EP2324115A1 - Procédé d'isolement et de purification d'acides nucléiques - Google Patents

Procédé d'isolement et de purification d'acides nucléiques

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Publication number
EP2324115A1
EP2324115A1 EP09811122A EP09811122A EP2324115A1 EP 2324115 A1 EP2324115 A1 EP 2324115A1 EP 09811122 A EP09811122 A EP 09811122A EP 09811122 A EP09811122 A EP 09811122A EP 2324115 A1 EP2324115 A1 EP 2324115A1
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EP
European Patent Office
Prior art keywords
volume
nucleic acids
butanediol
buffer
nacl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP09811122A
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German (de)
English (en)
Inventor
Ralf Himmelreich
Sabine Werner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qiagen GmbH
Original Assignee
Qiagen GmbH
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Filing date
Publication date
Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Priority to EP09811122A priority Critical patent/EP2324115A1/fr
Publication of EP2324115A1 publication Critical patent/EP2324115A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

  • the present invention relates to a method for isolating and purifying nucleic acids by separating the nucleic acids of nucleated acidic samples, as well as biological materials.
  • the present invention further relates to a kit for carrying out the method according to the invention.
  • a method for isolating and purifying nucleic acids which is already known in the prior art, is based on the adsorption of nucleic acids on glass or silica particles in the presence of chaotropic salts, followed by the recovery of the adsorbed nucleic acids (Vogelstein, B. and Gillespie, D. (1979) "Preparative and analytical purification of DNA from Agarose", Proc Natl Acad, U.S.A.
  • RNA isolated and purified by agarose using high concentrations of chaotropic salts, such as, for example, sodium iodide, Sodium perchlorate or guanidinium thiocyanate, DNA isolated and purified by agarose
  • chaotropic salts such as, for example, sodium iodide, Sodium perchlorate or guanidinium thiocyanate
  • DNA isolated and purified by agarose The RNA or DNA can also be isolated or purified from various mixtures (Boom, R 1 , 1990: "Rapid and Simple Method for Purification of Nucleic Acids", J. Clin. Microbiol. 28, 495-503).
  • nucleic acids are often used in the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the PCR amplifies nucleic acids in a sequence-specific manner and is therefore widely used in gene or DNA diagnosis.
  • inhibitory substances that have not been removed from the nucleic acid purification step can inhibit the PCR.
  • Such inhibitory substances are, for example, hemoglobin and surfactants used in the nucleic acid extraction process. From this background, it can be seen that the methods for extracting and purifying nucleic acids are very important and relevant (Oshima et al., JJCL A, 22 (2) 145-150 (1997)).
  • nucleic acids Methods for extraction and purification of nucleic acids are very often automated. There are already automated nucleic acid extraction methods in the prior art, such as described in JP-A-107854/1999 and JP-A-266864/1999. In most procedures For the isolation and purification of nucleic acids, a solution which contains salts at high concentration and contains alcohol at a high concentration, and in which the nucleic acids are present, is brought into contact with an adsorption surface. The adsorption surface may be in the form of a resin in a column. Then, the nucleic acids are adsorbed to this surface and later eluted with the aid of solutions containing less highly concentrated saline solutions.
  • ethanol-containing solutions are classified as hazardous materials (HAZMAT) in accordance with IATA regulations (International Air Transport Association). According to the IATA regulations, all products, materials and goods are classified into nine main classes. Classification as dangerous goods will incur additional charges and taxes for air transport. It was therefore an object of the present invention to substantially replace ethanol (or isopropanol) in the process for purifying and extracting nucleic acids in order to facilitate the isolation and purification of nucleic acids, to provide an ethanol-free process and to facilitate air freight transport.
  • solvents for wash buffers have been found for washing surface-immobilized nucleic acids which allow the wash buffer to be substantially free of alcohol, ie ethanol.
  • the present invention thus relates to a method for washing nucleic acids immobilized on surfaces, wherein nucleic acids immobilized on surfaces are brought into contact with a washing buffer and the washing buffer is substantially free from ethanol.
  • substantially free of etanianol in the context of the present invention means that ethanol is present in a concentration of less than 24% by volume, preferably less than 16% by volume and most preferably not at all in the wash buffer.
  • the present invention relates to a method for washing nucleic acids immobilized on surfaces, wherein the wash buffer contains solvents selected from the group comprising C3 to C5 alkyldiols, and short chain ethylene glycol derivatives and various water soluble polymeric compounds, wherein the wash buffer substantially is free of ethanol.
  • the present invention is a process for washing surface-immobilized nucleic acids, wherein the washing buffer contains solvents which are selected from the group comprising 1,2-butanediol, 1,2-propanediol, 1,3-butanediol, l -methoxy-2-propanol, 3-methyl ⁇ l, 3 ⁇ 5-pentanetriol, DBE-2 dibasic ester, DBE dibasic ester-3, DBE-4 dibasic ester, DBE dibasic ester 5, DBE-6 dimethyl adipate, diethylene glycol monoethyl ether ( DGME), diethylene glycol monoethyl ether acetate (DGMEA), ethyl lactate, ethylene glycol, poly (2-ethyl-2-oxazoline), poly (4-styrenesulfonic acid-co-maleic acid) sodium salt solution, tetraethylene glycol (TEG), tetraglycol (tetrahydro
  • the washing buffer is particularly preferably selected from the group comprising tetraglycol, tetraethylene glycol, 1,3-butanediol, 1,2-butanediol and diethylene glycol monoethyl ether.
  • the subject matter of the present invention is a method for extracting nucleic acids from a solution comprising the steps:
  • Chaotropic conditions are achieved by the addition of chaotropic substances.
  • chaotropic chemical substances are referred to dissolve the ordered hydrogen bonds in aqueous solutions. They thus reduce the hydrophobic effect and have a denaturing effect on proteins, since the driving force of protein folding is the assembly of the hydrophobic amino acids in the water.
  • Examples of chaotropic substances are barium salts, guanidinium hydrochloride, thiocyanates such as guanidinium thiocyanate, perchlorates or sodium chloride. Chaotropic salts can be used according to their solubility product in concentration ranges between 1 M and 8 M.
  • High salt conditions means highly concentrated salt solutions, the concentration of salts in the solution being at least 1 M, preferably WM.
  • the binding mediator is selected from the group consisting of diethylene glycol monoethyl ether, diethylene glycol monoethyl ether acetate, furfuryl alcohol, poly (I -vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate), poly (2-ethyl-2-oxazoline), poly (4-ammonium-styrenesulfonic acid) J Tetraethylene glycol dimethyl ether, tetraethylene glycol, tetrahydrofurfuryl polyethylene glycol 200 and triethylene glycol monoethyl ether.
  • the person skilled in the art can also successfully replace ethanol in the binding process in the nucleic acid preparation by mixtures of said binding mediators. Since ethanol-containing solutions up to 24% (vol / vol) are not classified as HAZMAT, mixtures of the binding mediators can be used with ethanol.
  • the binding mediators are preferably present in the following concentrations;
  • DGME Diethylene glycol monoethyl ether
  • DGMEA Diethylene glycol monoethyl ether acetate
  • Concentration range 3 ⁇ 5Vol%, preferred concentration 5Vol% poly (2-ethyl-2-oxazoline) [CAS 25805-17-] - Concentration range 9-15Vol% (w / v), preferred concentration 12Vol%; in combination with ethanol: 22.5% (w / v) and 16-24% (v / v) ethanol
  • Tetraethylene glycol dimethyl ether [CAS 143-24-] - Concentration range 70-98% by volume, preferred concentration 98% by volume; in combination with ethanol: 73.5Vol% and
  • Triethylene glycol monoethyl ether [CAS 112-50-5] - Concentration range 70-90% by volume, preferred concentration 90% by volume
  • Elution buffers are generally buffered low salt solutions with a neutral to slightly alkaline pH (eg buffer TE 10 mM Tris / Cl pH 8, 1 mM EDTA).
  • a neutral to slightly alkaline pH eg buffer TE 10 mM Tris / Cl pH 8, 1 mM EDTA.
  • the skilled artisan partly also uses distilled water,
  • the washing buffer is tetraglycol in a concentration of 45% by volume to 80% by volume, preferably 52% by volume to 72% by volume;
  • 2 to 4 M preferably 2.5 M of chaotrope, e.g. Guanidiniurnhydrochlorid is the preferred concentration of tetraglycol at 45 to 55 vol%, preferably at 50 vol%.
  • washing buffer tetraethylene glycol in a concentration of 45 to 80 vol%, preferably from 50 vol% to 70 vol%.
  • the preferred concentration of tetraethylene glycol is 45 to 55% by volume, preferably 50% by volume.
  • the washing buffer is 1,3-butanediol in a concentration of 45 to 85% by volume, preferably of 51% to 80% by volume.
  • the preferred concentration of 1,3-butanediol is 45 to 55% by volume, preferably 49% by volume.
  • the washing buffer is 1,2-butanediol in a concentration of 41% by volume to 71% by volume.
  • 2 to 4 M preferably 2.5 M of chaotrope, e.g. Guanidinium hydrochloride is the preferred concentration of 1, 2-butanediol at 41 to 55Vol%, preferably 49Vol%.
  • the washing buffer is present in a concentration of 38 to 98% by volume, preferably of 60% to 98% by volume.
  • 2 to 4 M preferably 2.5 M of chaotrope, e.g. Guanidmium hydrochloride is the preferred concentration of 1, 2-butanediol at 38 to 45% by volume, preferably 42% by volume.
  • the surface to which the nucleic acids are adsorbed may be based on materials which may be selected from the group consisting of silica materials, carboxylated surfaces, zeolites, and titanium dioxide.
  • the chaotropic and or high-salt conditions of reaction step b) are preferably achieved by the addition of chaotropic salts such as potassium iodide, guanidine hydrochloride, guanidine thiocyanate or sodium chloride to the nucleic acid-containing solution.
  • chaotropic salts such as potassium iodide, guanidine hydrochloride, guanidine thiocyanate or sodium chloride
  • the nucleic acid may be DNA such as genomic DNA.
  • the nucleic acid may also be RNA, such as total RNA.
  • the nucleic acid may be single-stranded or double-stranded nucleic acid, for example short double-stranded DNA fragments.
  • the nucleic acid-containing solution can be obtained by a lysis process from a nucleic acid-containing material.
  • the nucleic acid-containing material may be selected from the group consisting of blood, tissue, swabs, bacterial cultures, urine, cell suspensions and adherent cells, PCR reaction mixtures, and in vzYr ⁇ nucleic acid modification reaction mixtures.
  • the nucleic acid-containing material may include human, animal or plant material.
  • the nucleic acid-containing solution may be derived from a biochemical nucleic acid modification reaction.
  • surfactants are added to the nucleic acid-containing solution. These surfactants are preferably used in concentration ranges from 0.1% by volume to 10% by volume. Further, anti-foaming agents may be added, preferably in a range of 0.01 to 1% by weight.
  • the present invention also provides a reagent kit for washing surface-immobilized nucleic acids comprising a solution 1 comprising a washing buffer, wherein the washing buffer is substantially free of ethanol.
  • the present invention particularly relates to a reagent kit for washing nucleic acids immobilized on surfaces
  • a solution 1 comprising the wash buffer selected from the group consisting of C3- and C4-alkyldiols, as well as short-chain ethylene glycol derivatives and various water-soluble polymeric compounds and wherein the wash buffer is substantially free of ethanol.
  • the reagent kit comprises a solution 1 comprising the wash buffer selected from the group consisting of 1, 2-butanediol, 1,2-propanediol, 1,3-butanediol, 1-methoxy-2-propanol acetate, 3-methyl
  • DGMEA ethyl lactate
  • ethylene glycol ethylene glycol
  • poly (4-styrenesulfonic acid-co-maleic acid) sodium salt solution tetraethylene glycol (TEG)
  • Tetraglycol tetrahydrofurfuryl polyethylene glycol ether
  • tetrahydrofurfuryl-pol y-ethylene glycol 200 tetrahydrofurfuryl-pol y-ethylene glycol 200
  • tri (ethylene glycol) divinyl ether anhydrous triethylene glycol
  • Triethylene glycol monoethyl ether Triethylene glycol monoethyl ether.
  • the reagent kit for washing surface-immobilized nucleic acids, the reagent kit comprises
  • a solution 1 comprising the wash buffer selected from the group comprising tetraglycol, tetraethylene glycol, 1,3-butanediol, 1, 2-butanediol and diethyl englycol monoethyl ether.
  • the present invention also provides a reagent kit for the extraction of nucleic acids from a solution comprising the abovementioned reagent kit and additionally comprising a mixture 2 comprising binding mediator, and
  • the binding mediator is selected from the group comprising diethylene glycol monoethyl ether, diethylene glycol monoethyl ether acetate, furfuryl alcohol, poly (1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate), poly (2-ethyl-2-oxazoline),
  • the person skilled in the art can also replace mixtures of the binding mediators mentioned successfully with ethaiol in the binding process in the nucleic acid preparation. Since ethanol-containing solutions can not be classified as HAZMAT up to 24% (vol / vol), mixtures can also be classified the binding mediators are used with ethanol.
  • the binding mediators are preferably present in the following concentrations:
  • DGME Diethylene glycol monoethyl ether
  • DGMEA 16-24Vol% ethanol diethylene glycol monoethyl ether acetate
  • Poly (4-ammonium styrenesulfonic acid) [CAS 29965-34-] - Concentration range 8-22% by volume (w / v), preferred concentration 12% by volume; in combination with ethanol: 8-22 (w / v) and 24% (v / v) ethanol tetraethylene glycol dimethyl ether [CAS 143-24-] concentration range 70-98% by volume, preferred concentration 98% by volume; in combination with ethanol: 73.5% by volume and 24% by volume ethanol tetraglycol [CAS 9004-76] - preferred concentration with etlianol: 75% by volume and 16% by weight
  • Triethylene glycol monoethyl ether [CAS 112-50-5] - Concentration range 70-90% by volume, preferred concentration 90% by volume
  • Elution buffer are usually buffered low salt solutions with neutral to slightly alkaline pH (eg: buffer TE Fa. QIAGEN GmbH, Hilden). The skilled artisan sometimes also uses distilled water.
  • the present invention furthermore relates to a reagent kit for extracting nucleic acids from a solution comprising the abovementioned reagent kit and comprising a further solution 4 comprising a lysis buffer and a protease.
  • the chaotropic and / or high-salt conditions of reaction step b) are, as already explained above, preferably by the addition of chaotropic salts such as potassium iodide, guanidine hydrochloride, guanidine thiocyanate or sodium chloride to the nucleic acid-containing solution reached.
  • chaotropic salts such as potassium iodide, guanidine hydrochloride, guanidine thiocyanate or sodium chloride
  • D all is the subject of the present invention, a reagent kit according to the invention for the extraction of nucleic acids from a solution, wherein one of the solutions contains a chaotropic salt or high-salt conditions.
  • the chaotropic salt is preferably selected from a group comprising potassium iodide, guanidine hydrochloride, guanidine thiocyanate and sodium chloride,
  • the present invention also relates to the use of a reagent kit according to the invention for the extraction of nucleic acids from biological materials such as blood, tissue, smear preparations, bacteria, cell suspensions and adhering cells.
  • the present invention also provides the use of a reagent kit according to the invention for the purification of nucleic acids from biochemical reaction mixtures, PCR reaction mixtures and in vitro
  • Second wash buffer WB6: 69.8% by volume tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH
  • Second wash buffer WB7: 50.4 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Second wash buffer WB6: 72.0% by volume tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Second wash buffer WB 7: 52.0% by volume tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • First washing buffer WB10: 49.0% by volume 1, 3-butanediol; 2.5 M GuHCl
  • Second wash buffer WB5: 80.0 vol% 1,3-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Second wash buffer WB7: 51.0 vol% 1,3-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH
  • First washing buffer WB 10: 49.0% by volume 1, 2-butanediol; 2.5 M GuHCl
  • First Wash Buffer WB 11:42 by volume diethylene glycol monoethyl ether; 2.5 M GuHCl Second wash buffer: WB 1. 98% by volume diethylene glycol monoethyl ether Second wash buffer: WB6: 71% by volume diethylene glycol monoethyl ether; 10 mM NaCl;
  • First wash buffer WB 10: 48.5% by volume tetraethylene glycol; 2.5 M GuHCl
  • Second wash buffer WB6: 69.8 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Second wash buffer WB7: 50.4 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH
  • First wash buffer WB1 1: 50.0% by volume tetraglycol; 2.5 M GuHCl Second wash buffer: WB6: 72.0 vol% tetraglycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5
  • Second wash buffer WB7: 52 3 0Vol% tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 Fig S:
  • Second wash buffer WB5: 80% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Second wash buffer WB7: 51% by volume 1.3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • First Wash Buffer WB 11: 42% by volume diethylene glycol monoethyl ether; 2.5 M GuHCl Second wash buffer: WB 1. 98% by volume diethylene glycol monoethyl ether Second wash buffer: WB6: 71% by volume diethylene glycol monoethyl ether; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • the best wash buffers to replace the commercially available buffer PE contain 48-64% by volume tetraethylene glycol; 24% ethanol; 100 mM NaCl; 10 mM Tris pH 7.5
  • Second wash buffer WB5: 80.0 vol% 1,3-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5
  • Second wash buffer WB7: 51, 0% vol 1, 3-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • First wash buffer WBl 0: 49.0% by volume 1, 2-butanediol; 2.5 M GuHCl
  • Second wash buffer WB6: 71.0% by volume 1, 2-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • First wash buffer WB10: 48.5% by volume tetraethylene glycol; 2.5 M GuHCl
  • Second wash buffer WB6: 69.8 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Second wash buffer WB7: 50.4 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • First wash buffer WB1 1: 50.0% by volume tetraglycol; 2.5 M GuHCl Second wash buffer: WB6: 72.0 vol% tetraglycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5
  • Second wash buffer WB7: 52.0 vol% tetraglycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5
  • Second wash buffer WB6: 69.8 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Second wash buffer WB7: 50.4 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Second wash buffer WB6: 72.0% by volume tetraglycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5
  • Second wash buffer WB7: 52.0% by volume tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • First wash buffer WB10: 49.0% by volume 1,3-butanediol; 2.5 M GuHCl
  • Second wash buffer WB5: 80.0 vol% 1,3-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH
  • Second wash buffer WB7: 51.0 vol% 1,3-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • First washing buffer WB10: 49.0% by volume of 1,2-butanediol; 2.5 M GuHCl
  • Second wash buffer WB6: 71, 0% vol 1, 2-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH
  • First wash buffer 20% by volume tetraethylene glycol; 900mM GTC; 10 mM Tris / CL pH 7.5;
  • Second wash buffer 70% by volume tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Washing combination "b" First washing buffer: 20% by volume 1, 3-butanediol; 900mM GTC; 10 mM Tris / CL pH 7.5
  • Second wash buffer 80% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • RNeasy® excludes small RNAs (5.8S, tRNA, miRNA, ...) during purification.
  • the limit size is about 150 bases. This experiment shows that the size exclusion of the tested chemicals is comparable to ethanol as a reference.
  • Wa 20% by volume 1,3-butanediol; 900mM GTC; 10 mM Tris / CL pH 7.5 Wb: 60% by volume 1, 3 butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5 Fig. 15:
  • MWl replacement buffer according to the present invention 2 49% by volume 1,3-butanediol; 2.5 mg / hl
  • Buffer RPE Replacement buffer according to the present invention "2-60" 60% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 "2-55” 55% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 "2-50" 50% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 "2-45" 45% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • BioSprint ® 96 with log file "BS96_DNA_Blut_200"
  • Buffer AW2 replacement buffer according to the present invention
  • Buffer AW2 replacement solutions o 80% by volume of 1,3-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5
  • Buffer PM Replacement Solution According to the Present Invention • 64% by volume tetraethylene glycol; 24% ethanol; 100 mM NaCl; 10mM «Centrifuge dry
  • Carrier RNA is not required when using> 100 ng of DNA.
  • buffer BD deulfonation buffer
  • Buffer BW replacement solutions according to the present invention for protocol step 13 "60% by volume diethylene glycol monoethyl ether; 10 mM NaCl; 1 OmM Tris-Cl pH 7-5 • 72% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • test kits / test protocols according to the invention are shown in FIGS. 1-21.
  • the studies presented here focused on the ethanol-replacing chemicals in wash buffers for the silica-mediated preparation of nucleic acids.
  • the sample material tested ranged from blood to tissues as well as nucleic acid modification reaction-upregulation products.
  • the purification of various types of nucleic acids was tested (genomic DNA, total RNA, small double-stranded DNA fragments).
  • the identified chemicals were tested by methods using columns with silica-based membranes or silica-coated magnetic particles.
  • DGME Diethylene glycol monoethyl ether
  • DGMEA Diethylene glycol monoethyl ether acetate
  • DGMEA 50-80% by volume of DGMEA; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • TAG Tetraethylene glycol
  • TEG 50.4-60% by volume TEG; 10 mM NaCl; 10 mM Tris-Cl pH 7.5
  • Tri (ethylene glycol) divinyl ether [triethylene glycol divinyl ether]
  • Triethylene glycol anhydrous Triethylene glycol, anhydrous
  • Triethylene glycol monoethyl ether Triethylene glycol monoethyl ether

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Abstract

La présente invention concerne un procédé d'isolement et de purification d'acides nucléiques par élution des acides nucléiques à partir d'échantillons contenant des acides nucléiques, ainsi que de matières biologiques. La présente invention concerne en outre une trousse destinée à la mise en œuvre du procédé selon l'invention.
EP09811122A 2008-09-03 2009-09-02 Procédé d'isolement et de purification d'acides nucléiques Withdrawn EP2324115A1 (fr)

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EP08163624A EP2163621A1 (fr) 2008-09-03 2008-09-03 Procédé d'isolation et de nettoyage d'acides nucléiques
EP09811122A EP2324115A1 (fr) 2008-09-03 2009-09-02 Procédé d'isolement et de purification d'acides nucléiques
PCT/EP2009/061358 WO2010026167A1 (fr) 2008-09-03 2009-09-02 Procédé d'isolement et de purification d'acides nucléiques

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