EP1904633A1 - Procédé de purification de l'adn / arn de protéines et de résidus cellulaires dans du lysat cellulaire - Google Patents

Procédé de purification de l'adn / arn de protéines et de résidus cellulaires dans du lysat cellulaire

Info

Publication number
EP1904633A1
EP1904633A1 EP05819710A EP05819710A EP1904633A1 EP 1904633 A1 EP1904633 A1 EP 1904633A1 EP 05819710 A EP05819710 A EP 05819710A EP 05819710 A EP05819710 A EP 05819710A EP 1904633 A1 EP1904633 A1 EP 1904633A1
Authority
EP
European Patent Office
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.)
Withdrawn
Application number
EP05819710A
Other languages
German (de)
English (en)
Inventor
Vitali Dshemelinski
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.)
Dshemelinski Vitali
Original Assignee
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
Publication of EP1904633A1 publication Critical patent/EP1904633A1/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Withdrawn legal-status Critical Current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
    • 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 4 Selective flocculation of proteins and cellular residues without involvement of the DNA / RNA with task 3 and application of the composition from the polymer with negatively charged groups (Polly (acrylamide-co-sodium acrylate) with LD: 50-60 mol% ) and polymers with negatively charged and hydrophobic groups (polly (acrylamide-co-sodium acrylate) with LD: 2-4 mol%) through the takeover of proteins and cellular residues mostly from, acting as mediators, ionic (sodium lauryl sulfate) and non-ionic (Triton x-100) surfactants.
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (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.
EP05819710A 2005-02-07 2005-12-22 Procédé de purification de l'adn / arn de protéines et de résidus cellulaires dans du lysat cellulaire Withdrawn EP1904633A1 (fr)

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
PCT/AT2005/000519 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

Publications (1)

Publication Number Publication Date
EP1904633A1 true EP1904633A1 (fr) 2008-04-02

Family

ID=35985350

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05819710A Withdrawn 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

Country Status (3)

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

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2919288A1 (fr) * 2013-08-23 2015-02-26 Boehringer Ingelheim Rcv Gmbh & Co Kg Microparticules pour la rupture cellulaire et/ou la recuperation de biomolecules
FR3038616B1 (fr) * 2015-07-06 2020-11-06 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

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4333805A1 (de) * 1993-08-24 1995-03-02 Tosoh Corp Verfahren zur Extraktion von Nucleinsäuren und Verfahren zum Nachweis spezifizierter Nucleinsäuresequenzen
WO2002004555A1 (fr) * 2000-07-11 2002-01-17 Bayer Aktiengesellschaft Polymerisats en perles superparamagnetiques

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5128247A (en) * 1989-08-14 1992-07-07 Board Of Regents, The University Of Texas System Methods for isolation of nucleic acids from eukaryotic and prokaryotic sources
US5047511A (en) * 1989-08-28 1991-09-10 Pitman-Moore, Inc. Method for recovering recombinant proteins
CN1089727C (zh) * 1997-04-11 2002-08-28 广州市环境保护科学研究所 阳离子/两性接枝型聚丙烯酰胺絮凝剂的制备方法
SE0202074D0 (sv) * 2002-06-28 2002-07-02 Amersham Biosciences Ab Isolation of nucleic acids
EP1462519A1 (fr) * 2003-03-24 2004-09-29 Boehringer Ingelheim Austria GmbH Méthodes et appareilles pour la production de molécules biologiques
CA2567324C (fr) * 2003-05-30 2012-01-03 Advisys, Inc. Dispositifs et procedes de production de biomateriaux

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4333805A1 (de) * 1993-08-24 1995-03-02 Tosoh Corp Verfahren zur Extraktion von Nucleinsäuren und Verfahren zum Nachweis spezifizierter Nucleinsäuresequenzen
WO2002004555A1 (fr) * 2000-07-11 2002-01-17 Bayer Aktiengesellschaft Polymerisats en perles superparamagnetiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO2006081597A1 *

Also Published As

Publication number Publication date
WO2006081597A1 (fr) 2006-08-10
AT501380B1 (de) 2007-11-15
AT501380A1 (de) 2006-08-15

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