WO2001059098A9 - Verfahren zur aufreinigung von nukleinsäuren - Google Patents
Verfahren zur aufreinigung von nukleinsäurenInfo
- Publication number
- WO2001059098A9 WO2001059098A9 PCT/EP2001/001134 EP0101134W WO0159098A9 WO 2001059098 A9 WO2001059098 A9 WO 2001059098A9 EP 0101134 W EP0101134 W EP 0101134W WO 0159098 A9 WO0159098 A9 WO 0159098A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- membrane
- nucleic acids
- starting material
- binding buffer
- peg
- Prior art date
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
- C12N15/1017—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
Definitions
- the invention relates to a method for the purification of nucleic acids from nucleic acid-containing starting material using a porous membrane.
- the affinity of nucleic acids for the membranes used is generally determined in connection with a special configuration of the membrane surface certain conditions in the buffer system in which the starting material passes the membrane.
- nucleic acids are bound to hydrophilic membranes, the binding buffer system being an organic solvent, e.g. Isopropanol, is added in high concentration.
- organic solvent e.g. Isopropanol
- the object of the invention is to provide a method for the purification of nucleic acids which can be carried out in a simpler manner than the known method.
- the invention is intended to cover two variants.
- any membrane can be used.
- special membranes are used.
- the first variant provides that a binding buffer is added to the optionally pre-cleaned starting material, the buffer contains polyethylene glycol (PEG) and / or at least one salt in a concentration such that the final concentration in the mixture of starting material and binding buffer is above the value required for the precipitation of nucleic acids, then the mixture of starting material and binding buffer, for example under vacuum or by means of centrifugation can permeate through the membrane, the nucleic acids being selectively retained on the surface or in the pores (depth filter effect) of the membrane.
- PEG polyethylene glycol
- the membrane can then be washed in order to remove any non-specifically bound impurities. If desired, the washing step can then be followed by an elution step in which the bound nucleic acids are detached from the membrane again.
- PEG is particularly preferably used as a precipitant in a final concentration of more than 6% based on the mixture of binding buffer and starting material.
- the purification of nucleic acids in the presence of PEG 8000 on silica beads is described by Engelstein et al. in Microbial & Comparative Genomics, Vol. 3, No. 4, 1998.
- the binding buffer used in this publication contains 20% PEG 8000.
- the PEG 8000 is intended to replace the chaotropic reagents previously required in connection with purification of nucleic acids on silk carriers. At no point is it indicated or described that the mechanism described here would be possible in connection with other carrier materials than silica and also differently made carriers as beads.
- EP 0512767 mentioned at the beginning determines in the table shown on page 7 that none in the presence of PEG (10% or 20%) DNA was bound to membranes, which speaks against the results of the applicant.
- Plastic membranes are usually made of e.g. Polypropylene, polyamide, polyester, polysulfone or PVDF are used.
- the membranes can preferably have pores with a diameter of 0.2-10 ⁇ m, the yield being able to be optimized by the choice of the pore diameter. As the pore diameter decreases, the membrane retention rate increases. However, smaller pores also clog more quickly, so that the pore diameter should not be chosen too small.
- the membranes are preferably designed so that both surface filter and depth filter effects occur.
- the selected binding conditions should basically ensure that the nucleic acid molecule is in a compressed form, since this enables optimized retention on and in the membrane.
- the membranes used in the purification processes are functionalized with deprotonatable groups, in particular with sulfonic acid, carboxylic acid or phosphoric acid groups.
- the nucleic acid-containing starting material is allowed to pass through the membrane functionalized in this case in a binding buffer, the binding buffer containing a precipitant for nucleic acids.
- the membrane can then optionally be washed and the nucleic acids can then be eluted from the membrane. It is provided that the precipitant is present in the mixture of binding buffer and starting material in a concentration which is above the value required for the precipitation of nucleic acids.
- the nucleic acids precipitate in the presence of the binding buffer. If the binding buffer starting material mixture is then sucked through the membrane, the nucleic acids adhere to the membrane.
- a precipitant e.g. Salt, PEG or isopropanol are used, to name just a few particularly suitable examples which do not restrict the invention.
- nucleic acids are bound in the presence of PEG to magnetic microparticles whose surface is functionalized.
- the method described here is specially tailored to magnetic beads, which require more complicated handling than the membranes used according to the invention.
- Another disadvantage is that beads require higher elution volumes than membranes.
- the functionalized groups are bound to polymer chains, one end of which is fixed to the surface of the membrane and the other end of which is free to move.
- These polymer chains significantly increase the retention properties of membranes for nucleic acids.
- the polymer chains align differently with the membrane depending on the external environment; If the pH is rather alkaline or neutral and / or the ion concentration is low, the polymer chains protrude from the membrane to a greater or lesser extent. If the pH is reduced and / or the ion concentration is increased, the polymer chains attach to the membrane.
- the highest retention rate of the membrane for nucleic acids is when the polymer chains protrude from the membrane but are not fully stretched (tentacle structure). Under these conditions that arise when e.g. If there are higher ion concentrations or higher PEG concentrations, the nucleic acid molecule is also in a compressed form, in which it is retained particularly effectively in the tentacle structure.
- polymer chains e.g. Polyacrylic acid or the like can be used.
- the membranes preferably have pores with a diameter of 0.2-10 ⁇ m, the yield being able to be optimized by the choice of the pore diameter.
- the nucleic acids adhering to the membrane can be eluted with buffers of low ion concentration or with water at room temperature.
- the buffer used is sucked or pressed through the membrane.
- a possible washing step between binding and elution can be carried out in the same way with e.g. Ethanol or the like.
- conditions are preferably set in which the polymer chains protrude from the membrane as fully stretched as possible and the Small acid molecule is also in a stretched form. Under these conditions, which arise, for example, when using the elution buffer specified above, a particularly good detachment of the nuclear acid molecule from the membrane is possible.
- all buffers or mixtures used can be sucked through the membrane under vacuum or e.g. press through by centrifugation.
- the method is preferably carried out with membranes which are arranged in microtiter filter plates, spin columns or reaction vessels.
- Example 1 a) Modification of polypropylene membranes
- a nylon microfiltration membrane (Schleicher & Schüll, nominal pore size 0.45 ⁇ m, membrane thickness 127 ⁇ m) is modified under the same conditions as in a).
- the surface-modified membranes produced according to Examples 1 a and b were clamped in a 96 microfiltration plate.
- BP1 binding buffer
- the cleaning takes place by means of centrifugation steps (3000 rpm) according to the principle: bind-wash-elute.
- Surface-modified PP with a nominal pore size of 0.45 ⁇ m is used as the base material for this experiment.
- the semi-quantitative evaluation of 4 ⁇ l eluate is carried out by means of ethidium bromide gel electrophoresis (not shown).
- Example 3 A pDNA purification is carried out on the principle of alkaline lysis.
- bacteria from 1.5 ml of bacterial culture are centrifuged and the buffers P1-P3 (P 100 ⁇ l, P2: 300 ⁇ l, P3: 300 ⁇ l) from the “Perfect Prep Plasmid Midi” kit from Eppendorf are added. After centrifugation, the clear lysate is mixed with 700 ⁇ l binding buffer (see Table 1). The sample is worked up under the conditions listed in Example 1.
- a plasmid preparation is carried out by performing the complete purification procedure using the “Perfect Prep Plasmid Mini” kit from Eppendorf, from the identical 1.5 ml bacterial culture (data not shown).
- the mixture of clarified lysate and the respective binding buffer (see Table 1) is centrifuged for 30 minutes at 12000 g, room temperature and the supernatant is removed. This is followed by washing twice with 200 ⁇ l 70% ethanol, drying under vacuum, and addition of 30 ⁇ l Tris-HCl, pH 7.5 (data not shown).
- binding buffer pH 7.4, modified membrane 2.
- binding buffer pH 4.6 modified membrane 3.
- binding buffer pH 3.5 modified membrane 4.
- binding buffer pH 7.4 unmodified membrane 5.
- binding buffer pH 4.6 unmodified membrane 6.
- binding buffer pH 3.5 unmodified membrane
Landscapes
- 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)
- Separation Using Semi-Permeable Membranes (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10006591A DE10006591B4 (de) | 2000-02-11 | 2000-02-11 | Verfahren zur Aufreinigung von Nukleinsäuren |
DE10006591.0 | 2000-02-11 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2001059098A2 WO2001059098A2 (de) | 2001-08-16 |
WO2001059098A3 WO2001059098A3 (de) | 2002-05-23 |
WO2001059098A9 true WO2001059098A9 (de) | 2003-07-31 |
Family
ID=7630913
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2001/001134 WO2001059098A2 (de) | 2000-02-11 | 2001-02-02 | Verfahren zur aufreinigung von nukleinsäuren |
Country Status (3)
Country | Link |
---|---|
US (1) | US20030166916A1 (de) |
DE (1) | DE10006591B4 (de) |
WO (1) | WO2001059098A2 (de) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10113815A1 (de) * | 2001-03-21 | 2002-10-02 | Eppendorf Ag | Verfahren zur Isolierung von Plasmiden oder Proteinen aus suspendierten Bakterien- oder Hefezellen |
DE10222275A1 (de) * | 2002-05-18 | 2003-12-04 | Eppendorf Ag | Verfahren zur Isolierung von Nukleinsäuren aus einer flüssigen Probe |
US20040180445A1 (en) * | 2003-03-12 | 2004-09-16 | Domanico Michael J. | Methods and compositions for purification of nucleic acid from a host cell |
US7031802B2 (en) * | 2003-08-13 | 2006-04-18 | Hewlett-Packard Development Company, L.P. | Semi-autonomous operation of a robotic device |
JP2022532214A (ja) * | 2019-05-15 | 2022-07-13 | トランスレイト バイオ, インコーポレイテッド | メッセンジャーrnaの精製方法 |
Family Cites Families (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989004203A1 (en) * | 1987-11-10 | 1989-05-18 | North Carolina State University | Chromatography apparatus and method and material for making the same |
DD274676A1 (de) * | 1988-08-04 | 1989-12-27 | Akad Wissenschaften Ddr | Verfahren zur herstellung von festen phasen fuer molekularbiologische nachweis- und trennoperation |
US5187083A (en) * | 1990-11-13 | 1993-02-16 | Specialty Laboratories, Inc. | Rapid purification of DNA |
JPH04360686A (ja) * | 1991-06-04 | 1992-12-14 | Tosoh Corp | Dnaの精製方法 |
US5438128A (en) * | 1992-02-07 | 1995-08-01 | Millipore Corporation | Method for rapid purifiction of nucleic acids using layered ion-exchange membranes |
DE4321904B4 (de) * | 1993-07-01 | 2013-05-16 | Qiagen Gmbh | Verfahren zur chromatographischen Reinigung und Trennung von Nucleinsäuregemischen |
US5561064A (en) * | 1994-02-01 | 1996-10-01 | Vical Incorporated | Production of pharmaceutical-grade plasmid DNA |
US5483128A (en) * | 1994-09-06 | 1996-01-09 | Chunghwa Picture Tubes, Ltd. | Multi-mode, hybrid-type CRT and electron gun therefor with selectable different sized grid apertures |
US5705628A (en) * | 1994-09-20 | 1998-01-06 | Whitehead Institute For Biomedical Research | DNA purification and isolation using magnetic particles |
US5804684A (en) * | 1995-08-24 | 1998-09-08 | The Theobald Smith Research Institute, Inc. | Method for isolating nucleic acids |
US5707812A (en) * | 1996-08-06 | 1998-01-13 | Vical Incorporated | Purification of plasmid DNA during column chromatography |
EP0853123A1 (de) * | 1997-01-10 | 1998-07-15 | Roche Diagnostics GmbH | Reinigung von DNA durch Cross-Flow-Filtration |
US5958677A (en) * | 1997-07-28 | 1999-09-28 | The New York Blood Center, Inc. | Method for purifying viral nucleic acids |
DE19746874A1 (de) * | 1997-10-23 | 1999-04-29 | Qiagen Gmbh | Verfahren zur Isolierung und Reinigung von Nukleinsäuren an hydrophoben Oberflächen - insbesondere unter Verwendung hydrophober Membranen |
DE19958042A1 (de) * | 1999-12-03 | 2001-06-21 | Invitek Gmbh | Oberflächenmodifizierte Trägermaterialien zur Bindung biologischer Materialien, Verfahren zu ihrer Herstellung und ihre Verwendung |
-
2000
- 2000-02-11 DE DE10006591A patent/DE10006591B4/de not_active Expired - Fee Related
-
2001
- 2001-02-02 WO PCT/EP2001/001134 patent/WO2001059098A2/de active Application Filing
- 2001-02-02 US US10/182,957 patent/US20030166916A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
US20030166916A1 (en) | 2003-09-04 |
DE10006591B4 (de) | 2007-03-29 |
WO2001059098A2 (de) | 2001-08-16 |
DE10006591A1 (de) | 2001-08-23 |
WO2001059098A3 (de) | 2002-05-23 |
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