EP1155026A2 - Procede d'isolement d'acides nucleiques - Google Patents

Procede d'isolement d'acides nucleiques

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Publication number
EP1155026A2
EP1155026A2 EP00903676A EP00903676A EP1155026A2 EP 1155026 A2 EP1155026 A2 EP 1155026A2 EP 00903676 A EP00903676 A EP 00903676A EP 00903676 A EP00903676 A EP 00903676A EP 1155026 A2 EP1155026 A2 EP 1155026A2
Authority
EP
European Patent Office
Prior art keywords
weight
water
insoluble
particle size
monomer
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Withdrawn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
EP00903676A
Other languages
German (de)
English (en)
Inventor
Wolfgang Podszun
Rainer Neumann
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.)
Bayer AG
Original Assignee
Bayer AG
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 Bayer AG filed Critical Bayer AG
Publication of EP1155026A2 publication Critical patent/EP1155026A2/fr
Withdrawn legal-status Critical Current

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the invention relates to a method for separating and selectively releasing nucleic acids using special bead polymers.
  • gene diagnostics has become increasingly important. Genetic diagnostics has found its way into the diagnosis of human diseases (e.g. detection of infectious agents, detection of mutations in the genome, analysis of circulating tumor cells and identification of risk factors for the predisposition of a disease). But gene diagnostics is now also used in veterinary medicine, environmental analysis and food testing. Another area of application is investigations at pathological / cytological institutes or in the context of forensic questions. But also in the context of quality and process control (for example
  • nucleic acids In gene diagnostics, the acquisition of gene samples from biological material such as cells, blood, sputum, cerebrospinal fluid, serum or urine is an important sub-step.
  • the binding of nucleic acids to polyquaternary cationic polymers is known from US-A-4 046 750. However, the binding is irreversible, so that with this method the adsorbed nucleic acids cannot be released again.
  • US-A-4 055 469 discloses a method for purifying enzymes, wherein
  • Nucleic acids and unwanted proteins are precipitated using water-soluble cationic polymers.
  • US-A-4 839 231 discloses supports coated with vinylpyridine polymer which can adsorb proteins and nucleic acids. However, the capacity of
  • WO-A-91/05606 describes a silanized, porous support material with hydroxyalkylamino groups for the chromatographic separation of nucleic acids.
  • this material is for quick and as quantitative as possible
  • DE-A-4 139 664 describes a device and a method for isolating and purifying nucleic acids with the aid of anion exchangers.
  • a disadvantage of this method is that the desorption of the nucleic acids from
  • Anion exchangers are only successful with buffer solutions of high ionic strength and the separation of the salts from the buffer solution requires additional preparation steps.
  • DE-A-4 333 805 claims the extraction of nucleic acids from a sample with the aid of water-soluble carriers such as dextran, acrylamide or carboxymethyl cellulose and other reagents, the nucleic acids being precipitated.
  • EP-A-0 707 077 (corresponds to US-A-5 582 988) describes a method for
  • a disadvantage of the methods according to DE-A-4 333 805 and EP-A-0 707 077 is that handling, in particular the separation and purification of the precipitation product, is difficult and very time-consuming. These methods can also not be carried out or only under difficult conditions with the aid of automated analysis devices.
  • WO-A-96/18731 describes a method for isolating nucleic acids with the aid of a detergent and a solid support. Since solid supports without pores and without swellability are used, the binding capacity of the supports is relatively low.
  • WO-A-97/08547 describes a method for isolating nucleic acids in which the nucleic acids are bound to a solid hydrophilic organic polymer without an effective positive charge, for example to cellulose. With this method, the bond occurs via weak forces, such as Van der Waal's interactions.
  • WO-A-97/34909 describes a method for isolating nucleic acids using a special particulate polymer with a lower critical solubility temperature (LCST) of 25-45 ° C.
  • LCST critical solubility temperature
  • the invention relates to a method for isolating nucleic acids from a sample, comprising the following steps
  • the water-insoluble polymer is a bead polymer with an average particle size of 3 to 100 microns and from polymerized units of
  • the biological material is lysed in an intermediate step after process step A).
  • the present invention preferably relates to a method for isolating nucleic acids from a sample, comprising the steps
  • the water-insoluble polymer is a bead polymer with an average particle size of 3 to 100 ⁇ m and a specific surface area measured according to BET of 5 to 500 m / g and composed of polymerized units of
  • the present invention particularly preferably relates to a method for isolating nucleic acids from a sample, comprising the steps A), B) and C) defined above, characterized in that the water-insoluble polymer is a macroporous bead polymer having a particle size of 3 to 100 ⁇ m, one
  • the method according to the invention is suitable for the isolation and / or purification of nucleic acids of different origins, for example from cells, tissue materials, blood or infectious agents. Before isolating the nucleic acids, this is The material to be examined is disrupted by techniques known per se, such as digestion by protease digestion, a sample suitable for the further steps A to C, a lysate, being obtained. The biological material is optionally lysed in an intermediate step after process step A). Further suitable digestion processes have been described in DE-A-4 333 805.
  • the sample is mixed with a water-insoluble polymer at a pH of 7 or less, preferably in the range 2-6, particularly preferably in the range 2-3, at room temperature.
  • the water-insoluble polymer is separated off by e.g. Filtration or centrifugation.
  • the complex of nucleic acid and polymer thus obtained can now be washed by washing with suitable buffers, e.g. TE can be cleaned.
  • the pH of the complex is now adjusted to pH values above 7, preferably from 8 -
  • the bead polymers according to the invention provide higher adsorption and re-release rates than the soluble polymers according to EP-A-0 707 077.
  • the isolation is easier, i.e. perform with fewer steps and in shorter times.
  • the purity of the isolated nucleic acids is higher, in particular they receive less inhibiting by-products, so that amplification of the nucleic acids, for example by means of the so-called "PCR reaction” and "RT-PCR", is particularly successful.
  • the method according to the invention is also superior to the method described in EP-A-0 707 077 with regard to digestion of the nucleic acids obtained by means of restriction enzymes.
  • the present invention furthermore relates to the macroporous bead polymers, characterized in that they have an average particle size of 3 to 100 ⁇ m, a pore diameter of 10 to 1,000 nm and a specific upper Area measured according to BET from 5 to 500 m 2 / g and from polymerized units of
  • the water-insoluble but swellable bead polymers characterized in that they have an average particle size of 3 to 100 ⁇ m and from polymerized units of
  • the present invention furthermore relates to a process for the preparation of water-insoluble, macroporous bead polymers having an average particle size of 3 to 100 ⁇ m, a pore diameter of 10 to 1000 nm and a specific surface area measured according to BET of 5 to 500 m 2 / g, characterized in that one a mixture of
  • Amino monomers (a) in the context of the invention are polymerizable, ethylenically unsaturated compounds having at least one primary, secondary or tertiary amino group.
  • the secondary or tertiary amino group can also be part of a cycloaliphatic or aromatic ring. Examples include N-vinylimidazole, N-vinylbenzimidazole, 2-vinylpyridine and 4-vinylpyridine.
  • Highly suitable amino monomers are also the derivatives of acrylic acid and methacrylic acid, such as, for example, 2-aminoethyl methacrylate, N, N-dimethylaminoethyl methacrylate, N, N-dimethylaminopropyl methacrylate, N, N-dimethylaminoethyl acrylate, N-tert-butylaminopropyl methacrylate, N- (3-aminopropyl) methacrylamide, N- (3-imidazoyl-propyl-methacrylamide, N- (2-imidazoylethyl) methacrylamide, N- (3-aminopropyl) -acrylamide, N- (3-imidazoyl-pi-opyl) acrylamide, N- (2-imidazoylethyl) acrylamide, N- (l, l - Dimethyl-3-imidazoylpropyl) methacrylamide, N- (l, l
  • Highly suitable amino monomers are also the reaction products of isocyanatoethyl (meth) acrylate and imidazoylalkylamines, such as, for example, the amino monomer of the formula (I) which is new in the context of the present invention.
  • styrene and ⁇ -methylstyrene with amino groups are also well suited. Examples include: 4-N, N-dimethylaminostyrene, 2-N, N-dimethylaminostyrene, 4-N, N-diethylaminostyrene and 4-N, N-bis (2-hydroethyl) aminostyrene.
  • suitable crosslinking agents (b) are , Butanediol diacrylate, pentaerytritol diacrylate, 1, 3-glycerol diacrylate, triethylene glycol diacrylate, trimethylolpropane triacrylate, pentaerytritol triacrylate, pentaerytritol tetraacrylate,
  • Hydrophobic vinyl monomers (c1) which can be present in the bead polymer according to the invention and are suitable for the purposes of the present invention are C 1 -C 6 -alkyl acrylate, C 1 -C 6 -alkyl methacrylate, such as methyl methacrylate or butyl acrylate, acrylonitrile, methacrylonitrile, Vinyl chloride, vinylidene chloride, vinyl acetate and aromatic vinyl monomers, such as Styrene, vinyl naphthalene, vinyl toluene, ethyl styrene, ⁇ -methyl styrene, chlorostyrenes and vinyl benzyl chloride.
  • Hydrophilic vinyl monomers (c2) which are suitable in the context of the present invention are, for example: 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, triethylene glycol monomethacrylate,
  • Porogens are used for the process according to the invention for the production of water-insoluble macroporous bead polymers.
  • Water-immiscible compounds which dissolve the monomers used and precipitate the polymer formed are suitable.
  • Examples include aliphatic hydrocarbons such as hexane, heptane, octane, isooctane, isododecane and alcohols such as octanol.
  • the porogen is used in amounts of 10 to 150% by weight, preferably 20 to 100% by weight, based on the sum of the monomers and crosslinking agents used.
  • Suitable radical formers in the context of the present invention are preferably oil-soluble initiators.
  • examples include: peroxy compounds such as dibenzoyl peroxide, dilauryl peroxide, bis (p-chlorobenzoyl peroxide), dicyclohexyl peroxydicarbonate, tert-butyl peroctoate, 2,5-bis (2-ethylhexanoylpero ⁇ y) -2,5-dimethylhexane and tert-amylperoxy-2-ethylhexane, further azo compounds such as 2,2'-azobis (isobutyronitrile), 2,2'-azobis (2,4-dimethylvalereonitrile) and 2,2'-azobis (2-methylisobutyronitrile) .
  • the initiators are generally used in amounts of 0.05 to 2.5% by weight, preferably 0.2 to 1.5% by weight, based on the monomer mixture.
  • Protective colloids are optionally used in the aqueous phase in the preparation of the gel-shaped and macroporous bead polymers according to the invention.
  • Suitable protective colloids according to the present invention are natural and synthetic water-soluble polymers, such as gelatin, starch,
  • Cellulose derivatives in particular cellulose esters and cellulose ethers, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and copolymers of acrylic acid, methacrylic acid, methacrylic acid esters and / or acrylic acid esters. Copolymers of methacrylic acid and methacrylic acid ester neutralized with alkali metal hydroxide are particularly suitable.
  • the amount of protective colloids used is generally 0.05 to 2%, based on the aqueous phase, preferably 0.1 to 1%.
  • the aqueous phase can optionally also contain a buffer system.
  • Buffer systems are preferred which adjust the pH of the aqueous phase at the start of the polymerization to a value between 12 and 5, preferably between 10 and 6. Under these conditions, dispersants with carboxylic acid groups are wholly or partly present as salts. In this way, the effect of the protective colloids is influenced favorably.
  • Buffer systems that are particularly suitable contain phosphate or borate salts.
  • the amount of the water phase is generally 75 to 1200% by weight, preferably 100 to 500% by weight, based on the sum of monomers, crosslinking agents and porogen.
  • the stirring speed during the polymerization is important for the adjustment of the particle size.
  • the size of the bead polymers obtained decreases with increasing stirrer speed.
  • the exact stirring speed for setting a certain predetermined bead size depends in individual cases on the reactor size, the reactor geometry and the stirrer geometry. It has proven to be expedient to determine the necessary stirring speed experimentally.
  • bead sizes of 6 to 30 ⁇ m are generally achieved at speeds of 300 to 500 rpm.
  • the polymerization temperature of the production process according to the invention depends on the decomposition temperature of the initiator used. It is generally between 50 and 150 ° C, preferably between 55 and 100 ° C. The polymerization takes 0.5 to a few hours. It has proven useful to use a temperature program in which the polymerization is started at a low temperature, for example 70 ° C., and the reaction temperature is increased as the polymerization conversion progresses.
  • the polymer can be isolated using customary methods, for example by filtration or decanting, and optionally dried after one or more washes.
  • the porogen can be removed during drying.
  • low-boiling porogens such as, for example, hexane
  • the water-swellable bead polymers are prepared analogously to the preparation of the macroporous bead polymers, with hydrophilic vinyl monomers (c2) instead of the hydrophobic vinyl monomers (c2) and a instead of the porogen
  • Solvent is used. Suitable solvents are those which are immiscible with water, which dissolve the monomers and the crosslinking agent and which do not precipitate out, but rather dissolve or swell the polymer formed. Suitable solvents are toluene, xylene, tetrachloromethane, chloroform, methylene chloride, dichloroethane and. Ethyl acetate. The amount of
  • Auxiliary solvent is generally 10 to 200% by weight, preferably 10 to 150% by weight, particularly preferably 20 to 100% by weight, based on the sum of monomers and crosslinking agent. If desired, the auxiliary solvent can be separated off after the polymerization, for example by distillation. Toluene is particularly easy to remove by azeotropic distillation.
  • the water-swellable bead polymers according to the invention have swelling indices of 1.2 to 12, preferably 1.5 to 8, measured at 25 ° C. and pH 7.
  • the swelling index is the quotient of the volume of the bead polymer swollen in water until saturation and the Volume of the anhydrous polymer beads defined.
  • the present application furthermore relates to compositions for isolating nucleic acids containing water-insoluble macroporous bead polymers having an average particle size of 3 to 100 ⁇ m and a pore diameter of 10 to
  • water-insoluble but swellable bead polymers with an average particle size of 3 to 100 ⁇ m, consisting of polymerized units of a) 5 to 79.7% by weight of amino monomer b) 0.3 to 10% by weight of crosslinking agent and c2) 10 to 93% by weight of hydrophilic vinyl monomer.
  • aqueous dispersions with a solids content of 0.1-50%, particularly preferably 1-10%.
  • the aqueous phase can optionally contain buffers which are preferably effective in the range 2-6.
  • test kits Possible areas of application of an agent formulated, for example, as a test kit are the above-mentioned application examples, for example in the isolation of nucleic acids from cells, tissue materials, blood or infectious agents, all questions of diagnostics playing a role in particular.
  • the test kits also include the polymers described for routine nucleic acid tests in microtiter plates and / or test tubes or other formats such as, for example, in the context of chip technology.
  • Example 3 was repeated, an organic solution consisting of 23.71 g of amino monomer from Example 1, 13.04 g of styrene, 10.67 g of divinylbenzene, 0.71 g of 2,2'-azobis (2,4-dimethylvalereonitrile) and 38 g of hexane was used. 35 g of macroporous bead polymer with an average particle size of 25 ⁇ m and a specific surface area of 74 m 2 / g were obtained. Biological result
  • TE a suitable buffer
  • TE stands for 10 mmol of T ⁇ s HC1 and 1 mmol of ethylenediaminetetraacetic acid pH 7 4 in the final concentration
  • IGEPAL CA-630® is a non-ionic detergent, which can be obtained, for example, from Sigma, order number I 3021) were then added to the sediment. , given and incubated again for 5 mm at room temperature
  • IGEPAL CA-630® instead of IGEPAL CA-630®, other lysis buffers can also be used. Examples include classic methods such as with Protease K digestion and subsequent cleaning using phenol / chloroform or
  • Nat ⁇ umlaurylsulfat solutions called (Sigma order number L 6026), for example as a 0.5% water solution
  • the nucleic acid bound to the particles was released by adjusting the pH to> 12 by adding 1 ⁇ l of 0.5 normal NaOH
  • the concentration of the nucleic acid obtained was determined using a suitable method, for example by analysis in a gel system, particularly preferably the "Submerged Gel Nucleic Acid Electrophoresis System", order no. 170 4406 from BIO-RAD (webpage: www.bio-rad.com).
  • PCR polymerase chain reaction

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Cette invention concerne des acides nucléiques pouvant être efficacement adsorbés dans un milieu neutre ou acide sur des polymères en perles spécifiques qui sont insolubles dans l'eau et dont la taille moyenne des particules se situe entre 3 et 100 νm. Dans un milieu basique, ces acides sont libérés avec un débit plus important.
EP00903676A 1999-02-19 2000-02-09 Procede d'isolement d'acides nucleiques Withdrawn EP1155026A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19907023A DE19907023A1 (de) 1999-02-19 1999-02-19 Verfahren zur Isolierung von Nucleinsäuren
DE19907023 1999-02-19
PCT/EP2000/001028 WO2000049031A2 (fr) 1999-02-19 2000-02-09 Procede d'isolement d'acides nucleiques

Publications (1)

Publication Number Publication Date
EP1155026A2 true EP1155026A2 (fr) 2001-11-21

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ID=7898058

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00903676A Withdrawn EP1155026A2 (fr) 1999-02-19 2000-02-09 Procede d'isolement d'acides nucleiques

Country Status (6)

Country Link
EP (1) EP1155026A2 (fr)
JP (1) JP2002537306A (fr)
AU (1) AU2547100A (fr)
CA (1) CA2362979A1 (fr)
DE (1) DE19907023A1 (fr)
WO (1) WO2000049031A2 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9425138D0 (en) 1994-12-12 1995-02-08 Dynal As Isolation of nucleic acid
US6914137B2 (en) 1997-12-06 2005-07-05 Dna Research Innovations Limited Isolation of nucleic acids
US6958392B2 (en) * 1998-10-09 2005-10-25 Whatman, Inc. Methods for the isolation of nucleic acids and for quantitative DNA extraction and detection for leukocyte evaluation in blood products
US6383783B1 (en) * 1999-09-21 2002-05-07 3M Innovative Properties Company Nucleic acid isolation by adhering to hydrophobic solid phase and removing with nonionic surfactant
DE10261910A1 (de) * 2002-12-30 2004-07-15 Polymerics Gmbh Adsorbermaterial für Blut-, Blutplasma- und Albuminreinigungsverfahren
US7098253B2 (en) 2004-05-20 2006-08-29 3M Innovative Properties Company Macroporous ion exchange resins
US7683100B2 (en) 2005-12-21 2010-03-23 3M Innovative Properties Company Method of making macroporous cation exchange resins
US7674835B2 (en) 2005-12-21 2010-03-09 3M Innovative Properties Company Method of making macroporous anion exchange resins
US7674836B2 (en) 2006-07-28 2010-03-09 3M Innovative Properties Company Method of making macroporous cation exchange resins
DE102008063003A1 (de) 2008-12-23 2010-06-24 Qiagen Gmbh Nukleinsäureaufreinigungsverfahren
CN108431129B (zh) * 2015-12-25 2021-10-08 株式会社可乐丽 水性乳液和使用其得到的粘接剂

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0214909A1 (fr) * 1985-09-02 1987-03-18 Plant Genetic Systems N.V. Moyens et procedes de transfert de proteines et/ou d'acides nucleiques sur une surface receptrice supportee
DE3935098C2 (de) * 1989-10-21 1995-05-24 Macherey Nagel & Co Chem Chromatographisches Trägermaterial sowie seine Verwendung in einem Verfahren zur chromatographischen Trennung von Nukleinsäuren
DE69317154T2 (de) * 1992-05-29 1998-11-12 Rohm & Haas Verfahren zur Herstellung vernetzter Copolymere von Methacrylsäureanhydrid
US5804684A (en) * 1995-08-24 1998-09-08 The Theobald Smith Research Institute, Inc. Method for isolating nucleic acids
CA2222192A1 (fr) * 1996-03-20 1997-09-25 Bio Merieux Isolement de l'acide nucleique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0049031A2 *

Also Published As

Publication number Publication date
JP2002537306A (ja) 2002-11-05
WO2000049031A3 (fr) 2001-04-12
AU2547100A (en) 2000-09-04
WO2000049031A2 (fr) 2000-08-24
DE19907023A1 (de) 2000-08-24
CA2362979A1 (fr) 2000-08-24

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