EP1037973A2 - Procede d'isolement d'acides nucleiques a chaine courte et a chaine longue - Google Patents

Procede d'isolement d'acides nucleiques a chaine courte et a chaine longue

Info

Publication number
EP1037973A2
EP1037973A2 EP98966787A EP98966787A EP1037973A2 EP 1037973 A2 EP1037973 A2 EP 1037973A2 EP 98966787 A EP98966787 A EP 98966787A EP 98966787 A EP98966787 A EP 98966787A EP 1037973 A2 EP1037973 A2 EP 1037973A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acids
carrier
membranes
materials
carrier material
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.)
Withdrawn
Application number
EP98966787A
Other languages
German (de)
English (en)
Inventor
Timo Hillebrand
Peter Bendzko
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.)
Invitek Gesellschaft fuer Biotechnik und Biodesign mbH
Original Assignee
Invitek 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 Invitek GmbH filed Critical Invitek GmbH
Publication of EP1037973A2 publication Critical patent/EP1037973A2/fr
Withdrawn legal-status Critical Current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
    • C12N15/1017—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 simple and extremely fast method for isolating and purifying large amounts of short- and long-chain nucleic acids from different biological and other starting materials. It is of great importance for a large number of laboratories working biologically, molecular biologically, forensically, medically, analytically and biochemically.
  • the fields of application of the invention are forensic medicine, medical diagnostics, molecular biology, biochemistry, genetic engineering and all other related fields.
  • kits are based on the well-known principle of binding nucleic acids to mineral carriers in the presence of solutions of different chaotropic salts and use suspensions of finely ground glass powder (e.g. Glasmilk, BIO 101, La Jolla, CA), diatomaceous earth (Sigma) as carrier materials. or also silica gel. (Diagen, DE 41 39 664 AI).
  • a method for isolating nucleic acids that is practical for a large number of different applications is shown in US Pat. No. 5,234,809 (Boom).
  • a method for isolating nucleic acids from nucleic acid-containing starting materials by incubating the starting material with a chaotropic buffer and a DNA-binding solid phase is described there.
  • the chaotropic buffers implement both the lysis of the starting material and the binding of the nucleic acids to the solid phase.
  • the method is well suited for isolating nucleic acids from small sample quantities and is particularly useful in the area of isolating viral nucleic acids.
  • the object of the invention was to develop a method for isolating nucleic acids, in particular plasmid DNA, preferably from a wide variety of starting quantities, which leads to isolated nucleic acids which meet the high quality parameters of subsequent applications, extremely simple to carry out, quickly and is inexpensive.
  • the object was achieved according to the claims, according to the invention by a combined batch column process, in which the nucleic acid binding to mineral support materials is carried out as a batch process and the bound nucleic acids are then processed chromatographically.
  • the method according to the invention for the isolation and purification of short- and long-chain nucleic acids from different biological and other starting materials is characterized by
  • the method according to the invention is also suitable for the isolation and purification of short- and long-chain nucleic acids from other starting materials, e.g. from bacterial lysates, vegetable components, DNA fragments from PCR batches or from agarose gels.
  • Preferred carrier materials are silicon compounds, e.g. Silicon dioxide, silicas or silica gels.
  • the silicon compounds used according to the invention have an average particle size of 7 nm to 5000 ⁇ m, preferably 7-40 nm or 500-5000 nm.
  • Chaotropic salts are e.g. Guanidine isothiocyanate, guanidine hydrochloride, lithium chloride, sodium perchlorate, sodium iodide and / or urea, preferably in concentrations of 1 to 10 M.
  • Detergents such as e.g. TritonX-100, Tween, N-Lauryl-Sarcosyl, SDS and / or CTAB, optionally protein-degrading enzymes, such as e.g. Proteinases used.
  • membranes which have a pore size which is larger than that of the carrier materials used, preferably in centrifugation cartridges, the pore size of the membranes being specified as a function of the average particle size of the carrier material used.
  • nylon or polysulfone membranes are preferably used.
  • the carriers with the fixed nucleic acids are washed before or after their application to the membrane. The subsequent elution of the nucleic acid from the carrier is carried out professionally with buffers of low ionic strength.
  • centrifugation membranes which can effectively be used for nucleic acid purifications must have a pore size which is smaller than that of the carrier matrices used, since mineral particles which have particle diameters smaller than the pores of the centrifugation membranes always centrifuge through the pores during a centrifugation step become.
  • the invention is based on the surprising finding that a combined batch column method for isolating nucleic acids using mineral carrier particles, which can even be very much smaller than the pore diameter of commercially available centrifugation columns, is possible.
  • the process according to the invention of combining a batch process with a column process is not limited by the use of any mineral carrier materials, even very small mineral carrier particles and their extremely large specific surfaces, with regard to the volumes of the starting materials.
  • the method provides very high yields of high-purity DNA and is also very easy to carry out and can be compared to widely used anion exchange systems e.g. in the isolation of plasmid DNA can be realized in a much shorter time.
  • Chromosomal DNA and proteins are subsequently sedimented by a 5 minute centrifugation step at 14,000 rpm and the clear centrifuged supernatant is subsequently incubated with 20 ⁇ l of a suspension made of a mineral carrier material consisting of silica nanoparticles (average particle size 40 nm).
  • the solution is then transferred to a mini centrifugation column (e.g. Micro Spin Nylon or Micro Spin PSE; LIDA).
  • the carrier particles with the bound plasmid DNA are brought to the surface of the filter membrane by centrifugation for 1 minute at 12,000 rpm and washed there by adding a washing solution (60% ethanol; NaCl) and a further centrifugation step. The washing step is repeated one more time.
  • the remaining ethanol is finally removed from the carrier material by centrifugation at 12,000 rpm for 2 min.
  • the bound plasmid DNA is detached by adding 100 ⁇ l of a low salt buffer (e.g. 10 mm Tris-HCl) at 70 ° C and subsequent centrifugation for 1 minute at 12,000 rpm.
  • the centrifugate contains the isolated plasmid DNA.
  • Chromosomal DNA and proteins are subsequently sedimented by a 10 minute centrifuge step at 5000 rpm and the clear centrifuged supernatant is subsequently treated with 150 ⁇ l of a suspension made of a mineral carrier material consisting of silica nanoparticles (average particle size 40 nm) incubated.
  • the solution is then transferred to a centrifugation column (eg Macro Spin Nylon; LIDA).
  • the carrier particles with the bound plasmid DNA are brought to the surface of the filter membrane by centrifugation for 5 minutes at 5000 rpm and there by adding a washing solution (60%) ethanol; NaCl) and another centrifugation step. The washing step is repeated one more time.
  • the remaining ethanol is finally removed from the carrier material by centrifugation at 5000 rpm for 10 min or by incubation of the centrifugation column at 37 ° C. for 10 min.
  • the bound plasmid DNA is detached by adding 200 ⁇ l - 300 ⁇ l of a low salt buffer (eg 10 mm Tris-HCl) at 70 ° C. and subsequent centrifugation for 5 minutes at 5000 rpm.
  • the centrifugate contains the isolated plasmid DNA.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Saccharide Compounds (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Le procédé selon l'invention permet d'isoler et de purifier des acides nucléiques à chaîne courte et à chaîne longue à partir de différents matériaux de départ biologiques ou d'une autre nature. Ce procédé se caractérise par le fait qu'il comprend les étapes suivantes: lyse de matériaux contenant des acides nucléiques; incubation avec un matériau support minéral; application sur des membranes présentant une taille de pore supérieure à la taille de particule du matériau support; et séparation des acides nucléiques du matériau support par élution, les particules de matériau support restant, de façon surprenante, sur la membrane.
EP98966787A 1997-12-18 1998-12-18 Procede d'isolement d'acides nucleiques a chaine courte et a chaine longue Withdrawn EP1037973A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19758102 1997-12-18
DE19758102 1997-12-18
PCT/DE1998/003728 WO1999032616A2 (fr) 1997-12-18 1998-12-18 Procede d'isolement d'acides nucleiques a chaine courte et a chaine longue

Publications (1)

Publication Number Publication Date
EP1037973A2 true EP1037973A2 (fr) 2000-09-27

Family

ID=7853518

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98966787A Withdrawn EP1037973A2 (fr) 1997-12-18 1998-12-18 Procede d'isolement d'acides nucleiques a chaine courte et a chaine longue

Country Status (6)

Country Link
EP (1) EP1037973A2 (fr)
JP (1) JP2001526888A (fr)
AU (1) AU2509799A (fr)
CA (1) CA2315257A1 (fr)
DE (1) DE19858447A1 (fr)
WO (1) WO1999032616A2 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7148343B2 (en) * 2001-10-12 2006-12-12 Gentra Systems, Inc. Compositions and methods for using a solid support to purify RNA
DE10222133A1 (de) * 2002-05-17 2003-12-04 Gl Biotech Gmbh Verfahren zur Nukleinsäure-Extraktion und Nukleinsäure-Reinigung
US7482116B2 (en) 2002-06-07 2009-01-27 Dna Genotek Inc. Compositions and methods for obtaining nucleic acids from sputum
DE102008057317A1 (de) 2007-11-13 2009-09-10 Stratec Biomedical Systems Ag Vorrichtung und Verfahren zur Aufreinigung von Biomolekülen
KR101569832B1 (ko) * 2008-11-19 2015-11-18 삼성전자주식회사 시료 중의 게놈 ｄｎａ 및 플라스미드 ｄｎａ를 서로 분리하는 방법 및 그를 위한 키트
US9442046B2 (en) 2011-06-19 2016-09-13 Abogen, Inc. Device for sample collection
JP6670765B2 (ja) 2014-03-07 2020-03-25 ディーエヌエー ジェノテック インク 生物試料中の核酸を安定化する組成物および方法

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5075430A (en) * 1988-12-12 1991-12-24 Bio-Rad Laboratories, Inc. Process for the purification of DNA on diatomaceous earth
NL8900725A (nl) * 1989-03-23 1990-10-16 Az Univ Amsterdam Werkwijze en combinatie van middelen voor het isoleren van nucleinezuur.
US5155018A (en) * 1991-07-10 1992-10-13 Hahnemann University Process and kit for isolating and purifying RNA from biological sources
WO1995006652A1 (fr) * 1993-08-30 1995-03-09 Promega Corporation Compositions et procede de purification d'acides nucleiques
DE4422044A1 (de) * 1994-06-14 1995-12-21 Invitek Gmbh Verfahren zur Isolierung, Reinigung und ggf. Lagerung von Nukleinsäuren
DE4422040A1 (de) * 1994-06-14 1995-12-21 Invitek Gmbh Verfahren zur Isolierung und Reinigung von PCR-Produkten
ATE184013T1 (de) * 1994-06-14 1999-09-15 Invitek Gmbh Universelles verfahren zur isolierung und reinigung von nukleinsäuren aus extrem geringen mengen sowie sehr stark verunreinigten unterschiedlichsten ausgangsmaterialien
DE19506887C2 (de) * 1995-02-17 1999-10-14 Invitek Gmbh Verfahren zur simultanen Isolierung von genomischer DNS und hochreiner Total RNS
US5783686A (en) * 1995-09-15 1998-07-21 Beckman Instruments, Inc. Method for purifying nucleic acids from heterogenous mixtures
DE19717717B4 (de) * 1997-04-18 2007-08-02 InViTek Gesellschaft für Biotechnik & Biodesign mbH Verfahren zur nichtinvasiven Erkennung bösartiger Tumoren der Lunge

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9932616A3 *

Also Published As

Publication number Publication date
JP2001526888A (ja) 2001-12-25
WO1999032616A2 (fr) 1999-07-01
DE19858447A1 (de) 1999-07-01
WO1999032616A3 (fr) 1999-09-10
CA2315257A1 (fr) 1999-07-01
AU2509799A (en) 1999-07-12

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