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WO2006081597A1 - Procede de purification de l'adn / arn de proteines et de residus cellulaires dans du lysat cellulaire - Google Patents

Procede de purification de l'adn / arn de proteines et de residus cellulaires dans du lysat cellulaire Download PDF

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Publication number
WO2006081597A1
WO2006081597A1 PCT/AT2005/000519 AT2005000519W WO2006081597A1 WO 2006081597 A1 WO2006081597 A1 WO 2006081597A1 AT 2005000519 W AT2005000519 W AT 2005000519W WO 2006081597 A1 WO2006081597 A1 WO 2006081597A1
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WO
WIPO (PCT)
Prior art keywords
mol
polly
proteins
acrylate
acrylamide
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
PCT/AT2005/000519
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German (de)
English (en)
Inventor
Vitali Dshemelinski
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Individual
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Individual
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 Individual filed Critical Individual
Priority to EP05819710A priority Critical patent/EP1904633A1/fr
Publication of WO2006081597A1 publication Critical patent/WO2006081597A1/fr
Anticipated expiration legal-status Critical
Ceased 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
    • 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
    • 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 invention relates to a method for purifying the DNA / RNA from proteins and cellular residues in the cell lysate.
  • Process I Use of organic solvents.
  • Phenol / chloroform cleaves DNA / RNA; 2. Time-consuming process with three stages of phenol / chloroform extraction and separation of the solution phases; 3. Incomplete cleaning and losses when the solution phases are separated; 4. Required stage of ethanol precipitation of the DNA / RNA due to residues of phenol and / or chloroform in the solution; 5. Phenol and chloroform are harmful carcinogenic chemicals.
  • Method II Use of DNA-binding substances. Different surfaces with positive charges are used: 1. Silicate gel; 2. Clay sand, silica gel; 3rd anion column. Disadvantages: 1. Impure DNA / RNA associated with proteins is precipitated or positioned on the surface of the cell lysate and this causes rupture, cleavage and cross-connections on the DNA / RNA strand;
  • DNA / RNA is precipitated with salts and proteins / residues remain in solution or proteins / residues are precipitated with salts and DNA / RNA remains in solution.
  • DNA / RNA precipitation DNA is impure because proteins are not completely separated from DNA and the precipitation conditions of DNA / RNA and proteins / residues overlap.
  • Disadvantages of protein / residue precipitation DNA is impure because proteins are not completely separated from DNA and the yield is small because precipitation operations of proteins / residue and DNA / RNA overlap.
  • Task 1 Binding DNA / RNA in solution by low pH (3, o ⁇ pH ⁇ 5, o).
  • Deoxyribonucleic acid and ribonucleic acid with recognized names DNA and RNA are long chains and consist of nucleosides, which are linked via their sugars between the 5 '- and 3'-hydroxyl groups by a phosphodiester compound.
  • DNA / RNA Polyanions due to negatively charged phosphorus groups.
  • RNA also has polar hydroxyl groups.
  • Task 2 Unfolding proteins up to the primary structure using the composition of the ionic (sodium lauryl sulfate) and non-ionic (Triton x-100) surfactants in the buffer with low pH (3, o ⁇ pH ⁇ 5, o) and SS bridging substance (2- Mercaptoethanol).
  • ionic sodium lauryl sulfate
  • Triton x-100 non-ionic surfactants in the buffer with low pH (3, o ⁇ pH ⁇ 5, o) and SS bridging substance (2- Mercaptoethanol).
  • Proteins are polypeptide chains with negative, positively charged and hydrophobic groups, on average approx. 1000 amino acids long. Four stages of protein folding are known: primary structure, secondary structure, tertiary structure and quaternary structure. Task 3: Separation of DNA and solubilization of proteins in solution by: protons, at low pH (3, o ⁇ pH ⁇ 5, o), negatively charged and hydrophobic groups of the composition from the ionic (sodium lauryl sulfate) and non-ionic (Triton x- 100) surfactants. Protein hystones are generally positively charged, particularly at binding sites, and form so-called nucleosomes with the DNA, with the proportion of approximately 100% of proteins permanently connected to the DNA.
  • Hystones have from 200 to 280 amino acids and are positively charged at low pH, above a certain limit. DNA / RNA is less negatively charged at low pH.
  • Task 5 Intensification of the process and formation of large, heavy flakes through the use of microparticles from the three-dimensional polymer matrix with negatively charged, hydrophobic groups (Polly (acrylamide-co-N'N'-methylenebisacrylamide-co-sodium-acrylate) with LD : from 2 to 4 mol%) in the second and polycations (PoUy (aci ⁇ amid-co-N, N, N, -trimetylammoi ⁇ um- ethyl acrylate) chloride with LD: 50-60 mol%) in the third last step. Polycations now only bind very strongly negatively charged flakes to even larger conglomerates. 4. Solution of the task.
  • the object is achieved in that cleaning is carried out by means of controlled selective flocculation of proteins and cellular residues by the application of the composition from the polymers with negatively, positively charged and hydrophobic groups ((poly (acrylamide-co-sodium-acrylate) with LD: 50 -60 mol%), (Polly (acrilamid-co- N, N, N, -trimetylammonium ethyl acrylate) chloride with LD: 50-60 mol%) and (Polly (acrylamide-co-sodium acrylate) with LD: 2-4 mol%)), microparticles from the three-dimensional polymer matrix with negatively charged and hydrophobic groups (Polly (acrylamide-co-N'N'-methylenebisacrylamide-co-sodium-acrylate) with LD: from 2 to 4 mol%), the composition of the ionic (sodium lauryl sulfate) and nonionic (Triton X-ioo) surfactants and SS bridging substance
  • composition of the polyanions, polymers with negatively charged and hydrophobic groups ((Polly (acrylamide-co-sodium-acrylate) with LD: 50-60 mol%) and (Polly (acrylamide-co-sodium-acrylate) with LD: 2-4 mol%)), microparticles from the three-dimensional polymer matrix with negatively charged and hydrophobic groups (Polly (acrylamide-co-N'N'-methylenebisacrylamide-co-sodium acrylate) with LD: from 2 to 4 mol -%); c.
  • the method is suitable for cell lysates from all possible sources according to the specific precursor for each sample / source: human and animal tissues, cell cultures and blood, bacteria, viruses, yeast and bacterial questions, fungi, plants, foods and plasmid, cosmid and and BAC-DNA in various areas: transgenommics, genotyping, the genetic fingerprint in the evidence of perpetrator and paternity, in diagnoses of inherited diseases, infection diagnosis, breeding analysis and quality control in the food industry for all downstream applications: SNP analysis, Southern blots , PCR, real-time PCR, DNA arrays and sequencing.
  • Polly (acrylamide-co-sodium acrylate): M: 10.7XiO 3 XiO 3 , LD; from 2 to 4 mol% of hydrophobic groups should have at least about 20 acrilamide members without a negatively charged group.

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

Abstract

Procédé de purification de l'ADN / ARN de protéines et de résidus cellulaires dans du lysat cellulaire, selon lequel la purification est effectuée à l'aide de la floculation sélective commandée de protéines et de résidus cellulaires par l'utilisation de la composition constituée de polymères à groupes hydrophobes, chargés positivement et chargés négativement, de microparticules issus de la matrice polymère tridimensionnelle avec des groupes hydrophobes et chargés négativement, de la composition constituée de tensioactifs ioniques et non ioniques et de la substance clivant les ponts SS dans un tampon à pH bas, selon l'ordre d'ajout suivant: 1. tampon à pH bas, composition constituée des tensioactifs ioniques et non ioniques, substance clivant les ponts SS, 2. composition constituée des polymères à groupes chargés négativement et des polymères à groupes hydrophobes et chargés négativement, microparticules issus de la matrice polymère tridimensionnelle à groupes hydrophobes et chargés négativement, 3. polycations, ledit mélange étant ensuite centrifugé.
PCT/AT2005/000519 2005-02-07 2005-12-22 Procede de purification de l'adn / arn de proteines et de residus cellulaires dans du lysat cellulaire Ceased WO2006081597A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP05819710A EP1904633A1 (fr) 2005-02-07 2005-12-22 Procédé de purification de l'adn / arn de protéines et de résidus cellulaires dans du lysat cellulaire

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AT1952005A AT501380B1 (de) 2005-02-07 2005-02-07 Verfahren zur reinigung der dna/rna von proteinen und zellulären reststoffen im zellenlysat
ATA195/2005 2005-02-07

Publications (1)

Publication Number Publication Date
WO2006081597A1 true WO2006081597A1 (fr) 2006-08-10

Family

ID=35985350

Family Applications (1)

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PCT/AT2005/000519 Ceased WO2006081597A1 (fr) 2005-02-07 2005-12-22 Procede de purification de l'adn / arn de proteines et de residus cellulaires dans du lysat cellulaire

Country Status (3)

Country Link
EP (1) EP1904633A1 (fr)
AT (1) AT501380B1 (fr)
WO (1) WO2006081597A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017005754A1 (fr) * 2015-07-06 2017-01-12 Gl-Biocontrol Procede de purification et de concentration d'acides nucleiques
CN111518161A (zh) * 2020-06-02 2020-08-11 英文特生物技术(北京)有限公司 一种柱式法从细胞中分离蛋白质的方法
CN111875665A (zh) * 2013-08-23 2020-11-03 贝林格尔·英格海姆Rcv两合公司 用于细胞破碎和/或回收生物分子的微粒
US12193638B2 (en) 2007-04-27 2025-01-14 Intuitive Surgical Operations, Inc. Complex shape steerable tissue visualization and manipulation catheter

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991002740A1 (fr) * 1989-08-14 1991-03-07 Board Of Regents, The University Of Texas System Methodes et compositions pour isoler les acides nucleiques de sources eucaryotiques et procaryotiques
WO1991003486A1 (fr) * 1989-08-28 1991-03-21 Pitman-Moore, Inc. Methode de recuperation de proteines recombinantes a l'aide d'agents de floculation
US5990216A (en) * 1997-04-11 1999-11-23 Guangzhou Institute Of Environmental Protection Sciences Method for manufacturing grafted polyacrylamide flocculant of cationic/ampholytic ions
WO2004003200A1 (fr) * 2002-06-28 2004-01-08 Amersham Biosciences Ab Isolation d'acides nucleiques a l'aide d'un polymere polycationique utilise en tant qu'agent de precipitation
WO2004085643A1 (fr) * 2003-03-24 2004-10-07 Boehringer Ingelheim Austria Gmbh Procedes et dispositifs pour la production de biomolecules
US20050014245A1 (en) * 2003-05-30 2005-01-20 Advisys, Inc. Devices and methods for biomaterial production

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3451667B2 (ja) * 1993-08-24 2003-09-29 東ソー株式会社 核酸抽出及び特定核酸配列の検出方法
DE10033583A1 (de) * 2000-07-11 2002-01-24 Bayer Ag Superparamagnetische Perlpolymerisate

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991002740A1 (fr) * 1989-08-14 1991-03-07 Board Of Regents, The University Of Texas System Methodes et compositions pour isoler les acides nucleiques de sources eucaryotiques et procaryotiques
WO1991003486A1 (fr) * 1989-08-28 1991-03-21 Pitman-Moore, Inc. Methode de recuperation de proteines recombinantes a l'aide d'agents de floculation
US5990216A (en) * 1997-04-11 1999-11-23 Guangzhou Institute Of Environmental Protection Sciences Method for manufacturing grafted polyacrylamide flocculant of cationic/ampholytic ions
WO2004003200A1 (fr) * 2002-06-28 2004-01-08 Amersham Biosciences Ab Isolation d'acides nucleiques a l'aide d'un polymere polycationique utilise en tant qu'agent de precipitation
WO2004085643A1 (fr) * 2003-03-24 2004-10-07 Boehringer Ingelheim Austria Gmbh Procedes et dispositifs pour la production de biomolecules
US20050014245A1 (en) * 2003-05-30 2005-01-20 Advisys, Inc. Devices and methods for biomaterial production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1904633A1 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12193638B2 (en) 2007-04-27 2025-01-14 Intuitive Surgical Operations, Inc. Complex shape steerable tissue visualization and manipulation catheter
CN111875665A (zh) * 2013-08-23 2020-11-03 贝林格尔·英格海姆Rcv两合公司 用于细胞破碎和/或回收生物分子的微粒
WO2017005754A1 (fr) * 2015-07-06 2017-01-12 Gl-Biocontrol Procede de purification et de concentration d'acides nucleiques
FR3038616A1 (fr) * 2015-07-06 2017-01-13 Gl-Biocontrol Procede de purification et de concentration d'acides nucleiques.
CN111518161A (zh) * 2020-06-02 2020-08-11 英文特生物技术(北京)有限公司 一种柱式法从细胞中分离蛋白质的方法

Also Published As

Publication number Publication date
AT501380A1 (de) 2006-08-15
EP1904633A1 (fr) 2008-04-02
AT501380B1 (de) 2007-11-15

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