9) DNA diluent (R8), the component of R8 are TE damping fluid (10mmol/LTris-HCl pH7.5,1mmol/L EDTA);

(10) in addition some 50--100ml centrifuge tubes need be arranged.

3. in the material by the described purifying DNA plasmid of claim 1, be characterized as being formulated as of described reagent R5: 5 gram diatomite mix with 50ml 7mol/L Guanidinium hydrochloride; Necessarily shake up when taking, and diatomite necessarily requires acidification.

4. by in the material of the described purifying DNA plasmid of claim 1, be characterised in that 50% ethanol that is formulated as of described reagent R6,100mmol/L NaCl, 10mmol/L Tris-HCl (pH7.5), 5mmol/L EDTA.