+

WO2000040697A9 - Procedes et compositions permettant d'isoler des molecules d'acide nucleique - Google Patents

Procedes et compositions permettant d'isoler des molecules d'acide nucleique

Info

Publication number
WO2000040697A9
WO2000040697A9 PCT/US2000/000170 US0000170W WO0040697A9 WO 2000040697 A9 WO2000040697 A9 WO 2000040697A9 US 0000170 W US0000170 W US 0000170W WO 0040697 A9 WO0040697 A9 WO 0040697A9
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid molecules
matrix
molecular weight
cells
Prior art date
Application number
PCT/US2000/000170
Other languages
English (en)
Other versions
WO2000040697A1 (fr
Inventor
Robert W Blakesley
Original Assignee
Life Technologies Inc
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 Life Technologies Inc filed Critical Life Technologies Inc
Priority to EP00904210A priority Critical patent/EP1141234A4/fr
Priority to AU25998/00A priority patent/AU2599800A/en
Priority to JP2000592395A priority patent/JP2002534080A/ja
Publication of WO2000040697A1 publication Critical patent/WO2000040697A1/fr
Publication of WO2000040697A9 publication Critical patent/WO2000040697A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • 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
    • C12N15/101—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 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Definitions

  • the present invention is in the fields of molecular biology and genetics.
  • the invention relates generally to compositions, methods and kits for use in isolating nucleic acid molecules. More specifically, the invention relates to such compositions, methods and kits that are useful in the isolation of small molecular weight nucleic acid molecules (e.g.. vectors, plasmids. and the like) from cells via lysis and one or more additional isolation steps, such as one or more chromatography steps.
  • the compositions, methods and kits of the invention are suitable for isolating a variety of forms of nucleic acid molecules from cells.
  • plasmid DNA isolation from bacterial cells is based on alkaline lysis, followed by batch chromatography.
  • lysis procedure Birnboim. H. and Doly. J. ( 1979) . ucleic Acids Res. " . 1513) cells are completely ruptured, released proteins and nucleic acids are denatured, then plasmid DNA is preferentially renatured. Precipitated denatured proteins and chromosomal DNA are then separated from soluble plasmid DNA.
  • plasmid DNA is further purified from residual contaminating proteins, lipids and nucleic acids by selective binding and release from a chromatography matrix.
  • the mat ⁇ x may be any porous matrix that traps or binds high molecular weight and/or does not substantialK bind or trap low molecular weight nucleic acid molecules
  • matrix ma> include but is not limited to a polyester matrix, a polyolefin matrix, a scintered poK ethylene matrix, a nitrocellulose matrix, a cellulose acetate matrix, a cellulose mat ⁇ x.
  • a porous ceramic matrix a silica matrix, a polysaccha ⁇ de matrix (Sepharose. agarose. Sephadex. etc ).
  • a poK mer matrix (Sephacry 1. T ⁇ sacry 1. Toyopearl. Bio-Gel.
  • the invention combines such size separation chromatography/filtration with cell lysis/disruption (preferably such lysis/disruption is done when or approximately when the cellular source comes in contact with or after the cellular source is in contact with the chromatography/filtration matrix).
  • the composition or compound that disrupts the cellular membrane or cell wall integrity may comprise one or more detergents.
  • detergents such as sodium dodecylsulfate (SDS) or Sarkosyl. Triton X-100.
  • NP-40. deoxycholate or Brij 35 one or more chaeotropic agents such as sodium iodide, sodium perchlorate or guanidine or a salt thereof: one or more enzymes such as zymolyase. lyticase. lysozyme or lysostaphin: one or more inorganic salts such as sodium chloride, potassium chloride, or lithium chloride: one or more acids and/or bases or buffering agents (e.g..).
  • SDS sodium dodecylsulfate
  • Sarkosyl Triton X-100.
  • NP-40. deoxycholate or Brij 35 one or more chaeotropic agents such as sodium iodide, sodium perchlorate or guanidine or
  • the nucleic acid molecules of interest may be removed from the matrix by elution with an aqueous solution, such as a buffered salt solution or elution buffer.
  • an aqueous solution such as a buffered salt solution or elution buffer.
  • the unwanted molecules e.g.. chromosomal or genomic DNA
  • Such elution or removal of the desired nucleic acid molecules may be facilitated by centrifugation. gravity, vacuum. pressure, etc.. which provides flow of the desired nucleic acid sample from the matrix.
  • the isolated nucleic acid molecules of interest mav then be further purified by standard nucleic acid purification techniques and/or further manipulated by standard molecular biology techniques such as sequencing, amplification, endonuclease digestion (e.g.. restriction enzyme digestion), nucleic acid synthesis, transformation, transfection. and the like.
  • the methods according to the invention are suitable for isolation of low molecular weight nucleic acid molecules from any cell or cellular source, including bacterial cells (particularly Escherichia coli cells), yeast cells, fungal cells, animal cells (particularly insect cells, and mammalian cells including human cells, CHO cells. VERO cells. Bowes melanoma cells.
  • the invention also relates to isolated nucleic acid molecules produced by the methods of the invention, which are preferably low molecular weight nucleic acid molecules, such as the extrachromosomal nucleic acid molecules described herein and particularly plasmids and vectors.
  • isolated nucleic acid molecules produced by the methods of the invention, which are preferably low molecular weight nucleic acid molecules, such as the extrachromosomal nucleic acid molecules described herein and particularly plasmids and vectors.
  • the invention also relates to vectors
  • nucleic acid source any cell, tissues, organs, populations of cells, etc can be used as a nucleic acid source Preferably .
  • nucleic acid sources are used to isolate any giv en plasmid. v ector. or other extragenomic or extrachromosomal structure
  • a population of different v ectors for example, a cDNA hbran or genomic library contained by the cell population or culture
  • the inv netion allows isolation of such population of vectors/cDNA library /genomic library
  • Amplification refers to any in vitro method for increasing the number of copies of a nucleotide sequence with the use of a polymerase. Nucleic acid amplification results in the inco ⁇ oration of nucleotides into a nucleic acid (e.g.. DNA) molecule or primer thereby forming a new nucleic acid molecule complementary to the nucleic acid template. The formed nucleic acid molecule and its template can be used as templates to synthesize additional nucleic acid molecules. As used herein, one amplification reaction may consist of many rounds of nucleic acid synthesis. Amplification reactions include, for example, polymerase chain reactions (PCR). One PCR reaction may consist of 5 to 100 "cycles" of denaturation and synthesis of a nucleic acid molecule.
  • PCR polymerase chain reactions
  • a host or host cell may serve as the cellular source for the desired nucleic acid molecule to be isolated.
  • a vector is a nucleic acid molecule (preferably DNA) capable of replicating autonomously in a host cell. Such vectors may also be characterized by having a small number of endonuclease restriction sites at which such sequences may be cut without loss of an essential biological function and into which nucleic acid molecules may be spliced to bring about its replication and cloning. Examples include plasmids. autonomously replicating sequences ( ARS). centromeres, cosmids and phagemids. Vectors can further provide primer sites, e.g.. for PCR.
  • the vector can further contain one or more selectable markers suitable for use in the identification of cells transformed or transfected with the vector, such as kanamycin. tetracycline. amplicillin. etc.
  • vectors which have compatible replicons for use in combination in a single host e.g.. pACYC 184 and pBR322
  • eukaryotic episomal replication vectors e.g.. pCDM8
  • the vectors contemplated by the invention include vectors containing inserted or additional nucleic acid fragments or sequences (e.g.. recombinant vectors) as well as derivatives or variants of any of the vectors described herein.
  • isolated low molecular weight nucleic acid molecule or "isolated vector” refers to a low molecular weight nucleic acid molecule preparation or vector preparation which contains no more than about 50%. 45%. 40%. 35%. 30%. 25%. 20%. 15%. 10%. and 7%. preferably no more than 5%. 2.5%. and 2%. and most preferably less than 1 %. 0.5%. and 0.1 % (percentages by weight) of high molecular weight nucleic acid molecules (e.g.. chromosomal/genomic DNA).
  • a solution comprising an isolated nucleic acid molecule may comprise one or more buffer salts and/or a solvents, e.g.. water or an organic solvent such as acetone, ethanol, methanol. and the like, and yet the nucleic acid molecule may still be considered an "isolated" nucleic acid molecule with respect to its starting materials.
  • the methods, compositions and kits of the inv ention are suitable for isolation of low molecular weight nucleic acid molecules from any cellular source, including a v a ⁇ ety of cells, tissues, organs or organisms, hich may be natural or which may be obtained through any number of commercial sources (including American Ty pe Culture Collection (ATCC). Rockville. Maryland. Jackson
  • Cells that may be used as cellular nucleic acid sources may be prokaryotic (bacterial, including members of the genera Eschenchia (particularly E coli). Serratia. Salmonella. Staphylococcus, Streptococcus. Clostridium. Chlamydia. Neisseria. Treponema. Mycoplasma,
  • Any virus may also be used as a cellular source of nucleic acid molecules in accordance with the invention
  • eukaryotic including fungi or yeasts, plants, protozoans and other parasites, and animals including humans and other mammals
  • Any virus may also be used as a cellular source of nucleic acid molecules in accordance with the invention
  • mammalian tissues or organs such as those derived from brain, kidne . hv er.
  • pancreas blood, bone marrow, muscle, nervous, skin, genitourinary , circulatory , ly mphoid.
  • gastrointestinal and connective tissue sources as well as those deriv ed from a mammalian (including human) embry o or fetus
  • These cells, tissues and organs may be normal, transformed, or established cell lines, or they mav be pathological such as those inv olv ed in infectious diseases (caused by bacteria, fungi or v east, v lruses (including ⁇ IDS) or parasites), in genetic or biochemical pathologies (e g . cvstic fibrosis. hemophilia.
  • the methods of the invention are useful in the isolation of plasmid or v ector DNA from bacterial cells
  • Other cells, tissues, v iruses. organs and organisms that will be familiar to one of ordinary skill in the art may also be used as sources of nucleic acid molecules for the preparation of isolated nucleic acid molecules according to the present invention
  • the invention relates to methods for isolating nucleic acid molecules, particularly small molecular weight nucleic acid molecules such as plasmids. vectors, organellar nucleic acid molecules, and the like
  • Methods according to this aspect of the invention may comprise one or more steps which result in the isolation of one or more nucleic acid molecules or populations of nucleic acid molecules (e g . a cDNA library) from the natural environment in which the nucleic acid molecules are found
  • the methods of the invention may comprise
  • chromosomal or genomic nucleic acid molecules contained by the cellular source This separation may be facilitated by any means for moving the desired nucleic acid molecules through the mat ⁇ x including cent ⁇ fugation, v acuum. grav lty pressure and the like
  • mutanolysin or lysostaphin at a concentration of about 0.1 to 5 mg ml; one or more inorganic salts such as sodium chloride, potassium chloride, magnesium chloride, lithium chloride, or praseodymium chloride at a concentration of about 1 mM to 5M: one or more organic solvents such as toluene, phenol, butanol. isopropyl alcohol, isoamyl alcohol, ethanol. an ether (e.g.. diethyl ether, dimethyl ether, or ethylmethyl ether), or chloroform at a concentration of 25 to 60% (v/v); or any other compound which disrupts the integrity of (i.e..
  • kits of the invention may further comprise one or more additional components or reagents that may be useful in further processing, analysis, or use of the nucleic acid molecules isolated or purified according to the invention, for example components or reagents useful in nucleic acid amplification, sequencing. cloning, transfection. transcription, translation, and the like.
  • additional components or reagents that may be useful in further processing, analysis, or use of the nucleic acid molecules isolated or purified according to the invention, for example components or reagents useful in nucleic acid amplification, sequencing. cloning, transfection. transcription, translation, and the like.
  • Such reagents or components may. for example, include one or more restriction enzymes, one or more polypeptides having nucleic acid polymerase activity, one or more polypeptides having reverse transcriptase activity, one or more cells competent for transformation, one or more transfection reagents (e.g., lipids) and other reagents that will be familiar to one of ordinary skill in the art.
  • nucleic acid molecules that are isolated by the compositions, methods and kits of the present invention may be further characterized or manipulated, for example by cloning, sequencing, amplification, nucleic acid synthesis, endonuclease digestion and the like.

Landscapes

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

Abstract

La présente invention concerne, de manière générale, des compositions, des procédés et des kits destinés à être utilisés pour isoler des molécules d'acide nucléique. De manière plus spécifique, cette invention concerne des compositions, des procédés et des kits qui sont utiles pour isoler des molécules d'acide nucléique à partir de cellules (telles que par exemple des cellules bactériennes, des cellules animales, des cellules de champignons, des virus, des cellules de levure ou des cellules de plantes) au moyen d'une étape de lyse et d'au moins une étape additionnelle d'isolement, telle qu'au moins une étape d'analyse chromatographique. De manière plus spécifique, cette invention concerne des compositions, des procédés et des kits dans lesquels les molécules d'acide nucléique sont isolées au moyen d'une matrice mixte de lyse/chromatographie pouvant elle-même comprendre un ou plusieurs supports (par exemple en polyoléfine, en polyéthylène fritté, en nitrocellulose, en polypropylène, en polycarbonate, en acétate de cellulose, en silice et autres) qui ont été traités avec un ou plusieurs réactifs de lyse chimique (par exemple un ou plusieurs détergents, un ou plusieurs chaéotropes, une ou plusieurs enzymes et autres) ou bien qui ont été associés à ces derniers. Les compositions, les procédés et les kits de l'invention sont adaptés pour isoler diverses formes de molécules d'acide nucléique à partir de cellules, comprenant sans limitation aucune, des plasmides, des vecteurs, de l'ADN, de l'ADNc, de l'ARN, de l'ADN mitochondrial, de l'ADN chloroplastique, et autre, ces derniers pouvant être de type monocaténaire ou bicaténaire, linéaire ou circulaire. Les compositions, les procédés et les kits selon l'invention sont particulièrement adaptés pour isoler rapidement un plasmide soluble ou un ADN vecteur présent dans des cellules bactériennes.
PCT/US2000/000170 1999-01-06 2000-01-06 Procedes et compositions permettant d'isoler des molecules d'acide nucleique WO2000040697A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP00904210A EP1141234A4 (fr) 1999-01-06 2000-01-06 Procedes et compositions permettant d'isoler des molecules d'acide nucleique
AU25998/00A AU2599800A (en) 1999-01-06 2000-01-06 Methods and compositions for isolation of nucleic acid molecules
JP2000592395A JP2002534080A (ja) 1999-01-06 2000-01-06 核酸分子の単離のための方法および組成物

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11486599P 1999-01-06 1999-01-06
US60/114,865 1999-01-06

Publications (2)

Publication Number Publication Date
WO2000040697A1 WO2000040697A1 (fr) 2000-07-13
WO2000040697A9 true WO2000040697A9 (fr) 2001-08-02

Family

ID=22357869

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/000170 WO2000040697A1 (fr) 1999-01-06 2000-01-06 Procedes et compositions permettant d'isoler des molecules d'acide nucleique

Country Status (4)

Country Link
EP (1) EP1141234A4 (fr)
JP (1) JP2002534080A (fr)
AU (1) AU2599800A (fr)
WO (1) WO2000040697A1 (fr)

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5108399A (en) * 1998-07-16 2000-02-07 Invitrogen Corporation Archiving of vectors
US6750059B1 (en) 1998-07-16 2004-06-15 Whatman, Inc. Archiving of vectors
AU4312201A (en) * 1999-12-10 2001-06-18 Genespan Corporation Isolation and purification of nucleic acids
CA2415713A1 (fr) * 2000-07-13 2002-01-24 Invitrogen Corporation Methodes et compositions d'extraction et d'isolation rapides de proteines et de peptides au moyen d'une matrice de lyse
WO2002008368A1 (fr) * 2000-07-21 2002-01-31 Cargill, Incorporated Procede d'elimination de contaminants presents dans de l'huile vegetale et dans de la lecithine
KR20020028387A (ko) * 2000-10-09 2002-04-17 박제철 대량의 동물조직으로부터 미토콘드리아 디.엔.에이의순수분리 방법
DE10113815A1 (de) * 2001-03-21 2002-10-02 Eppendorf Ag Verfahren zur Isolierung von Plasmiden oder Proteinen aus suspendierten Bakterien- oder Hefezellen
US20050032105A1 (en) * 2001-10-12 2005-02-10 Bair Robert Jackson Compositions and methods for using a solid support to purify DNA
GB0127803D0 (en) * 2001-11-20 2002-01-09 Glaxo Group Ltd Processing nucleic acid
EP1388588B1 (fr) * 2002-07-29 2008-09-17 JSR Corporation Méthode de séparation d'acide nucléiques et solutions pour les extraire
US7214484B2 (en) 2002-12-17 2007-05-08 Sigma-Aldrich Co. Compositions and methods for nucleic acid extraction from biological samples
CA2586532C (fr) * 2004-11-05 2014-03-25 Qiagen North American Holdings, Inc. Compositions et procedes pour purifier des acides nucleiques provenant de reactifs de stabilisation
JP4699868B2 (ja) 2005-11-04 2011-06-15 株式会社日立ハイテクノロジーズ 核酸精製方法及び核酸精製器具
US7608399B2 (en) 2006-06-26 2009-10-27 Blood Cell Storage, Inc. Device and method for extraction and analysis of nucleic acids from biological samples
US8163535B2 (en) 2006-06-26 2012-04-24 Blood Cell Storage, Inc. Devices and processes for nucleic acid extraction
US7759112B2 (en) 2007-10-31 2010-07-20 Akonni Biosystems, Inc. Apparatus, system, and method for purifying nucleic acids
JP5328547B2 (ja) * 2009-07-31 2013-10-30 一般財団法人電力中央研究所 有機物の抽出方法、有機物の製造方法、有機物抽出装置組立体、湿潤材料の処理方法
JP6161205B2 (ja) * 2010-07-14 2017-07-12 キアゲン ゲーエムベーハー 生体分子の単離および／または精製のためのデバイス
WO2012159063A2 (fr) 2011-05-19 2012-11-22 Blood Cell Strorage, Inc. Dispositif fluidique d'écoulement par gravité pour extraction d'acide nucléique
CN108103057B (zh) * 2012-08-28 2021-09-03 阿科尼生物系统公司 用于纯化核酸的方法和试剂盒
TR201606310A2 (tr) 2016-05-12 2017-11-21 Akdeniz Ueniversitesi Genomik dna izolasyon yöntemi ve kiti.
CN111867728A (zh) * 2018-01-08 2020-10-30 摇篮基因组公司 分离核酸的方法和试剂盒
US11591591B2 (en) 2019-08-21 2023-02-28 New England Biolabs, Inc. Isolation of high molecular weight DNA using beads

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3639949A1 (de) * 1986-11-22 1988-06-09 Diagen Inst Molekularbio Verfahren zur trennung von langkettigen nukleinsaeuren
CA2319691C (fr) * 1998-02-02 2012-12-18 Gentra Systems, Inc. Reactifs d'elution, procedes et trousses permettant d'isoler un adn
JP5300112B2 (ja) * 1998-02-02 2013-09-25 キアジエン・ノース・アメリカン・ホールデイングス・インコーポレーテツド Ｄｎａを単離するための溶解マトリックスを使用するための組成物および方法

Also Published As

Publication number Publication date
WO2000040697A1 (fr) 2000-07-13
AU2599800A (en) 2000-07-24
EP1141234A1 (fr) 2001-10-10
JP2002534080A (ja) 2002-10-15
EP1141234A4 (fr) 2004-05-19

Similar Documents

Publication Publication Date Title
WO2000040697A9 (fr) Procedes et compositions permettant d'isoler des molecules d'acide nucleique
US20020127587A1 (en) Methods and compositions for isolation of biological macromolecules
US6383393B1 (en) Chromatographic purification and separation process for mixtures of nucleic acids
JP3115324B2 (ja) 核酸を単離する装置及び方法
US7264927B2 (en) Isolation and purification of nucleic acids
US5660984A (en) DNA isolating apparatus comprising a non-porous DNA binding, anion exchange resin and methods of use thereof
US6180778B1 (en) Process for the separation of double-stranded/single-stranded nucleic acid structures
US20020012982A1 (en) Methods and compositions for rapid protein and peptide extraction and isolation using a lysis matrix
EP2137309B1 (fr) Procédés de séparation de molécules biologiques utilisant du sulfolane
AU2002333673A1 (en) Isolation and purification of nucleic acids
US12247198B2 (en) Nucleic acid synthesis and purification device, use thereof, and nucleic acid synthesis and purification method
HUP0301869A2 (hu) DNS-tisztítási eljárások és a tisztított DNS
EP1719816A1 (fr) Mecanisme de separation et de purification d 'adn et ainsi de suite
EP0991456A1 (fr) Dispositif et procede de concentration et purification de molecules biologiques en une etape
US20080146789A1 (en) Methods for the separation of biological molecules using dioxolane
CA2394746C (fr) Materiau chromatographique et procedes d'utilisation d'un tel materiau
US20070275920A1 (en) Method for Chromatographic Separation of a Nucleic Acid Mixture
KR101380909B1 (ko) 핵산 정제용 흡착제 및 그 흡착제를 이용한 정제방법
EP2271756A1 (fr) Procédé de séparation d'acides nucléiques bicaténaires et monocaténaires
JP2012527990A (ja) 膜を保護する方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase in:

Ref country code: JP

Ref document number: 2000 592395

Kind code of ref document: A

Format of ref document f/p: F

AK Designated states

Kind code of ref document: C2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1/3-3/3, DRAWINGS, REPLACED BY NEW PAGES 1/3-3/3; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

WWE Wipo information: entry into national phase

Ref document number: 2000904210

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000904210

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载