CN101979541A - Rapid covalently closed circular DNA purification kit - Google Patents
Rapid covalently closed circular DNA purification kit Download PDFInfo
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Abstract
The invention relates to a rapid covalently closed circular DNA purification kit. The kit is finished by a method which comprises the following steps of: bacterial precipitation, non-alkaline cracking, combining circular DNA by a silicone membrane, column elution, and elution; on the basis of the method, components such as hexamminecobalt chloride and spermine are added; bacteria are cracked by adopting improved non-alkaline cracking liquid; linear genome DNA and RNA are highly concentrated by using polyamine to lose the capability of combining the silicone membrane selectively, so that the genome DNA and RNA can still combine the silicone membrane; and a rapid covalently closed circular DNA purification kit can be achieved by washing and eluting. The DNA purification technology of the invention replaces two-step DNA purification technology, completely saves a plasmid crude extraction step, does not depend on the insensitivity of the species of escherichia coli to the acuteness degree of an operation, has less steps, shortens the time of the purification operation of plasmid DNA, saves manpower and material resources, and is suitable to be used in high-flux plasmid DNA purification and industrial large-scale plasmid DNA purification technology.
Description
Technical field:
The present invention relates to a kind of quick covalently closed circular DNA purification kit.
Background technology:
Plasmid DNA is that molecular biology research almost all will be used plasmid DNA every day, also need frequent plasmid DNA purification, this external gene therapy and gene vaccine field, along with increasing gene therapy medicament and gene vaccine enter clinical trial and industrialization stage, also increasing to the demand of plasmid DNA.Because the clinical requirement difference of using plasmid DNA with the quality standard of plasmid DNA with molecular biology research, small scale experiments chamber purifying and technical grade large scale purification process be difference to some extent also, and therefore good plasmid DNA purification technique must satisfy the different demands of every field.Can reach at present this requirement only based on alkaline lysis plasmid DNA purification process, the molecular biology research field that is is mainly concentrated in the use of another method boiling method.
One, alkaline lysis plasmid DNA (covalently closed circular DNA) purification technique
Alkaline lysis be 1979 by Birnboim ﹠amp; Doly invention, its plasmid are slightly put forward part and be made up of FOUR EASY STEPS: the first step, bacterium is resuspended.Resuspended liquid composition is 25mM Tris-Cl (pH 8.0), 50mM glucose and 10mM EDTA.Wherein Tris-Cl is used for buffer pH with the stabilized DNA structure; Glucose is used to keep osmotic pressure and buffer pH, prevents the genomic dna overbreak; EDTA is used for Ao and closes divalent-metal ion, makes the easier fragmentation of cell, also makes the DNase inactivation, helps keeping the integrity of plasmid DNA.Second step, alkaline lysis.The composition of alkaline lysis liquid is 1%SDS and 0.2M NaOH.First effect of SDS and NaOH is to make lysis discharge the thalline content.Second effect of NaOH is that the pH of system is remained between the 12.0-12.5, in this pH scope, reversible denaturation only takes place because distinctive topological superhelix is arranged in genomic dna irreversible denaturation and plasmid DNA, can be in next step N-process natural renaturation; Second effect of SDS is solubilising protein (on average two amino acid in conjunction with a SDS molecule) and fat.The 3rd step, neutralization.The composition of neutralizer is 3M Potassium ethanoate/2M acetic acid.Its on the one hand in and NaOH, make the plasmid DNA renaturation and genomic dna still keeps the strand state; Salt concn in the raising system on the other hand makes protein, genomic dna and RNA that salt precipitation take place; Potassium of Jia Ruing and SDS reaction generates insoluble dodecyl sulphate potassium (PDS) in addition, PDS also with protein and genomic dna co-precipitation.So after the neutralization reaction, most of protein, genomic dna and RNA exist with the insolubles state, and most plasmid DNA does not form any precipitation, but exists with solution state because molecular weight is less.The 4th step, centrifugal.All insolubless that centrifugal process formed for the 3rd step are deposited to the pipe end, and supernatant is the plasmid DNA crude extract, can be directly used in the smart extracting method of various plasmids and carry out further purification process.
The great advantage of alkaline lysis is a technology maturation, can handle from 1mL to 500mL or more sample, both has been suitable for small-scale scientific research and has used, and can be used for the plasmid DNA of the clinical usefulness of technical grade scale operation again, and be applicable to colibacillary all bacterial strains.
Its main drawback is that operation is difficult to control, major cause be make genomic dna generation irreversible denaturation and cyclic plasmid DNA that the pH scope of reversible denaturation takes place is very narrow and small, between 12-12.5.If pH is lower than 12.0 then fully sex change of genomic dna will be polluted plasmid DNA; If pH is higher than 12.5, then plasmid DNA generation irreversible denaturation reduces plasmid DNA output.For pH being maintained this scope, necessary rapidly abundant mixing behind the adding alkaline lysis liquid, but discharge the genomic dna of bacterium at alkaline lysis after, the lysate very thickness that becomes, has only fully mixing of vigorous stirring, and vigorous stirring can make plasmid DNA be broken into linear DNA, equally irreversible denaturation takes place and loses reduction output with genomic dna; Vigorous stirring also can make big genomic dna fragment into small segment, is difficult to form precipitation, pollutes plasmid DNA at last.So the operation of alkaline lysis plasmid DNA purification extremely is difficult to optimize, handle all the more so to technical grade extensive (more than 10 liters).
Another shortcoming of alkaline lysis is to be not easy to remove RNA when handling on a large scale to pollute.Can use animal derived RNase when studying with small-scale plasmid DNA purifying, but purifying is clinical when using plasmid DNA at technical grade extensive (scale more than 10 liters), use animal derived RNase unrealistic on a large scale, because not only price is very expensive, but also introduce the virus of animal and other cause of diseases (as crazy heifer disease virus) easily, increase the trouble of a lot of quality examination aspects, cost is sharply risen.Generally take at present the way of non-enzyme to remove RNA, but need increase a lot of operation stepss, increased cost.
The 3rd shortcoming of alkaline lysis is that step is many, and the plasmid DNA loss is many, and ult rec has only about 50-70%.
Two, boiling method plasmid DNA purification technique
Present method the earliest by Holmes and Quigley at report in 1981, its process be earlier with bacterial suspension in the damping fluid that contains TritonX-100 and N,O-Diacetylmuramidase that can the peptic cell wall, be heated to 100 ℃ then and make its cracking.Heating also helps to untie the base pairing of DNA chain, and makes protein and chromosomal DNA sex change except destroying mantle.Because cyclic plasmid DNA has the topological framework of mutual winding, after temperature descended, on your marks again for the base of closed-circular DNA, forms the superhelix molecule.Centrifugal chromosomal DNA and the protein precipitation of removing sex change just can obtain containing the supernatant liquor of plasmid DNA.Boiling lysis is very effective for the miniplasmids less than 15kb, can be used for plasmid DNA purification from 1mL-250mL bacterium liquid, and most coli strain all is suitable for.
Its shortcoming is: (1) is not suitable for those coli strains that can discharge a large amount of carbohydrate after denaturing agent, N,O-Diacetylmuramidase and heat treated.Coli strain HB101 and derivative strain thereof (comprising TG1) can produce a large amount of carbohydrate, are unsuitable for using the boiling method cracking.(2) for the coli strain of expressing DNase, do not recommend to use the boiling method plasmid DNA purification yet.Because in boiling part, DNase can complete deactivation, and Mg is arranged in the operation steps of back unavoidably
2+Have (enzyme as restriction endonuclease is cut process), plasmid DNA can be degraded.When (3) extracting on a large scale, bacterial lysate is too sticky, is difficult to handle, and must add paraxin during culturing bacterium in substratum.Present method mainly is that use in the molecular biology research field at present, seldom is used for technical grade plasmid in large scale DNA purifying.
Three, Eppendorf single stage method plasmid DNA purification technique
A kind of single stage method plasmid DNA purification technique based on N,O-Diacetylmuramidase cracking bacterium has been invented by nearest German Eppendorf company, it is a kind of quick, anorganic high quality plasmid DNA patented technology of extracting from bacterium, and its technical data (comprising patent application) is not also published now.But according to its propaganda materials of publishing, this method is used the single lysate lysing cell that contains N,O-Diacetylmuramidase and stain remover, and the split product clarification is inviscid, and operation is very easy to.Dna direct is attached on the filtering membrane after the cracking.Obtain supercoiled plasmid DNA by quick centrifugal cleaning and wash-out.Its advantage is that operation steps is simple, the solution quantity that needs seldom, the used time is less than half of alkaline lysis, but the quality height of plasmid DNA, can be used for downstream tests as order-checking, enzyme is cut and clone.The shortcoming of this method is not to be suitable for all bacterial strains.
This method is compared with single step plasmid DNA purification technique of the present invention, operating process is quite similar, the maximum Eppendorf method that is not both need be used animal derived N,O-Diacetylmuramidase cracking bacterium, need to use animal derived RNase to remove RNA, because above-mentioned price and may introduce factor such as cause of disease, this method obviously is difficult to use technical grade plasmid in large scale DNA purifying and gets on.Secondly, the gentle cleavage method that is based on N,O-Diacetylmuramidase that the Eppendorf method is used, the chemical cracking liquid that contains guanidinium isothiocyanate and SDS that cracking bacterium effect is used not as the present invention certainly, the plasmid DNA rate of recovery can be very not high yet.Gentle in addition lysate is very weak to the inhibition of DNase, can not effectively prevent its destruction to plasmid DNA, is unsuitable for the EndA wild type strain.
Summary of the invention:
The objective of the invention is in order to overcome above-mentioned technical deficiency, the non-alkaline bleach liquor cleavage liquid that a kind of use is provided is exactly to have increased the high cobalt of six amminos and two kinds of compositions of spermine on the basis of above-mentioned prescription, adopt the non-alkaline lysis liquid cracking bacterium of improvement, optionally make the genomic dna of wire and RNA take place highly to concentrate to lose and pellosil bonded ability by polyamines, and cyclic plasmid DNA does not concentrate, still can combine with pellosil, process washing and wash-out just can reach a kind of quick covalently closed circular DNA purification kit of purpose of single step purification plasmid DNA.
The objective of the invention is to realize by following technical scheme:
A kind of quick covalently closed circular DNA purification kit, it is characterized in that: it is finished by following step and method, and the concrete steps method is as follows,
Covalently closed circular DNA purification technique step is as follows:
The first step: bacterial precipitation, because the bacterium volume at 10-50L, is mainly collected bacterial precipitation with filtration method, remove liquid nutrient medium, obtain thalline;
Second step: non-alkaline lysis, with non-alkaline lysis liquid cracking bacterium, need therebetween to use large stirrer fully to stir, make the bacterium cracking, polyamines makes genomic dna and RNA take place highly to concentrate and loses and pellosil bonded ability simultaneously, we optimize non-alkaline lysis liquid formula, have increased by two kinds of compositions of the high cobalt of spermine and six amminos;
The 3rd step: pellosil is in conjunction with cyclic DNA, the direct upper prop of step gained lysate in the use, lysate directly carries out the plasmid DNA purifying with silica gel adsorption, except that plasmid DNA, and protein and be the genomic dna of tight bulk and RNA can not combine with pellosil and enters and penetrate liquid;
The 4th step: wash post, residual protein, part RNA and small molecular weight impurity on the flush away pellosil washed post with the post liquid of washing of routine, for remove impurity as far as possible, may need repeatedly to repeat this step;
The 5th step: wash-out elutes plasmid DNA stand-by with low salts solution from pellosil; With the solution " as TE or ultrapure water " of less salt with pellosil bonded plasmid DNA wash-out,
Covalently closed circular DNA purification technique method is as follows:
1) bacterial precipitation, microorganism collection: be no more than the saturated bacterium liquid of 3mL incubated overnight with the plastic centrifuge tube collection, centrifugal half a minute of room temperature 12000-15000g, abandon supernatant;
2) non-alkaline lysis, the cracking thalline: add the 0.6-1mL lysate and " shake up with preceding need ", fully piping and druming or vibration cracking bacterium become clarification up to cracking, need half a minute usually; The centrifugal back of bacterium liquid adds high cobalt of six amminos and spermine, and the cobaltic final concentration of six amminos is 25mM, and the final concentration of spermine is 75mM;
3) pellosil is in conjunction with cyclic DNA, in conjunction with DNA: lysate is all transferred in the centrifugal adsorption column, room temperature leaves standstill plasmid DNA is combined with film, centrifugal half a minute of room temperature 12000-15000g, abandon and penetrate liquid, if once supernatant all can not be transferred in the centrifugal adsorption column, be carried out at twice;
4) wash post, prewashing: in centrifugal adsorption column, add the pre-washing lotion of 0.7mL, centrifugal half a minute of room temperature 12000-15000g, abandon and penetrate liquid, if being placed on low temperature, pre-washing lotion produced precipitation, make it to melt with before needing to be heated to 60 ℃, use again behind the mixing;
5) wash-out, prewashing again: in centrifugal adsorption column, add the pre-washing lotion of 0.3mL again, centrifugal half a minute of room temperature 12000-15000g, to abandon and penetrate liquid, this step prewashing is not preferably omitted, otherwise DNA purity is not high;
Clean: what add 0.7mL in centrifugal adsorption column washes post liquid, centrifugal half a minute of room temperature 12000-15000g, abandons and penetrates liquid;
Clean: what add 0.3mL again in centrifugal adsorption column washes post liquid again, centrifugal half a minute of room temperature 12000-15000g, and abandon and penetrate liquid, centrifugal half a minute of room temperature 12000-15000g, dry residual liquid;
Wash-out: place a new 1.5mL to provide plastic centrifuge tube for oneself on centrifugal post, add 30-100uL DNA elutriant, room temperature was placed 2 minutes, if the DNA elutriant is preheating to 65-80 ℃, had both got a kind of quick covalently closed circular DNA purification kit of the present invention; Described
Lysate: be 10%SDS+2M NaCl+10mM spermine;
Pre-washing lotion: be 2M NaCl;
Wash post liquid: be 50% ethanol;
Elutriant: be water;
Polyamines: be spermine: the high cobalt of six amminos=3: 1
The non-alkaline bleach liquor cleavage liquid that the technology of the present invention is used is exactly to increase polyamines and two kinds of compositions of Triton X-114 and get on the basis of above-mentioned prescription, after but the process polyamines concentrates, linear DNA and RNA form structure very closely, completely lose and pellosil bonded ability, and the cyclic plasmid DNA still keeps more open configuration, still can combine with pellosil, so by pellosil the difference of plasmid DNA being adsorbed just can be from containing genomic dna, remove wherein genomic dna and RNA in the mixed solution of RNA and plasmid DNA, reach the purpose of plasmid DNA purifying.
Beneficial effect of the present invention:
The present invention replaces dual-step type plasmid DNA purifying process with single step plasmid DNA purifying process, can directly use single bacterium colony to carry out the plasmid DNA purifying, do not need the incubated overnight bacterium, omit plasmid fully and slightly put forward step, the present invention is found to the high cobalt/spermine of a certain proportion of six amminos under the condition that guanidinium isothiocyanate exists, can optionally induce concentrating of linear DNA and RNA fully, and cyclic DNA still is lax configuration, illustrate that this process is not subjected to the influence of guanidinium isothiocyanate, on the basis of this discovery, we optimize non-alkaline lysis liquid formula, two kinds of compositions of the high cobalt of spermine and six amminos have been increased, with classical alkaline lysis liquid phase ratio, improvement, the non-alkaline lysis liquid that contains polyamines has following advantage:
(1) that genomic dna has been taken place is highly concentrated for polyamines, has greatly reduced the viscosity of lysate, makes no longer thickness of lysate, is convenient to the follow-up various places operation of carrying out.
(2) that genomic dna and RNA have been taken place is highly concentrated for polyamines, no longer combines with pellosil, removes assortment of genes DNA and RNA ten minutes easily.
(3) no longer need alkaline denaturation and, saved for 2/3 operating time with closely-related resuspended, the neutralization of alkaline denaturation and centrifugal three steps.
(4) RNA concentrates the back removal, no longer needs RNase to handle, and has greatly reduced the cost of large scale plasmid purification DNA.
(5) omitted the alkaline denaturation processing, made DNA avoid the alkali damage, the plasmid DNA quality is higher.
Single step plasmid DNA purifying process of the present invention is compared with traditional two-step process, has following advantage:
(1) more quick, make the whole plasmid DNA purification process time shorten 2/3, save the man power and material, be particularly suitable for high-throughput plasmid DNA purifying and technical grade plasmid in large scale DNA purifying.
(2) step is few, so the plasmid DNA loss also reduces, the rate of recovery improves, and therefore can directly carry out the purifying of micro-plasmid DNA with single bacterium colony, has removed the microbial culture step of spending the night from.
(3) non-alkaline lysis has been avoided the alkali damage of plasmid DNA, and the ratio of superhelix configuration is bigger, and quality improves.
(4) do not need to use animal derived RNase, reduced the possibility of polluting other pathogenies thus, also greatly reduced cost (especially when technical grade large scale plasmid purification DNA, it will be very expensive using RNase in a large number) simultaneously.
(5) insensitive to the operation severe degree, the plasmid DNA that is suitable for from trace (single bacterium colony) to the various scales of technical grade (more than 10 liters) exists, and operating process does not need to do great change.
(6) universality is wide, does not rely on colibacillary kind.
(7) lysate thickness not, the chromatography column that uses when being not easy to stop up subsequent purification.
Description of drawings:
Fig. 1 is a polyamines concentration of DNA theory structure synoptic diagram of the present invention;
Fig. 2 is a DNA-pellosil combination principle structural representation of the present invention;
Fig. 3 of the present inventionly extracts plasmid DNA from a bacterium colony that contains the pUC19 plasmid, and half is used for electrophoresis (sample A) DNA that obtains, second half with the linearizing of EcoRI restriction enzyme after agarose electrophoresis (sample B) synoptic diagram again;
Fig. 4 of the present inventionly extracts plasmid DNA with present method from 1.5mL contains the bacteria samples of incubated overnight of pUC19 plasmid, DNA uses 200uL elutriant wash-out at last, get 20uL and be used for agarose electrophoresis, the DNA of front is the superhelix configuration, the back for open loop configuration synoptic diagram;
Fig. 5 is that the present invention extracts the pUC19 plasmid DNA with this product from the saturated bacterium liquid of 100mL, uses 2mL elutriant eluted dna at last, gets 20uL and goes up sample and carry out agarose electrophoresis, and the DNA of front is the superhelix configuration, the back be open loop configuration synoptic diagram;
Embodiment: the invention will be further described below in conjunction with embodiment:
Embodiment 1: a kind of quick covalently closed circular DNA purification kit, and it is characterized in that: it is finished by following step and method, and the concrete steps method is as follows,
Covalently closed circular DNA purification technique step is as follows:
The first step: bacterial precipitation, because the bacterium volume at 10-50L, is mainly collected bacterial precipitation with filtration method, remove liquid nutrient medium, obtain thalline;
Second step: non-alkaline lysis, with non-alkaline lysis liquid cracking bacterium, need therebetween to use large stirrer fully to stir, make the bacterium cracking, polyamines makes genomic dna and RNA take place highly to concentrate and loses and pellosil bonded ability simultaneously, we optimize non-alkaline lysis liquid formula, have increased by two kinds of compositions of the high cobalt of spermine and six amminos;
The 3rd step: pellosil is in conjunction with cyclic DNA, the direct upper prop of step gained lysate in the use, lysate directly carries out the plasmid DNA purifying with silica gel adsorption, except that plasmid DNA, and protein and be the genomic dna of tight bulk and RNA can not combine with pellosil and enters and penetrate liquid;
The 4th step: wash post, residual protein, part RNA and small molecular weight impurity on the flush away pellosil washed post with the post liquid of washing of routine, for remove impurity as far as possible, may need repeatedly to repeat this step;
The 5th step: wash-out elutes plasmid DNA stand-by with low salts solution from pellosil; With the solution " as TE or ultrapure water " of less salt with pellosil bonded plasmid DNA wash-out,
Covalently closed circular DNA purification technique method is as follows:
1) bacterial precipitation, microorganism collection: be no more than the saturated bacterium liquid of 3mL incubated overnight with the plastic centrifuge tube collection, centrifugal half a minute of room temperature 12000-15000g, abandon supernatant;
2) non-alkaline lysis, the cracking thalline: add the 0.6-1mL lysate and " shake up with preceding need ", fully piping and druming or vibration cracking bacterium become clarification up to cracking, need half a minute usually; The centrifugal back of bacterium liquid adds high cobalt of six amminos and spermine, and the cobaltic final concentration of six amminos is 25mM, and the final concentration of spermine is 75mM;
3) pellosil is in conjunction with cyclic DNA, in conjunction with DNA: lysate is all transferred in the centrifugal adsorption column, room temperature leaves standstill plasmid DNA is combined with film, centrifugal half a minute of room temperature 12000-15000g, abandon and penetrate liquid, if once supernatant all can not be transferred in the centrifugal adsorption column, be carried out at twice;
4) wash post, prewashing: in centrifugal adsorption column, add the pre-washing lotion of 0.7mL, centrifugal half a minute of room temperature 12000-15000g, abandon and penetrate liquid, if being placed on low temperature, pre-washing lotion produced precipitation, make it to melt with before needing to be heated to 60 ℃, use again behind the mixing;
5) wash-out, prewashing again: in centrifugal adsorption column, add the pre-washing lotion of 0.3mL again, centrifugal half a minute of room temperature 12000-15000g, to abandon and penetrate liquid, this step prewashing is not preferably omitted, otherwise DNA purity is not high;
Clean: what add 0.7mL in centrifugal adsorption column washes post liquid, centrifugal half a minute of room temperature 12000-15000g, abandons and penetrates liquid;
Clean: what add 0.3mL again in centrifugal adsorption column washes post liquid again, centrifugal half a minute of room temperature 12000-15000g, and abandon and penetrate liquid, centrifugal half a minute of room temperature 12000-15000g, dry residual liquid;
Wash-out: place a new 1.5mL to provide plastic centrifuge tube for oneself on centrifugal post, add 30-100uL DNA elutriant, room temperature was placed 2 minutes, if the DNA elutriant is preheating to 65-80 ℃, had both got a kind of quick covalently closed circular DNA purification kit of the present invention; Described
Lysate: be 10%SDS+2M NaCl+10mM spermine;
Pre-washing lotion: be 2M NaCl;
Wash post liquid: be 50% ethanol;
Elutriant: be water;
Polyamines: be spermine: the high cobalt of six amminos=3: 1
1) plasmid DNA is extracted:
The plasmid DNA purifying is to carry out requisite technology such as molecular biology research, producer gene medicine and producer gene vaccine.Plasmid DNA only accounts for below 1% of host bacteria gross weight, so the essence of plasmid purification is exactly to remove the impurity that accounts for gross weight 99%, comprises genomic dna, RNA, protein, intracellular toxin etc.Because genomic dna and plasmid DNA homogeneity (all are double-stranded DNAs too by force, be the configuration difference), the most difficult removal, the pollution of how effectively to remove genomic dna is the difficult point and the key of plasmid DNA purifying always, at present general alkaline lysis and additive method all are exactly to utilize both configuration differences and it is separated, and reach the purpose of plasmid DNA purification.
Single step plasmid DNA purification technique of the present invention also is to utilize genomic dna and Plasmid DNA and It ' difference, but the method for using do not appear in the newspapers, and belongs to original technology.Its process is with the non-alkaline lysis liquid cracking bacterium of improvement, optionally makes the genomic dna of wire and RNA take place highly to concentrate to lose and pellosil bonded ability by polyamines, and cyclic plasmid DNA does not concentrate, still can combine with pellosil, process washing and wash-out just can reach the purpose of single step purification plasmid DNA.The whole process of above-mentioned purifying on molecular level, be actually by three independently the reaction form, right and wrong alkalescence bacterium cracking respectively, polyamines concentrate linear DNA and RNA and pellosil-DNA combination, and their roles can be summarized as follows with the variation of various molecules in the cell:
One, non-alkaline bacterium cracking principle
Non-alkaline bacterium cracking technique is reported in 1979 by Piotr Chomczynski and Nicoletta Sacchi the earliest, is used for RNA and extracts.This non-alkaline bacterial lysate singly is not used for the cracking bacterium, also is used for the histocyte (need carry out under the homogenate condition certainly) of cracking animal and plant, and its major ingredient is SDS, guanidinium isothiocyanate, 2 mercapto ethanol and EDTA.Owing to do not contain highly basic, so can damage dna.The non-alkaline bleach liquor cleavage liquid that the technology of the present invention is used is exactly to increase polyamines and two kinds of compositions of Triton X-114 and get on the basis of above-mentioned prescription, and the effect of each composition is as follows:
1, SDS is a reinforcing yin essence ion stain remover, and it utilizes amphiphilic dissolution of bacteria cytolemma, makes the bacterium cracking.SDS can also make protein (comprising DNase and RNase) sex change and dissolving, helps reducing the enzymolysis of DNA, keeps its integrity.Protein is dissolved state and also helps being eliminated when follow-up pellosil adsorbs.
2, guanidinium isothiocyanate is a kind of strong protein denaturant, not only can make the membranin sex change and destroys cytolemma, and then make the bacterium cracking, and protein is present in the lysate with solution state, is removed when the follow-up centrifugal fractionation by adsorption of pellosil.When 2 mercapto ethanol existed, the effect of guanidinium isothiocyanate denatured protein was better.Using another major reason of guanidinium isothiocyanate is the combination that it can promote the DNA-pellosil, can directly use the centrifugal adsorption method purifying of pellosil DNA wherein so contain the bacterial lysate of guanidinium isothiocyanate.
3,2 mercapto ethanol is a kind of reductive agent, and it destroys the S-S key in the natural protein, and protein (comprising DNase and RNase) is lost activity, the integrity of DNA and RNA in the assurance leaching process.It also has enhancement to the protein denaturation and the dissolving power of guanidinium isothiocyanate.
4, EDTA can Ao closes the Mg on the cytolemma
2+, Ca
2+Deng divalent-metal ion, make the easier cracking of cell, and (DNase needs Mg to suppress the DNase activity thus indirectly
2+As cofactor), the integrity of maintenance DNA.Another important effect of EDTA is exactly that it can stop RNA to combine with pellosil, has removed from and has used expensive RNase to remove the RNA pollution.
5, Triton X-114 is a kind of stain remover, and energy specificity ground form small ball with intracellular toxin and make it to lose and pellosil bonded ability, is eliminated when the plasmid DNA purifying easily.Remove no intracellular toxin and be the standard that important use the is arranged plasmid DNA of (as cell transformation experiment, gene therapy and gene vaccine) must reach.
6, polyamines can make DNA and RNA concentrate, and its ultimate principle sees next section.It is one of innovative point in the present technique that polyamines is incorporated in the bacterial lysate, will describe in detail in the innovation part.
Two, polyamines concentration of DNA principle (as shown in Figure 1)
In the aqueous solution, it is acid that DNA and RNA molecule are, electronegative, polyamines molecules such as spermine, spermidine, the high cobalt of six amminos are positively charged then, they not only can carry out embedded the combination with the major groove of dna molecular, can also carry out combining and striding in the chain chain combination with base and the phosphate on DNA and the RNA chain, the height that 4-6 the order of magnitude takes place for DNA and RNA molecule is concentrated, form very closely structure (Arscott, Biopolymers 30,619-630,1990) relevant (Hoopes of Zi length, NAR9,5493-5504,1981; Murphy, Analytical Biochemistry 295,143,2.
The degree of polyamines concentration of DNA and RNA not only divides 001 with DNA and RNA), and the configuration (wire or ring-type) with the kind of polyamines and nucleic acid is relevant, therefore suitably select the kind of polyamines can optionally make the not DNA of isomorphism type (as genomic dna and plasmid DNA) generation concentrated (Murphy in various degree, Analytical Biochemistry 295,143,2001), but what use in the report of Murphy is the gentle lysate that does not contain high salt, and the configuration selectivity of polyamines concentration of DNA is not very strong with this understanding.Optionally concentrate linear DNA and RNA in the lysate that contains high density guanidinium isothiocyanate (more than the 2M) in order to be implemented in, they damp gene studies personnel have carried out nearly 1 year groping and studying, finally find in lysate, to add high cobalt of a certain proportion of six amminos and spermine first, the selectivity that just can realize linear DNA and RNA concentrates, and it is lost and pellosil bonded ability, and the cyclic plasmid DNA does not concentrate under same condition, still can combine with pellosil.Domestic and foreign literature does not all have the report of similar discovery at present, so this discovery has originality.
Three, the centrifugal adsorption column purify DNA of pellosil principle
In the aqueous solution, it is acid, electronegative that dna molecular is; The silicon on pellosil surface-oxygen key aquation forms silanol (silanol group) and also is slightly acidic, and is electronegative, so can produce Coulomb repulsion between the two, can not mutually combine.But, when having certain density positively charged ion simultaneously in the solution, positively charged ion can be by forming positively charged ion bridge (cation bridge) in and the negative charge (being electrostatic shielding) on DNA and pellosil surface and make the two combination.However, DNA and the main mode of pellosil bonded are not the positively charged ion bridge, but the hydrogen bond that forms between DNA phosphate group and the pellosil surface silanol groups.The effect of positively charged ion bridge just reduces intermolecular Coulomb repulsion, help both near and form hydrogen bond.Though each of DNA and the formation of pellosil surface is fainter to the hydrogen bonded force rate, because its enormous amount still can make DNA be adsorbed on the pellosil surface securely.The DNA-pellosil combination of positively charged ion bridge mediation and hydrogen bond mediation is as shown in Figure 2:
Because hydrogen bond has played vital role in the combination of DNA-pellosil, so the every DNA-of influence pellosil forms the bonding strength that the factor of hydrogen bond ability and quantity can both influence the DNA-pellosil, these factors comprise the pH of salt concn (concentration that comprises guanidinium isothiocyanate) in conjunction with liquid, solution and DNA configuration etc.Generally speaking, be genomic dna and other linear DNA two end dissociatives of wire configuration, be subjected to the obstruction of space structure little, with the number maximum of pellosil surface formation hydrogen bond, in conjunction with the most firm; And in the form of a ring the DNA of configuration (comprising the plasmid DNA that is open loop and two kinds of configurations of superhelix) owing to there is not a free end, limited closely by space structure, form the linear DNA of the number of hydrogen bond with pellosil, so bonding strength also is weaker than the linear DNA of equal length far fewer than equal length.
After but the process polyamines concentrates, linear DNA and RNA form structure very closely, completely lose and pellosil bonded ability, and the cyclic plasmid DNA still keeps more open configuration, still can combine with pellosil, so by pellosil the difference of plasmid DNA is adsorbed the genomic dna and the RNA that just can remove wherein from the mixed solution that contains genomic dna, RNA and plasmid DNA, reaches the purpose of plasmid DNA purifying.
Major technique and performance index:
To being used for the plasmid DNA of molecular biology research, the whole world does not all have unified quality standard at present, and the present invention intends carrying out the standard of the famous plasmid DNA purified product developer Qiagen company of Germany.
To being used for the plasmid DNA of gene therapy and gene vaccine, the present invention intends carrying out the standard that proposes in " the prevention dna vaccination preclinical study technical director principle " and " people's gene treatment research and quality of the pharmaceutical preparations control techniques governing principle " of State Food and Drug Administration's promulgation in 2003.
One, the technological standard that is used for the plasmid DNA of molecular biology research:
| Interventions Requested | Calibration method | Standard |
| Telling test | A kind of restriction enzyme rear electrophoresis | With the expected results unanimity |
| The plasmid configuration | Agarose electrophoresis | Superhelix or open loop configuration |
| Plasmid purity | Ultraviolet spectrophotometer | OD260/280＝1.75-1.85 |
| RNA is residual | Agarose electrophoresis | Can't check |
| GDNA is residual | Agarose electrophoresis | Can't check |
Two, the technological standard that is used for the plasmid DNA of gene therapy and gene vaccine:
| Interventions Requested | Calibration method | Standard |
| Telling test | Restriction enzyme rear electrophoresis more than three kinds | With the expected results unanimity |
| The plasmid configuration | Agarose electrophoresis | ＞90% is the superhelix configuration |
| Plasmid purity | Ultraviolet spectrophotometer | OD260/280＝1.75-1.85 |
| Protein contamination | ELISA or amino acid analysis method | ＜1ug/mg |
| RNA is residual | Agarose electrophoresis | Can't check |
| GDNA is residual | Southern?Blot | ＜2ug/mg |
| Aseptic | UPS23 cultivates | No bacterial growth |
| Intracellular toxin is residual | The LAL test | ＜10EU/mg |
Technological innovation, process innovation:
Technological innovation
The present invention optionally concentrates linear DNA and RNA with polyamines at home and abroad first, and makes it forfeiture and pellosil bonded ability.Project is incorporated into a certain proportion of two polyamine species (spermine and the high cobalt of six amminos) in the non-alkaline lysis liquid and makes it to bring into play the effect of concentration of DNA.It is generally acknowledged that polyamines is the static combination with combining of DNA, during salt ion will pass through and DNA institute is electrically charged suppresses combining of polyamines and DNA, that is to say that salt ionic concentration is high more in the system, polyamines is just low more to the thickening efficiency of DNA.But people such as Murphy report, different polyamines are different to the sensitivity of salt ion, and under identical conditions, concentrated effect also is subjected to the kind (DNA or RNA) of nucleic acid molecule and the influence (Murphy of configuration (wire or ring-type), Analytical Biochemistry 295,143,2001).According to this clue, researchist of the present invention is through groping meticulously, be found to the high cobalt/spermine of a certain proportion of six amminos first under the condition that guanidinium isothiocyanate exists, can optionally induce concentrating of linear DNA and RNA fully, and cyclic DNA still is lax configuration, illustrates that this process is not subjected to the influence of guanidinium isothiocyanate.On the basis of this discovery, we optimize non-alkaline lysis liquid formula, have increased by two kinds of compositions of the high cobalt of spermine and six amminos.With classical alkaline lysis liquid phase ratio, non-alkaline lysis liquid improvement, that contain polyamines has following advantage:
(1) that genomic dna has been taken place is highly concentrated for polyamines, has greatly reduced the viscosity of lysate, makes no longer thickness of lysate, is convenient to the follow-up various places operation of carrying out.
(2) that genomic dna and RNA have been taken place is highly concentrated for polyamines, no longer combines with pellosil, removes assortment of genes DNA and RNA ten minutes easily.
(3) no longer need alkaline denaturation and, saved for 2/3 operating time with closely-related resuspended, the neutralization of alkaline denaturation and centrifugal three steps.
(4) RNA concentrates the back removal, no longer needs RNase to handle, and has greatly reduced the cost of large scale plasmid purification DNA.
(5) omitted the alkaline denaturation processing, made DNA avoid the alkali damage, the plasmid DNA quality is higher.
Process innovation
The present invention replaces dual-step type plasmid DNA purifying process with single step plasmid DNA purifying process.In till now nearly 30 years appear in molecule clone technology, the plasmid DNA purification technique that people use is all slightly carried by plasmid and was carried for two steps with the plasmid essence and form, the thick formulation of present most popular plasmid is an alkaline lysis, the smart extracting method of the most popular plasmid of tradition is the pellosil absorption method, and both are as follows in conjunction with the key step of plasmid DNA purification:
1. bacterial precipitation is removed liquid nutrient medium.
2. bacterium is resuspended, makes alkali cracking liquid and bacterium mixing fast, avoids the appearance of extreme pH and avoids thermal agitation
3. alkaline lysis makes the bacterium cracking with SDS and NaOH acting in conjunction, and NaOH also makes genomic dna generation irreversible denaturation, makes plasmid DNA generation reversible denaturation.
4. neutralization with in Potassium ethanoate/acetic acid and NaOH, makes the plasmid DNA renaturation and genomic dna can not renaturation, and protein is saltoutd, and makes SDS generate insoluble precipitation and forms polymkeric substance with protein and genomic dna.
5. centrifugal, make at the bottom of the macromole insolubles (comprising genomic dna) that N-process forms is deposited to the centrifuge tube pipe and separate with the plasmid DNA in the supernatant.
Plasmid DNA in the supernatant liquor combine with pellosil and residual protein, RNA and small molecular weight impurity can not in conjunction with and enter and penetrate liquid.
7. wash post, residual protein, part RNA and small molecular weight impurity on the flush away pellosil.
8. wash-out elutes plasmid DNA stand-by with low salts solution from pellosil.
Single step plasmid DNA purifying process of the present invention revolutionaryly has omitted plasmid fully and has slightly put forward step, directly carries out the pellosil adsorption and purification with cell lysate, and a step obtains purified plasmid DNA, and its key step is as follows:
Single step plasmid DNA purifying process of the present invention is compared with traditional two-step process, has following advantage:
(1) more quick, make the whole plasmid DNA purification process time shorten 2/3, save the man power and material, be particularly suitable for high-throughput plasmid DNA purifying and technical grade plasmid in large scale DNA purifying.
(2) step is few, so the plasmid DNA loss also reduces, the rate of recovery improves, and therefore can directly carry out the purifying of micro-plasmid DNA with single bacterium colony, has removed the microbial culture step of spending the night from.
(3) non-alkaline lysis has been avoided the alkali damage of plasmid DNA, and the ratio of superhelix configuration is bigger, and quality improves.
(4) do not need to use animal derived RNase, reduced the possibility of polluting other pathogenies thus, also greatly reduced cost (especially when technical grade large scale plasmid purification DNA, it will be very expensive using RNase in a large number) simultaneously.
(5) insensitive to the operation severe degree, the plasmid DNA that is suitable for from trace (single bacterium colony) to the various scales of technical grade (more than 10 liters) exists, and operating process does not need to do great change.
(6) universality is wide, does not rely on colibacillary kind.
(7) lysate thickness not, the chromatography column that uses when being not easy to stop up subsequent purification.
Research contents and the gordian technique that relates to and technical indicator are described
One, to only needing the molecular biology research field of (single bacterium colony-500mL bacterium liquid) plasmid DNA purifying on a small scale, present technique is developed to single step plasmid DNA purification kit series product
1, exploitation single step plasmid DNA trace (single bacterium colony) extracts test kit
This product mainly utilize single step with the plasmid DNA extractive technique in from the sample (the single bacterium colony that incubated overnight obtains on as solid medium) of trace, extracting plasmid DNA, the restriction endonuclease analysis that is used for PCR, conversion and limited number of times is judged and whether is inserted fragment.The benefit of using this product is the time that can save as the researchist one day.The alkaline denaturation plasmid DNA purification technique that uses is because the rate of recovery is limited at present, therefore must be earlier with single colony inoculation overnight incubation in the liquid nutrient medium, just can obtain abundant bacterium uses for plasmid purification, also do not have directly to extract the analogous products of plasmid DNA in the market, belong to blank with single bacterium colony.And single step plasmid DNA purification technique rate of recovery height so can directly use single bacterium colony to carry out the plasmid DNA purifying, does not need the incubated overnight bacterium.This product will be filled up the blank of this series products.
At present, this product successfully has been used to contain single bacterium colony of high copy number plasmid, but also be not used in the bacterium colony sample that contains low copy and middle copy plasmid, but can predict, because the absolute magnitude of the plasmid DNA in single bacterium colony seldom, be very easy to experimentation in the vessel and the pellosil adsorption column that use non-specific irreversible fixation takes place, cause and lose, influence output.Therefore the gordian technique that relates to this product development will be how to reduce non-special absorption and improve output.One of method that preparation is taked is to add non-specific nucleic acids such as a certain amount of poly r A or yeast tRNA, non-special the losing that they reduce the plasmid DNA sample by self and combining of non-specific combination site in lysate.Two of method is before the cracking bacterium, increases a short period of time microbial culture step of 1-3 hour, and the absolute magnitude that increases bacterium and plasmid DNA is in the hope of obtaining the plasmid DNA that enough electrophoresis detection are used.
2, the little extraction reagent kit of exploitation single step plasmid DNA
This product mainly utilize single step with the plasmid DNA extractive technique in from the saturated bacterium liquid of the incubated overnight of 0.2-5mL, extracting plasmid DNA (usually said puies forward for a short time), because the DNA that obtains amount can be used for kinds of experiments (as PCR, conversion, restriction endonuclease analysis, order-checking) at 1-5ug.
This product development has key issue to be solved is how to guarantee endotoxic effective removal, because the transfection experiment in the molecular biology research need not contain endotoxic plasmid DNA, though contained in the non-alkaline lysis liquid that present technique is used and can remove endotoxic Triton X-114, but the endotoxic effect of actual in the method removal has only preliminary data, also needs further experiment to collect more data.This work is wished to solve the term of execution of the present invention.
3, the big extraction reagent kit of exploitation single step plasmid DNA
This product mainly utilize single step with the plasmid DNA extractive technique in plasmid DNA purification (usually said carries greatly) from the saturated bacterium liquid of the incubated overnight of 100-500mL, the DNA that obtains is because amount is big, can be used for kinds of experiments simultaneously, as PCR, conversion, restriction endonuclease analysis, order-checking etc.
It is to determine the thickness of pellosil in the centrifugal adsorption column and the relation of bacterial treatment amount that this product development has key issue to be solved, because the ability of pellosil adsorption of DNA can be directly proportional by its thickness, and the amount of bacterium is many more, and the amount of plasmid DNA is also high more.To a certain specific plasmid DNA amount,, cause losing of plasmid DNA if the pellosil thickness low LCL that uses then can reach capacity owing to the ability of pellosil adsorption of DNA.If the pellosil thickness that uses is blocked up, then can produce non-special absorption to pollution molecules such as protein, reduce the purity of plasmid DNA.
4, the present invention is used for the technological standard of the plasmid DNA of molecular biology research:
| Interventions Requested | Calibration method | Standard |
| Telling test | A kind of restriction enzyme rear electrophoresis | With the expected results unanimity |
| The plasmid configuration | Agarose electrophoresis | Superhelix or open loop configuration |
| Plasmid purity | Ultraviolet spectrophotometer | OD260/280＝1.75-1.85 |
| RNA is residual | Agarose electrophoresis | Can't check |
| GDNA is residual | Agarose electrophoresis | Can't check |
Two, gene therapy and the gene vaccine field to needing technical grade extensive (more than 10 liters) to use is developed to the complete set technology that can transfer the possession of with present technique, need finish following research contents.
1, gordian technique
(1) because the industrial application of plasmid DNA mainly is meant clinical application, so its endotoxin content is reduced to the basic demand that medicine supervisory and management department specified standards is this series products or technology.Present technique is removed intracellular toxin by add this composition of TritonX-114 in the single step lysate, under the condition of small-scale plasmid DNA purifying, the preliminary experiment result is more satisfactory, but also be a brand-new problem high-volume, need the present invention to solve in carrying out to technical grade scale operation.
(2) to the plasmid DNA of clinical usefulness, general requirement its have 90% be the superhelix configuration.In the practice of using the alkaline lysis plasmid DNA purification, people have accumulated a large amount of about how improving the experience of superhelix configuration plasmid DNA ratio.Wherein one is the host bacteria of selecting no endogenous DNase for use, makes the native configurations of plasmid DNA that transformation from the superhelix to the open loop can not take place because of the effect of DNase in the cell.Secondly, be strict control bacterium cracked condition, especially agitation condition, make it fully cracking bacterium (cracking reaches more than 95%), but too violent and make plasmid DNA generation mechanical breaking and cause the configuration change.Single step technical grade plasmid in large scale DNA purifying also is a unknown number but can these methods migrate, and need solve in implementation of the present invention.
(3) the pellosil absorption and the purification step that use during the small scale purification plasmid DNA, the general desk centrifuge that uses just can be finished, but during the large-scale industrialization operation, owing to need the lysate volume of processing too big, use centrifugation method infeasible (whizzer that needs vast capacity), replace the pellosil centrifugal method so need use the pellosil chromatography method certainly instead.How the pellosil chromatography method effectively being combined with present technique also needs from the beginning to grope and optimize.
2, the present invention is used for the technological standard of the plasmid DNA of gene therapy and gene vaccine:
| Interventions Requested | Calibration method | Standard |
| Telling test | Restriction enzyme rear electrophoresis more than three kinds | With the expected results unanimity |
| The plasmid configuration | Agarose electrophoresis | ＞90% is the superhelix configuration |
| Plasmid purity | Ultraviolet spectrophotometer | OD260/280＝1.75-1.85 |
| Protein contamination | ELISA or amino acid analysis method | ＜1ug/mg |
| RNA is residual | Agarose electrophoresis | Can't check |
| GDNA is residual | Southern?Blot | ＜2ug/mg |
| Aseptic | UPS23 cultivates | No bacterial growth |
| Intracellular toxin is residual | The LAL test | ＜10EU/mg |
One, the single step plasmid DNA purification kit series product structure iron in research oriented field
Two, towards the extensive single step plasmid DNA purifying process process step in gene therapy and gene vaccine field:
The first step: bacterial precipitation, because the bacterium volume at 10-50L, is mainly collected bacterial precipitation with filtration method.
Second step: non-alkaline lysis with non-alkaline lysis liquid cracking bacterium, needs to use large stirrer fully to stir therebetween.
The 3rd step: pellosil goes on foot the direct upper prop of gained lysate in conjunction with cyclic DNA in the use.
The 4th step: wash post, wash post,, may need repeatedly to repeat this step for remove impurity as far as possible with the conventional post liquid of washing.
The 5th step: wash-out, the solution (as TE or ultrapure water) of using less salt is with pellosil bonded plasmid DNA wash-out.
The foundation that gordian technique realizes
Gordian technique of the present invention is optionally to concentrate the genomic dna of wire and make it forfeiture and pellosil bonded ability in the system of high salt with polyamines, and under similarity condition, the cyclic plasmid DNA still keeps loose configuration, still can follow the pellosil combination.Researchist of the present invention is through groping meticulously and large-scale screening, find first when the high cobalt of six amminos and spermine concentration sum are 100mM, though under the condition that the high density guanidinium isothiocyanate exists, still can induce concentrating and losing and pellosil bonded ability of glm gene group DNA and RNA, and the cyclic plasmid DNA still is lax configuration, still can combine with pellosil.And when the ratio of high cobalt of six amminos and spermine was 1: 3, the content of plasmid DNA was the highest among the final DNA that reclaims, and reach 95%, and the rate of recovery is also the highest, reaches 90%, and the genomic dna and the RNA that do not have electrophoresis to detect to obtain pollute.The data of correlative study are summarized as follows:
| The polyamines total concn | 100mM | 100mM | 100mM | 100mM | 100mM |
| The high cobalt final concentration of six amminos | 100mM | 75mM | 50mM | 25mM | 0mM |
| The spermine final concentration | 0mM | 25mM | 50mM | 75mM | 100mM |
| The RNA ratio | 20％ | ND | ND | ND | ND |
| The genomic dna ratio | 30％ | 25％ | 10％ | ND | ND |
| The plasmid DNA ratio | 50％ | 70％ | 85％ | 95％ | 95％ |
| The plasmid DNA rate of |
40％ | 60％ | 90％ | 90％ | 50％ |
Annotate: ND represent with electrophoresis detection less than.
On the basis of this discovery, we optimize the non-alkaline lysis liquid formula of classics, have increased by two kinds of compositions of the high cobalt of spermine and six amminos.With the non-alkaline lysis liquid phase ratio of classics, the lysate of improvement has following advantage:
(1) that genomic dna has been taken place is highly concentrated for polyamines, has greatly reduced the viscosity of lysate, makes no longer thickness of lysate, is convenient to the follow-up various places operation of carrying out.
(2) that genomic dna and RNA have been taken place is highly concentrated for polyamines, no longer combines with pellosil, removes assortment of genes DNA and RNA ten minutes easily.
(3) RNA concentrates the back removal, no longer needs RNase to handle the cost when greatly reducing scale operation.
One, present technique has been used for plasmid DNA trace (single bacterium colony) extraction, illustrates that the rate of recovery of its plasmid DNA is higher than traditional alkaline denaturation, because never the people successfully is used for alkaline denaturation extracting plasmid DNA from single bacterium colony.
As shown in Figure 3: extract plasmid DNA with present method from a bacterium colony that contains the pUC19 plasmid, half is used for electrophoresis (sample A) DNA that obtains, second half with the linearizing of EcoR I restriction enzyme after agarose electrophoresis (sample B) again.The DNA of A sample front is the superhelix configuration, the back for the open loop configuration.
Two, present technique has been used for plasmid DNA (the saturated bacterium liquid of 0.2-1.5mL) extraction on a small scale.
As shown in Figure 4: extract plasmid DNA with present method from 1.5mL contains the bacteria samples of incubated overnight of pUC19 plasmid, DNA uses 200uL elutriant wash-out at last, gets 20uL and is used for agarose electrophoresis.The DNA of front is the superhelix configuration, the back for the open loop configuration.
Three, present technique has been used for plasmid DNA large scale (the saturated bacterium liquid of 100mL) extraction.
As shown in Figure 5: from the saturated bacterium liquid of 100mL, extract the pUC19 plasmid DNA with this product, use 2mL elutriant eluted dna at last, get the last sample of 20uL and carry out agarose electrophoresis.The DNA of front is the superhelix configuration, the back for the open loop configuration.
Embodiment 2: animal mitochondria DNA extracts and divided for 2 steps:
Animal mitochondria DNA extracts (the first step is a total DNA extraction)
(1) cell lysis buffer solution:
Tris(pH8.0)100mmol/L
EDTA(pH8.0)500mmol/L
NaCl?20mmol/L
SDS?10％
Pancreatic RNase 20ug/ml
(2) Proteinase K: take by weighing the 20mg Proteinase K and be dissolved in the distilled water of 1ml sterilization ,-20 ℃ standby
(3) TE damping fluid (pH8.0): autoclaving, room temperature storage
(4) phenol: chloroform: primary isoamyl alcohol (25: 24: 1)
(5) primary isoamyl alcohol, cold dehydrated alcohol, 70% ethanol, aqua sterilisa
(1) gets the fresh or freezing piece 0.1g (0.5cm of animal tissues
3), shred as far as possible.Place glass homogenizer, the cell lysis buffer solution homogenate that adds 1ml changes in the 1.5ml centrifuge tube to loseing tissue block, adds Proteinase K (500ug/ml) 20ul, mixing.Water-bath 30min in 65 ℃ of thermostat water baths also can change 37 ℃ of water-bath 12-24h over to, and the interrupted oscillation centrifuge tube for several times., get supernatant liquor and add in another centrifuge tube with the centrifugal 5min of 12000rpm in desk centrifuge.
(2) add 2 times of volume Virahols, behind the reversing mixing, can see filament, choose, dry, dissolve again with 200ulTE with the 100ul suction nozzle.(can carry out PCR reaction etc., following these steps to of need being further purified carried out)
(3) add the phenol of equivalent: chloroform: primary isoamyl alcohol vibration mixing, the centrifugal 5min of 12000rpm.
(4) get upper solution and manage, add isopyknic chloroform: primary isoamyl alcohol, vibration mixing, centrifugal 12000rpm, 5min to another.
(5) get upper solution and manage, add the 7.5mol/L ammonium acetate of 1/2 volume and the dehydrated alcohol of 2 times of volumes, precipitation at room temperature 2min behind the mixing, the centrifugal 10min of 12000rpm to another.
(6) carefully outwell supernatant liquor, centrifuge tube is inverted on the thieving paper, the residual droplets that invests tube wall is removed.
(7) use 1ml 70% washing with alcohol throw out 1 time, the centrifugal 5min of 12000rpm.
(8) carefully outwell supernatant liquor, centrifuge tube is inverted on the thieving paper, the residual droplets that invests tube wall is removed.
(9) add 200ul deionized water dissolution precipitation thing again.
(second one for extracting Mitochondrial DNA (with plasmid DNA extraction step) in total DNA in the animal mitochondria DNA extraction
1) in conjunction with DNA: lysate is all transferred in the centrifugal adsorption column, and room temperature leaves standstill plasmid DNA is combined with film.Centrifugal half a minute of room temperature 12000-15000g, abandon and penetrate liquid.If once supernatant all can not be transferred in the centrifugal adsorption column, can be carried out at twice.
2) prewashing: in centrifugal adsorption column, add the pre-washing lotion of 0.7mL, centrifugal half a minute of room temperature 12000-15000g, abandon and penetrate liquid.If pre-washing lotion is placed on low temperature and has produced precipitation, make it about 60 ℃ to melt with before needing to be heated to, use again behind the mixing.
3) prewashing again: in centrifugal adsorption column, add the pre-washing lotion of 0.3mL again, centrifugal half a minute of room temperature 12000-15000g, abandon and penetrate liquid.This step prewashing is not preferably omitted, otherwise DNA purity is not high.
Clean: what add 0.7mL in centrifugal adsorption column washes post liquid, centrifugal half a minute of room temperature 12000-15000g, abandons and penetrates liquid.
Clean: what add 0.3mL again in centrifugal adsorption column washes post liquid, centrifugal half a minute of room temperature 12000-15000g, abandons and penetrates liquid again.Centrifugal half a minute of room temperature 12000-15000g, dry residual liquid.
Wash-out: place a new 1.5mL to provide plastic centrifuge tube for oneself on centrifugal post, add 30-100uL DNA elutriant, room temperature was placed 2 minutes.If the DNA elutriant is preheating to 65-80 ℃, then better effects if.
Embodiment 3: the plant chloroplast Mitochondrial DNA extracts in two steps, the first step plant total DNA extraction, and second step was extracted the plastosome chloroplast DNA from total DNA.
The plant mitochondria chloroplast DNA extracts (the first step)
SDS improved method DNA extraction liquid
(1)SolutionⅠ：NaCl(100mmol/L)，Tris-HCl(50mmol/LpH?8.0)，EDTA(50mmol/LpH8.0)。
(2)SolutionⅡ：NaCl(100mmol/L)，Tris-HCl(50mmol/LpH?8.0)，EDTA(50mmol/LpH8.0)，SDS(1％)
(3) PCA: the saturated phenol of Tris and chloroform, primary isoamyl alcohol are mixed by 25: 24: 1 volume ratio, place brown reagent bottle, 4 ℃ of preservations.
(4) CA: chloroform, primary isoamyl alcohol are mixed by 24: 1 volume ratio, place brown reagent bottle, 4 ℃ of preservations.
(5)5M?NaCl
(6) dehydrated alcohol
(7) 1 * TE damping fluids (pH 8.0): 1mol/LTris-HCl (pH 8.0) 1ml adds 0.5mol/LEDTA (pH 8.0) 0.2ml, is settled to 100ml, and the sterilization back is standby.
The SDS improved method extracts the total DNA step of plant:
(1) gets blade and take by weighing 0.5g, put in the mortar, quick grind into powder behind liquid nitrogen freezing.
(2) change over to rapidly in the 1.5mL centrifuge tube, add the Solution I solution of 800ul, put upside down mixing, the centrifugal 5min of 12000rpm abandons supernatant.
(3) the Solution II solution of adding 800ul, mixing is placed on 65 ℃ of water-bath 30min, shakes mixing frequently lightly.12000rpm then, centrifugal 10min gets supernatant.
(4) add equal-volume PCA (phenol/chloroform/primary isoamyl alcohol), shake up the liquid phase clarification to lower floor gently, room temperature leaves standstill 5min, and the centrifugal 10min of 12000rpm gets supernatant.
(5) add isopyknic CA (chloroform/primary isoamyl alcohol), shake up gently, room temperature leaves standstill 5min, and the centrifugal 10min of 12000rpm observes separation surface and has or not precipitation, gets supernatant.As required, supernatant liquor can extract repeatedly until no middle layer precipitation repeatedly with repeating (4)～(5) step.
(6) the 5M NaCl that adds the dehydrated alcohol of 2 times of volumes and 1/20 volume to supernatant liquid mixing gently places-20 ℃, 30min then.
(7) the centrifugal 10min of 12000rpm removes supernatant, precipitates 2～3 times with 70% washing with alcohol, and inversion drains.
(8) room temperature is dissolved in 200 μ L TE solution or aseptic ddH
2Among the O ,-20 ℃ of preservations are standby.
The plant mitochondria chloroplast DNA extracts (second step) and extracts with animal mitochondria DNA.
Claims (2)
1. quick covalently closed circular DNA purification kit, it is characterized in that: it is finished by following step and method, and the concrete steps method is as follows,
Covalently closed circular DNA purification technique step is as follows:
The first step: bacterial precipitation, because the bacterium volume at 10-50L, is mainly collected bacterial precipitation with filtration method, remove liquid nutrient medium, obtain thalline;
Second step: non-alkaline lysis, with non-alkaline lysis liquid cracking bacterium, need therebetween to use large stirrer fully to stir, make the bacterium cracking, polyamines makes genomic dna and RNA take place highly to concentrate and loses and pellosil bonded ability simultaneously, we optimize non-alkaline lysis liquid formula, have increased by two kinds of compositions of the high cobalt of spermine and six amminos;
The 3rd step: pellosil is in conjunction with cyclic DNA, the direct upper prop of step gained lysate in the use, lysate directly carries out the plasmid DNA purifying with silica gel adsorption, except that plasmid DNA, and protein and be the genomic dna of tight bulk and RNA can not combine with pellosil and enters and penetrate liquid;
The 4th step: wash post, residual protein, part RNA and small molecular weight impurity on the flush away pellosil washed post with the post liquid of washing of routine, for remove impurity as far as possible, may need repeatedly to repeat this step;
The 5th step: wash-out elutes plasmid DNA stand-by with low salts solution from pellosil; With the solution " as TE or ultrapure water " of less salt with pellosil bonded plasmid DNA wash-out,
Covalently closed circular DNA purification technique method is as follows:
1) bacterial precipitation, microorganism collection: be no more than the saturated bacterium liquid of 3mL incubated overnight with the plastic centrifuge tube collection, centrifugal half a minute of room temperature 12000-15000g, abandon supernatant;
2) non-alkaline lysis, the cracking thalline: add the 0.6-1mL lysate and " shake up with preceding need ", fully piping and druming or vibration cracking bacterium become clarification up to cracking, need half a minute usually; The centrifugal back of bacterium liquid adds high cobalt of six amminos and spermine, and the cobaltic final concentration of six amminos is 25mM, and the final concentration of spermine is 75mM;
3) pellosil is in conjunction with cyclic DNA, in conjunction with DNA: lysate is all transferred in the centrifugal adsorption column, room temperature leaves standstill plasmid DNA is combined with film, centrifugal half a minute of room temperature 12000-15000g, abandon and penetrate liquid, if once supernatant all can not be transferred in the centrifugal adsorption column, be carried out at twice;
4) wash post, prewashing: in centrifugal adsorption column, add the pre-washing lotion of 0.7mL, centrifugal half a minute of room temperature 12000-15000g, abandon and penetrate liquid, if being placed on low temperature, pre-washing lotion produced precipitation, make it to melt with before needing to be heated to 60 ℃, use again behind the mixing;
5) wash-out, prewashing again: in centrifugal adsorption column, add the pre-washing lotion of 0.3mL again, centrifugal half a minute of room temperature 12000-15000g, to abandon and penetrate liquid, this step prewashing is not preferably omitted, otherwise DNA purity is not high;
Clean: what add 0.7mL in centrifugal adsorption column washes post liquid, centrifugal half a minute of room temperature 12000-15000g, abandons and penetrates liquid;
Clean: what add 0.3mL again in centrifugal adsorption column washes post liquid again, centrifugal half a minute of room temperature 12000-15000g, and abandon and penetrate liquid, centrifugal half a minute of room temperature 12000-15000g, dry residual liquid;
Wash-out: place a new 1.5mL to provide plastic centrifuge tube for oneself on centrifugal post, add 30-100uL DNA elutriant, room temperature was placed 2 minutes, if the DNA elutriant is preheating to 65-80 ℃, had both got a kind of quick covalently closed circular DNA purification kit of the present invention.
2. according to claims 1 described a kind of quick covalently closed circular DNA purification kit, it is characterized in that: described
Lysate: be 10%SDS+2M NaCl+10mM spermine;
Pre-washing lotion: be 2M NaCl;
Wash post liquid: be 50% ethanol;
Elutriant: be water;
Polyamines: be spermine: the high cobalt of six amminos=3: 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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2010
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| CN113840615A (en) * | 2019-03-14 | 2021-12-24 | Om药物公司 | Method for preparing stable bacterial extracts and their use as medicaments |
| CN113755488A (en) * | 2021-09-30 | 2021-12-07 | 天津津科生物科技有限责任公司 | Method for rapidly extracting high-purity plasmid DNA |
| WO2023125592A1 (en) * | 2021-12-28 | 2023-07-06 | 南京金斯瑞生物科技有限公司 | Magnetic bead, and preparation method therefor and use thereof in nucleic acid extraction |
| CN114657174A (en) * | 2022-03-29 | 2022-06-24 | 湖南科技学院 | Kit for extracting bacterial plasmid by alkaline cracking method and method thereof |
| CN114657174B (en) * | 2022-03-29 | 2023-07-25 | 湖南科技学院 | Kit for extracting bacterial plasmid by alkaline lysis method and method thereof |
| CN115369112A (en) * | 2022-08-24 | 2022-11-22 | 广州美基生物科技有限公司 | Binding liquid for endotoxin-removing plasmid extraction, kit and plasmid extraction method |
| CN115369112B (en) * | 2022-08-24 | 2023-10-17 | 广州美基生物科技有限公司 | Binding solution and kit for extracting endotoxin-removing plasmid and plasmid extraction method |
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