FR2620706A1 - Process for solvent treatment of DNA in a heterogeneous phase - Google Patents
Process for solvent treatment of DNA in a heterogeneous phase Download PDFInfo
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- FR2620706A1 FR2620706A1 FR8713010A FR8713010A FR2620706A1 FR 2620706 A1 FR2620706 A1 FR 2620706A1 FR 8713010 A FR8713010 A FR 8713010A FR 8713010 A FR8713010 A FR 8713010A FR 2620706 A1 FR2620706 A1 FR 2620706A1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
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Abstract
Description
La pressente invention a pour objet un procédé de traitement par solvants dtADN en phase hétérogéne. The subject of the present invention is a method of treatment with dtDNA solvents in the heterogeneous phase.
On sait que 1'ADN est soluble presqu exclusivement dans l'eau, ce qui rend très difficile, voire impossible, cie lui faire subir des transformations chimiques faisant intervenir des réactions dans des solvants non miscibles à l'eau. It is known that DNA is soluble almost exclusively in water, which makes it very difficult, if not impossible, to subject it to chemical transformations involving reactions in solvents immiscible with water.
On connaît également les difficultés -rencontrées dans l'obtention d'ADN purifié, les procédés mis au point à ce jour faisant appel à une succession d'extractions en phases liquides qui présentent l'inconvénient d'être longues et délicates. We also know the difficulties encountered in obtaining purified DNA, the processes developed to date using a succession of extraction in liquid phases which have the drawback of being long and delicate.
Parmi ces procédés, l'un des plus récents consiste à fixer 1'ADN en -solution aqueuse sur les particules d'une poudre de verre au moyen d'un agent chaiotropique tel que l'iodure de sodium ou le perchlorate de potassium. Cette fixation se faisant sélectivement permet de séparer 1'ADN des protéines et de 1'ARN en solution qui l'accompagnent généralement, et conduit à l'obtention d'un produit de fixation de 1'ADN sur la poudre de verre, dont on sépare 1'ADN par une élution pratiquée au moyen d'une solution aqueuse. La solution d'ADN ainsi obtenue doit être ensuite soumise à une succession d'opérations de purification, qui consistent généralement en des traitements successifs à la RNase, à la protéinase K, au phénol et au chloroforme, tous opérés en phases liquides. Among these methods, one of the most recent consists in fixing the DNA in aqueous solution on the particles of a glass powder by means of a chaiotropic agent such as sodium iodide or potassium perchlorate. This binding takes place selectively to separate the DNA from the proteins and the RNA in solution which generally accompany it, and leads to the production of a DNA binding product on the glass powder, of which separates the DNA by an elution carried out using an aqueous solution. The DNA solution thus obtained must then be subjected to a succession of purification operations, which generally consist of successive treatments with RNase, proteinase K, phenol and chloroform, all operated in liquid phases.
I1 ressort de ce qui précéde que le traitement de 1'ADN se heurte à des difficultés non négligeables, dont la moindre n'est pas le nombre d'opérations successives mises en oeuvre, qui nuit à la rentabilité du traitement, empêchant en particulier la mise au point d'appareillages automatiques susceptibles de faciliter les manipulations et d'en accroître le rendement. It appears from the foregoing that the treatment of DNA faces significant difficulties, the least of which is not the number of successive operations implemented, which affects the profitability of the treatment, preventing in particular the development of automatic devices capable of facilitating handling and increasing performance.
La présente invention a pour but de remédier à ces divers inconvénients en proposant un procédé de traitement de 1'ADN qui rend aisée sa manipulation, que celle-ci vise à la modification chimique ou enzymatique de sa molécule ou à l'obtention d'ADN purifié. The object of the present invention is to remedy these various drawbacks by proposing a process for the treatment of DNA which makes its manipulation easy, whether this is aimed at the chemical or enzymatic modification of its molecule or in obtaining DNA. purified.
Le procédé selon l'invention consiste à traiter par au moins un solvant 1'ADN fixé de façon non covalente et réversible sur un support insoluble à la fois dans ledit solvant et dans 1 'eau. The method according to the invention consists in treating, with at least one solvent, the DNA fixed noncovalently and reversibly on a support insoluble both in said solvent and in water.
Le solvant mis en oeuvre dans le procédé selon l'invention peut être n'importe quel solvant organique ou minéral présentant la particularité de ne pas solubiliser 1'ADN. I1 peut être additionné d'eau ou d'une solution aqueuse d'au moins un sel minéral pouvant renfermer également au moins un agent tampon,un agent chélatant et/ou un agent antioxydant. The solvent used in the process according to the invention can be any organic or inorganic solvent having the particularity of not dissolving the DNA. It can be added with water or an aqueous solution of at least one mineral salt which may also contain at least one buffering agent, a chelating agent and / or an antioxidant agent.
A titre d'exemple de solvants utilisables dans le procédé selon l'invention, on peut citer le chloroforme, l'éthanol, l'isopropanol, le butanol ou encore une solution aqueuse saturée de phénol. By way of example of solvents which can be used in the process according to the invention, mention may be made of chloroform, ethanol, isopropanol, butanol or else a saturated aqueous solution of phenol.
L'eau susceptible d'être ajoutée au solvant peut être additionnée par exemple d'une faible quantité de chlorure de sodium, d'EDTA ou de Tris HC1. The water capable of being added to the solvent can be added for example with a small amount of sodium chloride, EDTA or Tris HCl.
Le support utilisé pour fixer 1'ADN peut être n'importe quel support finement divisé susceptible de fixer 1'ADN de façon réversible et présentant la particularité de n'être soluble ni dans l'eau ni dans le solvant. The support used to fix the DNA may be any finely divided support capable of reversibly fixing the DNA and having the particularity of being soluble neither in water nor in the solvent.
A titre d'exemple de tels supports, on peut citer la poudre de verre, et de préférence celle obtenue par broyage de flacons de scintillation, les particules retenues étant celles sédimentant à moins de 0,25 cm/minute, de dimensions égales ou inférieures à 5 microns. As an example of such supports, mention may be made of glass powder, and preferably that obtained by grinding scintillation vials, the particles retained being those which sediment at less than 0.25 cm / minute, of equal or smaller dimensions. at 5 microns.
L'ADN est fixé sur le support insoluble dans les conditions imposées par la nature dudit support : ainsi, dans le cas où le support est de la poudre de verre, la fixation se fera au moyen d'un agent chaiotropique tel que l'iodure de sodium ou le perchlorate de potassium. The DNA is fixed on the insoluble support under the conditions imposed by the nature of said support: thus, in the case where the support is glass powder, the fixing will be done by means of a chaiotropic agent such as iodide sodium or potassium perchlorate.
Dans un but de simplification, le produit de fixation de 1'ADN sur le support sera désigné dans tout ce qui suit par "complexe ADN - support". For the purpose of simplification, the product for fixing the DNA on the support will be designated in the following text as "DNA-support complex".
Dans un premier mode de réalisation du procédé selon l'invention, le complexe ADN - support est soumis à un traitement par solvant en vue d'une modification chimique ou enzymatique de sa molécule au moyen d'un réactif convenable. In a first embodiment of the method according to the invention, the DNA-support complex is subjected to a treatment by solvent with a view to chemical or enzymatic modification of its molecule by means of a suitable reagent.
Dans ce cas, le traitement par solvant conduit à l'obtention d'une suspension, dans ledit solvant, du complexe ADNsupport. La suspension obtenue présente l'avantage de pouvoir être facilement mise en oeuvre dans des réactions chimiques ou enzymatiques visant à modifier la molécule d'ADN. In this case, the treatment with solvent leads to the production of a suspension, in said solvent, of the DNA support complex. The suspension obtained has the advantage of being able to be easily used in chemical or enzymatic reactions aiming to modify the DNA molecule.
Dans un second mode de réalisation du procédé selon l'invention, le complexe ADN - support est soumis à un traitement par solvant en vue de l'obtention d'ADN purifié. In a second embodiment of the method according to the invention, the DNA-support complex is subjected to a solvent treatment with a view to obtaining purified DNA.
Dans ce cas, le traitement par solvant vise essentiellement à séparer de l'ADN les molécules contaminantes qui s'y trouvent fixées. Ces molécules contaminantes peuvent être entre autres des protéines, de l'agarose, des corps gras ou des agents fluorescents tels que le bromure d'éthidium, utilisés pour le marquage des molécules d'ADN. In this case, the solvent treatment essentially aims at separating from the DNA the contaminating molecules which are attached to it. These contaminating molecules can be among others proteins, agarose, fatty substances or fluorescent agents such as ethidium bromide, used for the labeling of DNA molecules.
Le choix du solvant est dans ce cas fonction de l'origine de l'ADN à purifier : ainsi dans le cas où l'ADN est contaminé en majeure partie par des protéines, on utilisera avantageusement du phénol saturé d'eau, tandis que dans le cas où le contaminant est essentiellement constitué de c-orps gras, le solvant sera de préférence du chloroforme. The choice of solvent in this case depends on the origin of the DNA to be purified: thus in the case where the DNA is contaminated for the most part with proteins, phenol saturated with water will advantageously be used, while in if the contaminant consists essentially of fatty c-orps, the solvent will preferably be chloroform.
Dans ce second mode de réalisation du procédé selon l'invention, le traitement par solvant du complexe ADN - support est suivi d'une élution de l'ADN du support, opérée au moyen d'eau éventuellement additionnée d'au moins un sel minéral, au moins un agent tampon, au moins un agent chélatant et/ou au moins un agent antioxydant. In this second embodiment of the method according to the invention, the solvent treatment of the DNA-support complex is followed by an elution of the DNA from the support, carried out by means of water optionally added with at least one mineral salt , at least one buffering agent, at least one chelating agent and / or at least one antioxidant agent.
L'ADN ainsi obtenu est d'une grande pureté, et peut être conservé dans la solution d'élution. The DNA thus obtained is of high purity, and can be stored in the elution solution.
Le procédé selon l'invention se trouve illustré par l'exemple non limitatif qui suit, et qui se rapporte à son second mode de réalisation. The method according to the invention is illustrated by the nonlimiting example which follows, and which relates to its second embodiment.
EXEMPLE
Cet exemple décrit l'obtention d'ADN plasmidique de la bactérie "ESCHERICHIA COLI".EXAMPLE
This example describes the obtaining of plasmid DNA from the bacterium "ESCHERICHIA COLI".
1) On prend 1,5 ml de culture de la bactérie que l'on centrifuge et on reprend le culot dans 100 ul de TGE (Tris HCl 25 mM pH 7,5 ; EDTA l0mM ; Glucose 50 mM). Toutes les étapes ultérieures sont réalisées à 4 C. 1) 1.5 ml of the culture of the bacteria are taken which is centrifuged and the pellet is taken up in 100 μl of TGE (25 mM Tris HCl pH 7.5; 10 mM EDTA; 50 mM Glucose). All subsequent steps are carried out at 4 C.
2) On rajoute 200 > il de solution NaOH 0,2 M SDS 1% et on mélange. 2) 200 μl of 0.2 M 1% SDS NaOH solution are added and mixed.
3) On rajoute 150 ul d'acétate de potassium 5 M pH 5, on mélange et on laisse agir 5 minutes. 3) 150 μl of 5 M potassium acetate, pH 5, are added, mixed and left to act for 5 minutes.
4) On centrifuge pendant 2 à 5 minutes (centrifugeuse type microfuge), et on récupère 400 yl de surnageant. 4) Centrifuge for 2 to 5 minutes (microfuge type centrifuge), and 400 μl of supernatant are recovered.
5) On rajoute 800 ,ul de solution aqueuse de NaI 8 M et environ 2,5 mg de poudre de verre. On mélange 5 minutes. 5) 800 μl of 8 M aqueous NaI solution and approximately 2.5 mg of glass powder are added. Mix for 5 minutes.
6) On centrifuge pendant 1 à 5 minutes et on élimine le surnageant. 6) Centrifuge for 1 to 5 minutes and remove the supernatant.
7) On reprend le culot dans 400 ul de phénol saturé en eau et on agite pendant 5 minutes. 7) The pellet is taken up in 400 μl of water-saturated phenol and the mixture is stirred for 5 minutes.
8) On centrifuge pendant 1 à 5 minutes et on élimine le surnageant (l'étape 7 peut être réalisée deux fois si nécessaire). 8) Centrifuge for 1 to 5 minutes and remove the supernatant (step 7 can be carried out twice if necessary).
9) On reprend le culot dans 400 ul de chloroforme et on mélange pendant 5 minutes. 9) The pellet is taken up in 400 μl of chloroform and mixed for 5 minutes.
l0-) On centrifuge pendant 1 à 5 minutes et on élimine le surnageant. 10-) Centrifuge for 1 to 5 minutes and remove the supernatant.
11) On reprend le culot dans 1 ml de solution de lavage à 50% d'éthanol, TE, NaCl 0,3M (TE = Tris HCl 10 m M - pH 7,5 ; EDTA 1 mM). On mélange pendant 5 minutes. 11) The pellet is taken up in 1 ml of washing solution at 50% ethanol, TE, 0.3M NaCl (TE = 10 m M Tris HCl - pH 7.5; 1 mM EDTA). Mix for 5 minutes.
12) On centrifuge pendant 1 à 5 minutes et on récupère le surnageant. 12) Centrifuge for 1 to 5 minutes and recover the supernatant.
13) On reprend le culot dans 50 à 100 )il de TE et cn mélange 5 minutes. 13) The pellet is taken up in 50 to 100) of TE and mixed for 5 minutes.
14) On centrifuge pendant 5 minutes et on récupère le surnageant. 14) Centrifuge for 5 minutes and recover the supernatant.
L'ADN plasmidique obtenu est extrêmement pur, les étapes de traitement à la RNase, à la protéinase K, au phénol, au chloroforme ne sont plus nécessaires. The plasmid DNA obtained is extremely pure, the steps of treatment with RNase, with proteinase K, with phenol, with chloroform are no longer necessary.
L'PDN ainsi préparé a été testé dans différentes réactions de biologie moléculaire et les résultats obtenus ont confirmé sa haute pureté. The PDN thus prepared was tested in various molecular biology reactions and the results obtained confirmed its high purity.
Le procédé selon l'invention, ainsi qu'il a déjà té dit, offre l'avantage de permettre le traitement chimique de l'ADN sans recourir à une multiplicité d'opérations délicates et de faible rendement. The method according to the invention, as has already been said, offers the advantage of allowing the chemical treatment of DNA without resorting to a multiplicity of delicate operations and of low yield.
Il rend également possible l'obtention d'ADN de grande pureté par une suite d'opérations simples, facilement automatisables. It also makes it possible to obtain DNA of high purity by a series of simple operations, easily automated.
Il trouve ainsi une application particulièrement intéressante dans l'élution d'ADN de gels d'agarose, l'isolement d'ADN de diverses origines et la modification chimique de molécules d'ADN. It thus finds a particularly interesting application in the elution of DNA from agarose gels, the isolation of DNA from various origins and the chemical modification of DNA molecules.
Claims (8)
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8713010A FR2620706A1 (en) | 1987-09-17 | 1987-09-17 | Process for solvent treatment of DNA in a heterogeneous phase |
| JP50786188A JPH02501353A (en) | 1987-09-17 | 1988-09-16 | Method of binding or separating molecules containing at least one piece of natural or synthetic DNA |
| PCT/FR1988/000461 WO1989002436A1 (en) | 1987-09-17 | 1988-09-16 | Method for fixing and separating molecules comprising at least one natural or synthetic dna fragment |
| EP19880908250 EP0333813A1 (en) | 1987-09-17 | 1988-09-16 | Method for fixing and separating molecules comprising at least one natural or synthetic dna fragment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8713010A FR2620706A1 (en) | 1987-09-17 | 1987-09-17 | Process for solvent treatment of DNA in a heterogeneous phase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| FR2620706A1 true FR2620706A1 (en) | 1989-03-24 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| FR8713010A Withdrawn FR2620706A1 (en) | 1987-09-17 | 1987-09-17 | Process for solvent treatment of DNA in a heterogeneous phase |
Country Status (1)
| Country | Link |
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| FR (1) | FR2620706A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59227744A (en) * | 1983-06-08 | 1984-12-21 | Rikaken Kk | Granular and powdery glass for recovering dna |
-
1987
- 1987-09-17 FR FR8713010A patent/FR2620706A1/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59227744A (en) * | 1983-06-08 | 1984-12-21 | Rikaken Kk | Granular and powdery glass for recovering dna |
Non-Patent Citations (3)
| Title |
|---|
| CHEMICAL ABSTRACTS, vol. 102, 1985, pages 321, no. 200711u, Columbus, Ohio, US; & JP-A-59 227 744 (RIKAKEN K.K.) 21-12-1984 * |
| CHEMICAL ABSTRACTS, vol. 103, 1985, page 284, no. 50762r, Columbus, Ohio, US; B.MARUO et al.: "Alkaline extraction-glas powder treatment method for isolation and purification of plasmid DNA" & NIHON DAIGAKU NO-JUIGAKUBU GAKUJUTSU KENKYU HOKOKU 1985, (42), 57-61 * |
| CHEMICAL ABSTRACTS, vol. 96, 1982, page 375, no. 177435d, Columbus, Ohio, US; M.A.MARKO et al.: "A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder" & ANAL. BIOCHEM. 1982, 121(2), 382-7 * |
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