EP1299531A2 - Procede pour isoler des acides nucleiques - Google Patents

Procede pour isoler des acides nucleiques

Info

Publication number
EP1299531A2
EP1299531A2 EP01971766A EP01971766A EP1299531A2 EP 1299531 A2 EP1299531 A2 EP 1299531A2 EP 01971766 A EP01971766 A EP 01971766A EP 01971766 A EP01971766 A EP 01971766A EP 1299531 A2 EP1299531 A2 EP 1299531A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acids
solution
nacl
isopropanol
concentration
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.)
Ceased
Application number
EP01971766A
Other languages
German (de)
English (en)
Inventor
Martin Weber
Thorsten Singer
Sarah Cosaert
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
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Publication of EP1299531A2 publication Critical patent/EP1299531A2/fr
Ceased legal-status Critical Current

Links

Classifications

    • 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 nucleic acids from a solution, the nucleic acids being adsorbed on a Si0 2 -containing surface.
  • the present invention relates to the use of a buffer solution for isolating nucleic acids on a support containing SiO 2 and to a kit for carrying out a method for isolating nucleic acids from a solution.
  • EP 0389063 B1 relates to a method for isolating nucleic acids from a biological source.
  • the biological sources containing nucleic acids such as blood, cells, plasma, etc.
  • the nucleic acids are then attached bound a silica surface. These are then washed and eluted.
  • the biological sample is acidified and treated with a chaotropic agent, e.g. mixed with a guanidinium salt.
  • a chaotropic agent e.g. mixed with a guanidinium salt.
  • Silicate particles are added to the sample and, under the given conditions, RNA binds to the silicate particles. Subsequently, the RNA is also separated from the particles here.
  • Colpan et al. in WO 95/01359 disclose a process for the purification and separation of nucleic acid mixtures by adsorption of the nucleic acid from an alcohol-containing solution with a high ionic strength.
  • the adsorption solution contains salts in a concentration of 1 to 10 M, the chaotropic salts, e.g. Guanidinium thiocyanate, sodium perchlorate, or guanidinium hydrochloride are preferred.
  • WO 95/21849 relates to a method for separating double-stranded and / or single-stranded nucleic acids from sources which contain these nucleic acids.
  • the nucleic acids are adsorbed on mineral carriers under conditions which allow binding of the desired nucleic acid species, while the undesired nucleic acid species does not bind on this mineral carrier.
  • the non-adsorbed double-stranded nucleic acid can then be further purified or isolated using known methods.
  • nucleic acids that are used for molecular biological applications are subject to very high requirements in terms of purity and integrity.
  • the use of nucleic acids in molecular diagnostics or molecular medicine presupposes that they must be free of toxic substances, which can lead, for example, to pathogenic effects in the organisms to be treated.
  • chaotropic salts are used in high concentrations for the isolation of nucleic acids on silica surfaces.
  • Chaotropic substances such as guanidinium hydrochloride, guanidinium thiocyanate or sodium perchlorate are highly toxic substances. It cannot be ruled out that the nucleic acids isolated in the presence of these substances will be contaminated with them and thus cannot be used or can only be used to a limited extent for use in molecular biological applications.
  • chaotropic substances also represents a high health risk for the user, so that the handling of these substances must be carried out under certain protective measures.
  • the technical problem on which the present invention is based is to provide an improved method which uses known disadvantages overcome.
  • the substances used in this method for binding the nucleic acid should not be toxic.
  • the method should be as inexpensive as possible, for example by using inexpensive chemicals, and the isolated nucleic acids should be isolated quantitatively and qualitatively purely.
  • the present invention provides a method for isolating nucleic acids from a solution which achieves this object.
  • the invention consists in the fact that in a first step the binding of nucleic acids to Si0 2 -containing surfaces in the presence of alkali halides in a concentration of 0.1-3M, preferably 0.25-1.5M, and alcohol in a concentration from 37 to 70 vol.% is carried out.
  • the nucleic acids adsorbed on the Si0 2 -containing surface are then optionally washed with an alcohol-containing washing buffer and the nucleic acid is eluted with an aqueous salt solution or with water.
  • Aqueous adsorption solutions are used to bind the nucleic acids to the Si0 2 -containing surface, the alkali halides such as NaCl, KCl and LiCl in a concentration of 0.1-3M, preferably 0.25-1.5M, particularly preferably 0.5 - 1, 25 M and in particular 0.5 - 1, 0 M included.
  • Alkali halides are non-toxic substances and handling the salt solutions in the concentration used is harmless to health.
  • the aqueous adsorption solutions contain lower aliphatic, branched or unbranched alcohols with a chain length of 1 to 5 carbon atoms.
  • the aliphatic alcohols present in the solution are preferably methanol, ethanol, propanol, isopropanol and butanol in a concentration of 37-70% by volume, preferably 37-50% by volume.
  • ethanol and / or isopropanol in a concentration of 37-70% by volume are particularly preferred.
  • Si0 2 -containing surfaces can, for example, be porous or non-porous silicon oxides or metal-silicon mixed oxides, silica gels, materials
  • Base of glasses e.g. modified or unmodified glass particles or glass powder, quartz, zeolites or mixtures of one or more of the substances mentioned above.
  • a surface is understood to mean any microporous separating layer.
  • the Si0 2 -containing surface is a porous membrane or a process
  • Filters made of silica gel, glass or quartz fibers made of silica gel, glass or quartz fibers.
  • the term surface in the broader sense also includes a layer of particles or also a granulate and also fibers, such as, for. B. silica gel nonwovens.
  • water or aqueous salt solutions are suitable as eluents for eluting the bound nucleic acid.
  • Buffer solutions known from the prior art are used as salt solutions, such as, for example, morpholinopropanesulfonic acid (MOPS), tris (hydroxymethyl) aminomethane (TRIS), 2- [4- (2-hydroxyethyl) -1-piperazino] ethanesulfonic acid (HEPES) in a concentration of 0.001 to 0.5 mol / liter, preferably 0.01 to 0.2 mol / liter, particularly preferably 0.01 to 0.05 molar solutions.
  • MOPS morpholinopropanesulfonic acid
  • TMS tris (hydroxymethyl) aminomethane
  • HEPES 2- [4- (2-hydroxyethyl) -1-piperazino] ethanesulfonic acid
  • the nucleic acids in the eluate can preferably be isolated by alcoholic precipitation.
  • the nucleic acids isolated with this method are free of toxic substances and are therefore suitable for use in molecular biology.
  • the term "nucleic acid” is to be understood in its broadest sense, that is to say ribonucleic acids (RNA) as well as deoxyribonucleic acids (DNA) in all lengths and configurations, such as double-stranded, single-stranded, circular and linear, branched, etc.
  • nucleic acids such as monomeric nucleotides, oligomers, plasmids, cosmids, viral and bacterial DNA and RNA, as well as genomic and non-genomic DNA and RNA from animal and plant cells or other eukaryotes, mRNA in processed and unprocessed form, tRNA, hn-RNA, rRNA, cDNA and include all other conceivable nucleic acids.
  • the method according to the invention allows nucleic acids of any origin to be isolated from solutions.
  • the sample containing nucleic acids comes, for example, from animal or vegetable tissues, tissue or cell cultures, bone marrow, human and animal body fluids such as blood, serum, plasma, urine, sperm, cerebrospinal fluid, sputum and smears, plants, plant parts and extracts, for example Juices, mushrooms, prokaryotic or eukaryotic microorganisms such as bacteria or yeast, fossil or mummified samples, soil samples, sewage sludge, waste water and food (especially processed, ie technically processed food).
  • Nucleic acids which have arisen from chemical reactions such as, for example, those which have been obtained by polymerase chain reaction (PCR) or plasmid DNA, genomic DNA and RNA and / or nucleic acids which come from microorganisms, can also be isolated according to the invention.
  • the method according to the invention is particularly suitable for isolating plasmid DNA from bacteria, such as, for example, E. coli, for subsequent cloning, transfection or sequencing.
  • the bacteria are lysed using known lysis methods, such as alkaline lysis according to Bimboim and Doly (1979) or lysis by heating according to Holmes and Quigley.
  • the cell debris as well as the precipitated proteins and the genomic DNA are removed from the viscous lysate by centrifugation or filtration, and a clarified lysate is obtained which contains the plasmid DNA.
  • the plasmid DNA can be purified, for example, by means of ion exchange chromatography, and the plasmid DNA pre-purified in this way can then be isolated using the method according to the invention.
  • Another object of the present invention is a kit for isolating nucleic acids from a solution, comprising a) an adsorption solution containing 0.25-1.5 M NaCl, KCI or a mixture thereof and ethanol or isopropanol in one
  • the Si0 2 -containing surface can be a porous membrane or a filter made of silica gel, glass or quartz fibers and arranged in a suitable device.
  • the kit preferably also contains solutions suitable for lysis and, as described above, washing and elution buffers.
  • nucleic acids isolated according to the invention are free from ; nucleic acid degrading enzymes and have such a high purity, that they can be processed and processed immediately in a variety of ways.
  • the nucleic acids produced according to the invention can be used for cloning and serve as substrates for a wide variety of enzymes, such as, for example, DNA polymerases, DNA restriction enzymes, DNA ligase and reverse transcriptase.
  • enzymes such as, for example, DNA polymerases, DNA restriction enzymes, DNA ligase and reverse transcriptase.
  • the nucleic acids provided by the method according to the invention are particularly well suited for amplification, in particular for PCR, strand displacement amplification, rolling circle methods, ligase chain reaction (LCR) and similar methods.
  • the method according to the invention is furthermore particularly suitable for providing nucleic acids for use in diagnostics, in particular for a diagnostic method, which is characterized in that the nucleic acid purified by the method according to the invention is amplified in a subsequent step and subsequently and / or simultaneously the nucleic acid amplified in this way is detected (e.g. Holland, PM et al., 1991. Proc. Natl. Acad. Sci. 88, 7276-7280. Livak, KJ et al., 1995. PCR Methods Applic. 4, 357 - 362; Kievits, T. et al., 1991. J. Virol. Meth. 35, 273-286; Uyttendaele, M. et al., 1994. J. Appl. Bacteriol. 77, 694-701).
  • each of the individual buffers (0.25-1.5 M NaCl; 50 mM Tris, pH 8.5; 15% (w / v) isopropanol) were mixed with 10 ⁇ g plasmid DNA (pCMVß; Fa.
  • the batches were transferred to a column containing a silica membrane (QIAquick, QIAGEN GmbH, # 28104) and under vacuum (approx. 600 mbar; use of the QIAvac 6S from QIAGEN GmbH) through the silica membrane.
  • the mixture was then washed with 750 ⁇ l of buffer PE (10 mM Tris, pH 7.5; 80% ethanol) and drawn through the membrane until the air was dry. Elution was carried out by adding 100 ⁇ l of buffer EB (10 mM Tris, pH 8.5) and the yield was determined photometrically at 260 nm.
  • the batches were transferred to a column containing a silica membrane and passed through the silica membrane under vacuum (approx. 600 mbar; use of the QIAvac 6S from QIAGEN GmbH).
  • the mixture was then washed with 750 ⁇ l of buffer PE (10 mM Tris, pH 7.5; 80% ethanol) and drawn through the membrane until the air was dry.
  • Elution was carried out by adding 100 ⁇ l of buffer EB (10 mM Tris, pH 8.5) and the yield was determined photometrically at 260 nm.
  • 15 ml buffer Q1 1, 25 M NaCl; 50 mM Tris, pH 8.5; 15 vol% Isopropanol
  • isopropanol was added to a final concentration of 49.8% by volume, mixed thoroughly and incubated for 5 minutes on the laboratory bench.
  • the DNA / Q1 / isopropanol mixture was transferred to a 20 ml syringe, which was equipped with a syringe filter containing a silica membrane (from Sartorius, Minisart series).
  • the recovery rate of the DNA is between 80 and 90% even with higher amounts of plasmid DNA.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour isoler des acides nucléiques contenus dans une solution. Selon ce procédé, les acides nucléiques sont adsorbés sur une surface contenant du SiO2 en présence d'halogénures alcalins et d'alcool. L'invention concerne en outre l'utilisation d'une solution tampon contenant des halogénures alcalins pour isoler des acides nucléiques sur un support contenant du SiO2, ainsi qu'un kit pour mettre en oeuvre ledit procédé.
EP01971766A 2000-07-12 2001-07-12 Procede pour isoler des acides nucleiques Ceased EP1299531A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10033991 2000-07-12
DE10033991A DE10033991A1 (de) 2000-07-12 2000-07-12 Verfahren zur Isolierung von Nukleinsäuren
PCT/EP2001/008066 WO2002004620A2 (fr) 2000-07-12 2001-07-12 Procede pour isoler des acides nucleiques

Publications (1)

Publication Number Publication Date
EP1299531A2 true EP1299531A2 (fr) 2003-04-09

Family

ID=7648743

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01971766A Ceased EP1299531A2 (fr) 2000-07-12 2001-07-12 Procede pour isoler des acides nucleiques

Country Status (5)

Country Link
US (1) US20040091875A1 (fr)
EP (1) EP1299531A2 (fr)
JP (1) JP2004502458A (fr)
DE (1) DE10033991A1 (fr)
WO (1) WO2002004620A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10000751B2 (en) 2011-12-22 2018-06-19 General Electric Company Method and apparatus for isolating nucleic acids

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Publication number Priority date Publication date Assignee Title
DE10253351B4 (de) * 2002-11-08 2007-02-22 InViTek Gesellschaft für Biotechnik & Biodesign mbH Neuartige Pufferfomulierungen zur Isolierung, Reinigung und Rückgewinnung lang- und kurzkettiger Nukleinsäuren
WO2004042058A2 (fr) * 2002-11-08 2004-05-21 InViTek Gesellschaft für Biotechnik & Biodesign mbH Nouvelles formulations de tampons pour isoler, purifier et recuperer des acides nucleiques a chaine longue et a chaine courte
WO2005054466A2 (fr) † 2003-07-25 2005-06-16 Ambion, Inc. Procedes et compositions de preparation d'une arn a partir d'un echantillon fixe
JP2008220380A (ja) * 2003-10-31 2008-09-25 Fujifilm Corp 核酸の分離精製方法
EP1690938A1 (fr) * 2005-02-11 2006-08-16 Qiagen GmbH Méthode pour l'isolation d'acides nucléiques dans laquelle les acides nucléiques sont immobilisés à haute température sur une matrice
DE102005047736B4 (de) 2005-09-29 2008-08-14 Aj Innuscreen Gmbh Verfahren und System zur Isolierung von Nukleinsäuren aus beliebigen komplexen Ausgangsmaterialien
DE102005059217B4 (de) * 2005-12-07 2011-03-17 Aj Innuscreen Gmbh Verfahren und Testkit zur Trennung, Aufreinigung und Wiedergewinnung von lang- und kurzkettigen Nukleinsäuren
JP5463492B2 (ja) * 2008-10-08 2014-04-09 島根県 微生物細胞からのプラスミドｄｎａ抽出法
DE102012012523B4 (de) 2012-06-26 2015-02-12 Magnamedics Gmbh Reinigung von Nukleinsäuren

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US5155018A (en) * 1991-07-10 1992-10-13 Hahnemann University Process and kit for isolating and purifying RNA from biological sources
US5329000A (en) * 1991-10-31 1994-07-12 Becton, Dickinson And Company Purification of DNA with silicon tetrahydrazide
DE4321904B4 (de) * 1993-07-01 2013-05-16 Qiagen Gmbh Verfahren zur chromatographischen Reinigung und Trennung von Nucleinsäuregemischen
ATE302208T1 (de) * 1994-02-11 2005-09-15 Qiagen Gmbh Verfahren zur trennung von doppelstrang/einzelstrangnukleinsäurestrukturen
US5576196A (en) * 1995-01-13 1996-11-19 Vical Incorporated Process for reducing RNA concentration in a mixture of biological material using diatomaceous earth
JP4196227B2 (ja) * 1997-05-20 2008-12-17 東洋紡績株式会社 核酸またはタンパク質抽出用シリカ粒子組成物
JPH11196869A (ja) * 1998-01-19 1999-07-27 Toyobo Co Ltd リボ核酸の単離方法
US6194562B1 (en) * 1998-04-22 2001-02-27 Promega Corporation Endotoxin reduction in nucleic acid purification
DE19856064C2 (de) * 1998-12-04 2000-11-30 Invitek Gmbh Universelles Verfahren zur Isolierung von DNA aus beliebigen Ausgangsmaterialien
JP4551568B2 (ja) * 1999-05-14 2010-09-29 プロメガ コーポレイション 常磁性粒子を用いた集細胞及びライセート清澄化

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10000751B2 (en) 2011-12-22 2018-06-19 General Electric Company Method and apparatus for isolating nucleic acids
US10683496B2 (en) 2011-12-22 2020-06-16 General Electric Company Method and apparatus for isolating nucleic acids

Also Published As

Publication number Publication date
WO2002004620A3 (fr) 2002-07-18
JP2004502458A (ja) 2004-01-29
US20040091875A1 (en) 2004-05-13
WO2002004620A2 (fr) 2002-01-17
DE10033991A1 (de) 2002-01-24

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