EP1904633A1 - Method for the purification of dna/rna of proteins and cellular residual substances in cell lysate - Google Patents

Method for the purification of dna/rna of proteins and cellular residual substances in cell lysate

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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
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mol
polly
proteins
acrylate
acrylamide
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Vitali Dshemelinski
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Dshemelinski Vitali
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    • 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.

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Abstract

The invention relates to a method for the purification of DNA/RNA of proteins and cellular residual substances in cell lysate. In order to create an advantageous method, purification is carried out by means of controlled selective flocculation of proteins and cellular residual substances by using a composition consisting of polymers with negatively, positively charged and gydrophobic groups, microparticles of a three-dimensional polymer matrix with negatively charged and gydrophobic groups, a composition consisting of ionic and non-ionic surfactants and a substance cleaving SS bridges in a buffer with a low pH in the following addition sequence: 1) a buffer with a low pH, the composition consisting of ionic and non-ionic surfactants and a substance cleaving SS bridges; 2) the composition consisting of polymers with negatively charged groups, polymers with negatively charged and gydrophobic groups, polymers with negatively charged and gydrophobic groups, microparticles of the three-dimensional polymer matrix with negatively charged and gydrophobic groups; 3) polycations, with subsequent centrifuging. .

Description

Verfahren zur Reinigung der DNA/RNA von Proteinen und zellulären Reststoffen im Zellenlysat.Process for the purification of DNA / RNA from proteins and cellular residues in the cell lysate.
Beschreibung.Description.
l.Beschreibungseinleitung.l. Introduction to the description.
Die Erfindung betrifft ein Verfahren zur Reinigung der DNA/RNA von Proteinen und zellulären Reststoffen im Zellenlysat.The invention relates to a method for purifying the DNA / RNA from proteins and cellular residues in the cell lysate.
2. Stand der Technik.2. State of the art.
Verfahren I: Anwendung von organischen Lösungsmitteln.Process I: Use of organic solvents.
Es werden zwei Lösungsmittel in bestimmter Reihenfolge angewendet: Phenol, Chloroform. Nachteile: i. Phenol/Chloroform spaltet DNA/RNA; 2. Zeitaufwendige Vorgang mit drei Stufen Phenol/Chloroform Extragierung und Trennung der Lösungsphasen; 3. Nicht vollkommene Reinigung und Verluste bei Trennung der Lösungsphasen; 4. Notwendige Stufe der Ethanolfällung der DNA/RNA wegen Resten von Phenol und/oder Chloroform in der Lösung; 5. Phenol und Chloroform sind gesundheitsschädliche krebserregende Chemikalien. Verfahren II: Anwendung von DNA bindenden Stoffen. Es werden verschiedene Oberflächen mit positiven Ladungen angewendet: 1. Silikatgel; 2. Tonsand, Kieselgel; 3. Anionensäule. Nachteile: 1. Es wird unreine, mit Proteinen verbundene, DNA/RNA aus dem Zellenlysat an Oberfläche gefallt oder positioniert und das verursacht Zerreißung, Spaltung und Kreuz-Verbindungen an dem DNA/RNA-Strang;Two solvents are used in a specific order: phenol, chloroform. Disadvantages: i. 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;
2. Komplexe Waschvorgang in drei Schritten: zwei Stufen Proteinen/Reststoffen- und eine Stufe DNA/RNA-Auswaschen; 3. Bindende Konstruktionen verursachen Mehrkosten. Verfahren III: Fällung.2. Complex washing process in three steps: two stages of protein / residues and one stage of DNA / RNA washing out; 3. Binding structures cause additional costs. Procedure III: Precipitation.
Es wird DNA/RNA mit Salzen gefällt und Proteinen/Reststoffen bleiben im Lösung oder Proteinen/Reststoffen werden mit Salzen gefällt und DNA/RNA bleibt im Lösung. Nachteile der DNA/RNA Fällung: DNA ist unrein, weil Proteinen sind nicht vollkommen abgetrennt von DNA und Fällungsbedingungen von DNA/RNA und Proteinen/Reststoffen sich überschneiden. Nachteile der Proteinen/Reststoffen Fällung: DNA ist unrein, weil Proteinen sind nicht vollkommen abgetrennt von DNA und Ausbeute ist klein weil Fällungsbedienungen von Proteinen/Reststoffen und DNA/RNA sich überschneiden.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. Disadvantages of 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.
3. Aufgabe der Erfindung.3. Object of the invention.
Aufgabe 1: Bindung DNA/RNA im Lösung durch niedrige pH (3,o<pH<5,o). Desoxyribonukleinsäure und Ribonukleinsäure mit anerkannten Namen DNA und RNA sind lange Ketten und bestehen aus Nucleosiden, die über ihre Zucker zwischen den 5 '- und 3'- Hydroxylgruppen durch eine Phosphodiesterverbindung verknüpft sind. DNA/RNA sind Polyanionen durch negativ geladene Phosphorgruppen. RNA hat auch polare Hydroxylgruppen.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. Are DNA / RNA Polyanions due to negatively charged phosphorus groups. RNA also has polar hydroxyl groups.
Aufgabe 2: Entfaltung Proteinen bis Prünärstruktur durch Anwendung der Zusammensetzung aus der ionischen (Natriumlaurylsulfat) und nichtionischen (Triton x-100) Tensiden im Puffer mit niedriger pH (3,o<pH<5,o) und SS Brücken spaltender Substanz (2-Merkaptoethanol).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).
Proteinen sind Polypeptidketten mit negativ, positiv geladenen und gydrophoben Gruppen, durchschnittlich ca. 1000 Aminosäuren lang. Es wird vier Stufen der Zusammenfaltung von Proteinen bekannt: Primärstruktur, Sekundärstruktur, Tertiärstruktur und Quartärstruktur. Aufgabe 3: Trennung von DNA und Solubilisierung von Proteinen im Lösung durch: Protonen, bei niedriger pH (3,o<pH<5,o), negativ geladenen und gydrophoben Gruppen der Zusammensetzung aus der ionischen (Natriumlaurylsulfat) und nichtionischen (Triton x-100) Tensiden. Proteinen-Hystonen sind allgemein, auch besonders an Bindungsstellen, positiv geladen und bilden mit der DNA sog. Nukleosomen mit der Anteil von ca. 100% von Proteinen dauerhaft verbundenen mit der DNA. Verhältnis positiv geladenen Gruppen zu negativ geladenen Gruppen bei diesem Proteinen sind: Hi: 5,4, H2A : 1,4, H2B : 1,7, H3 : 1,8, H4: 2, 5. Hystonen haben von 200 bis 280 Aminosäuren und sind bei niedriger pH, ab bestimmter Grenze, positiv geladen. DNA/RNA ist, bei niedriger pH, weniger negativ geladen. Aufgabe 4: Selektive Flockung von Proteinen und zellulären Reststoffen ohne Involwierung der DNA/RNA mit Aufgabe 3 und Anwendung der Zusammensetzung aus der Polymeren mit negativ geladenen Gruppen (Polly(acrylamid-co-Natrium-acrylat)mit LD: 50-60 mol-%) und Polymeren mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-Natrium- acrylat) mit LD: 2-4 mol-%) durch die Übernahme von Proteinen und zellulären Reststoffen großteils von, fungirenden als Vermittlern, ionischen (Natriumlaurylsulfat) und nichtionischen (Triton x-100) Tensiden. Hystonen, als lange Polypeptidketten, haben bei niedriger pH nur positive und gydrophobe Gruppen offen und bilden mit zugefügten Polymeren die Flocken.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. The ratio of positively charged groups to negatively charged groups in this protein are: Hi: 5.4, H2A: 1.4, H2B: 1.7, H3: 1.8, H4: 2, 5. 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. Hystones, as long polypeptide chains, only have positive and hydrophobic groups open at low pH and form the flakes with added polymers.
Aufgabe 5: Intensivierung des Prozesses und Bildung großen schweren Flocken durch die Anwendung von Mikropartikel aus dem dreidimensionalen Polymer- Matrix mit negativ geladenen, gydrophoben Gruppen (Polly(acrylamid-co-N'N'- methylenbisacrylamid-co-Natrium-acrylat) mit LD: von 2 bis 4 mol-%) im zweiten und Polykationen (PoUy(aciΗamid-co-N,N,N,-trimetylammoiύum- ethylacrylat)-chlorid mit LD: 50-60 mol-%) im drittem letzten Schritt. Polykationen binden jetzt nur sehr stark negativ geladene Flocken zu noch größeren Konglomeraten. 4. Lösung der gestellten Aufgabe. Die Aufgabe wird dadurch gelöst, dass Reinigung wird mittels gesteuerter selektiven Flockung von Proteinen und zellulären Reststoffen durch die Anwendung der Zusammensetzung aus der Polymeren mit negativ, positiv geladenen und gydrophoben Gruppen ((Polly(acrylamid-co-Natrium-acrylat) mit LD: 50-60 mol-%), (Polly(acrilamid-co- N,N,N,-trimetylammonium-ethylacrylat)-chlorid mit LD: 50-60 mol-%) und (Polly(acrylamid-co-Natrium-acrylat) mit LD: 2-4 mol-%)), Mikropartikel aus dem dreidimensionalen Polymer-Matrix mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-N'N'-methylenbisacrylamid-co-Natrium-acrylat) mit LD: von 2 bis 4 mol-%), der Zusammensetzung aus dem ionischen (Natriumlaurylsulfat) und nichtionischem (Triton X-ioo) Tensiden und SS Brücken spaltender Substanz (2-Merkaptoethanol) im Puffer mit niedriger pH (3,o<pH<5,o) in der Zugabereihenfolge: a. Puffer mit niedriger pH (3,o<pH<5,o), ionischer ( Natriumlaurylsulfat) und nichtionischer (Triton x-100) Tensiden, SS Brücken spaltende Substanz (2-Merkaptoethanol); b. Die Zusammensetzung aus der Polyanionen, Polymeren mit negativ geladenen und gydrophoben Gruppen ((Polly(acrylamid-co-Natrium-acrylat) mit LD: 50-60 mol-%) und (Polly(acrylamid-co- Natrium-acrylat) mit LD: 2-4 mol-%)), Mikropartikel aus dem dreidimensionalen Polymer- Matrix mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-N'N'- methylenbisacrylamid-co-Natrium-acrylat) mit LD: von 2 bis 4 mol-%); c. Polykationen (Polly(acrilamid-co-N,N,N,-trimetylammonium-ethylacrylat)-chlorid mit LD: 50-60 mol-%) mit der anschließender Zentrifugierung, durchgeführt. 5. Effekte der Erfindung und Unteransprüche.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 (aciΗamid-co-N, N, N, -trimetylammoiύum- 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 (2-mercaptoethanol) in the buffer with low pH (3, o <pH <5, o) in the order of addition: a. Buffer with low pH (3, o <pH <5, o), ionic (sodium lauryl sulfate) and non-ionic (Triton x-100) surfactants, SS bridging substance (2-mercaptoethanol); b. The 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. Polycations (Polly (acrilamid-co-N, N, N, -trimetylammonium-ethyl acrylate) chloride with LD: 50-60 mol%) with the subsequent centrifugation. 5. Effects of the invention and dependent claims.
1. Reine DNA/RNA durch bessere Trennung von Proteinen und bessere Entfernung von zellulären Reststoffen gelöst durch bestimmte Reihenfolge von Schritten, besonderes Flockungsprozess.1. Pure DNA / RNA through better separation of proteins and better removal of cellular residues solved by certain sequence of steps, special flocculation process.
2. Intakte, hochmolekulare DNA/RNA durch fehlende Operationen mit der.2. Intact, high molecular DNA / RNA due to missing operations with the.
3. Große Ausbeute der DNA/RNA durch ausreichende Trennungsbedienungen bei Zentrifugierung.3. Large yield of DNA / RNA through sufficient separation operations during centrifugation.
4. Minimale Anzahl von Schritten, kurze Behandlungszeiten.4. Minimum number of steps, short treatment times.
5. Verfahren ist geeignet für Zellenlysate aus allen möglichen Quellen nach der speziellen für jede Probe/Quelle Vorstufe: menschliche und tierische Gewebe, Zellkulturen und Blut, Bakterien, Viren, Hefen und Bakteriafagen, Pilzen, Pflanzen, Nahrungsmittel sowie von Plasmid-, Cosmid- und BAC-DNA in verschiedenen Bereichen: Transgenommics, Genotypisierung, der genetische Fingerabdruck im Täter- und Vaterschaftsnachweis, in Diagnosen erblich bedingter Krankheiten, Infektions-Diagnose, die Züchtungsanalyse und Qualitätskontrolle in der Nahrungsmittelindustrie für alle Downstream Applikationen: SNP- Analyse, Southern-Blots, PCR, Real-Time PCR, DNA-Arrays und Seqenzierung.5. 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.
6. Handliche und umweltfreundliche Ablauf durch verzieht auf gesudheitschädliche oder krebserregende Chemikalien. 7. Verfahren ist kostengünstig durch Verwendung von breitverwendbaren preisgünstigen6. Handy and environmentally friendly process due to warping or harmful chemicals. 7. The method is inexpensive through the use of widely used inexpensive
Stoffen. l. Natriumlaurylsulfat: CH3(CH2)iiSO4Na, M: 288,4Fabrics. l. Sodium lauryl sulfate: CH3 (CH2) iiSO4Na, M: 288.4
2. 2-Merkaptoethanol: HSCH2CH2OH, M: 78,12. 2-mercaptoethanol: HSCH2CH2OH, M: 78.1
3. Triton x-100: 4-(C8Hi7)C6H4(OCH2CH2)nOH, n=~io, M:6253. Triton x-100: 4- (C8Hi7) C6H4 (OCH2CH2) nOH, n = ~ io, M: 625
4. Polly(acrylamid-co-Natrium-acrylat): M: io,τxio3xio3, LD: von 50 bis 60 mol-%4. Polly (acrylamide-co-sodium acrylate): M: io, τxio 3 xio 3 , LD: from 50 to 60 mol%
5. Polly(acrylamid-co-Natrium-acrylat): M: 10,7XiO3XiO3, LD; von 2 bis 4 mol-% Gydrophoben Gruppen sollen mindestens ca. 20 Acrilamid-Glieder ohne negativ geladene Gruppe haben.5. 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.
7. Polly(acrilamid-co-N,N,N?-trimetylammonium-ethylacrylat)-chlorid: M: 6,ooxio3xio3, LD: von 50 bis 60 mol-%7. Polly (acrilamide-co-N, N, N ? -Trimetylammonium ethyl acrylate) chloride: M: 6, ooxio 3 xio 3 , LD: from 50 to 60 mol%
8. Polly(acrylamid-co-N'N'-methylenbisacrylamid-co-Natrium-acrylat) mit LD: von 2 bis 4 mol-% 8. Polly (acrylamide-co-N'N'-methylenebisacrylamide-co-sodium acrylate) with LD: from 2 to 4 mol%

Claims

Patentansprüche Claims
1. Verfahren zur Reinigung der DNA/RNA von Proteinen und zellulären Reststoffen im Zellenlysat, dadurch gekennzeichnet, dass Reinigung wird mittels gesteuerter selektiven Flockung von Proteinen und zellulären Reststoffen durch die Anwendung der Zusammensetzung aus der Polymeren mit negativ, positiv geladenen und gydrophoben Gruppen ((PoUy(acrylamid-co-Natrium-acrylat) mit LD: 50-60 mol-%), (Polly(acrilamid-co- N,N,N,-trimetylammonium-ethylacrylat)-chlorid mit LD: 50-60 mol-%) und (Polly(acrylamid-co-Natrium-acrylat) mit LD: 2-4 mol-%)), Mikropartikel aus dem dreidimensionalen Polymer-Matrix mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-N'N'-methylenbisacrylamid-co-Natrium-acrylat) mit LD: von 2 bis 4 mol-%), der Zusammensetzung aus der ionischen (Natriumlaurylsulfat) und nichtionischen (Triton X-100) Tensiden und SS Brücken spaltender Substanz (2-Merkaptoethanol) im Puffer mit niedriger pH (3,o<pH<5,o) in der Zugabereihenfolge: a. Puffer mit niedriger pH (3,o<pH<5,o), ionischen ( Natriumlaurylsulfat) und nichtionischen (Triton X-100) Tensiden, SS Brücken spaltende Substanz (2-Merkaptoethanol); b. Die Zusammensetzung aus der Polyanionen (Polly(acrylamid-co-Natrium-acrylat) mit LD: 50-60 mol-%), Polymeren mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-Natrium-acrylat) mit LD: 2-4 mol-%) , Mikropartikel aus dem dreidimensionalen Polymer-Matrix mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-N'N'-methylenbisacrylamid-co- Natrium-acrylat) mit LD: von 2 bis 4 mol-%); c. Polykationen (Polly(acrilamid-co-N,N,N,- trimetylammonium-ethylacrylat)-chlorid mit LD: 50-60 mol-%) mit der anschließender Zentrifugierung, durchgeführt.1. Process for the purification of the DNA / RNA from proteins and cellular residues in the cell lysate, characterized in that purification 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 (( PoUy (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-100) surfactants and SS bridging substance (2-mercaptoethanol) in the buffer low pH (3, o <pH <5, o) in the order of addition: a. Buffer with low pH (3, o <pH <5, o), ionic (sodium lauryl sulfate) and non-ionic (Triton X-100) surfactants, SS bridging substance (2-mercaptoethanol); b. The composition of the polyanions (Polly (acrylamide-co-sodium-acrylate) with LD: 50-60 mol%), polymers with negatively charged and hydrophobic groups (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. Polycations (Polly (acrilamid-co-N, N, N, - trimetylammonium ethyl acrylate) chloride with LD: 50-60 mol%) with the subsequent centrifugation.
2. Verfahren zur Reinigung der DNA/RNA von Proteinen und zellulären Reststoffen im Zellenlysat, dadurch gekennzeichnet, dass Proteinen und zellulären Reststoffen werden geflockt.2. Process for the purification of the DNA / RNA from proteins and cellular residues in the cell lysate, characterized in that proteins and cellular residues are flocculated.
3. Verfahren zur Reinigung der DNA/RNA von Proteinen und zellulären Reststoffen im Zellenlysat, dadurch gekennzeichnet, dass Flockung von Proteinen und zellulären Reststoffen wird durch Anwendung der Zusammensetzung aus der Polymeren mit negativ, positiv geladenen und gydrophoben Gruppen ((Polly(acrylamid-co-Natrium-acrylat) mit LD: 50-60 mol-%), (Polly(acrilamid-co-N,N,N,-trimetylammonium-ethylacrylat)-chlorid mit LD: 50-60 mol-%) und (Polly(acrylamid-co-Natrium-acrylat) mit LD: 2-4 mol-%)) durchgeführt.3. Process for the purification of the DNA / RNA from proteins and cellular residues in the cell lysate, characterized in that flocculation of proteins and cellular residues is achieved by using the composition of the polymers with negatively, positively charged and hydrophobic groups ((Polly (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%)).
4. Verfahren zur Reinigung der DNA/RNA von Proteinen und zellulären Reststoffen im Zellenlysat, dadurch gekennzeichnet, dass Flockung von Proteinen und zellulären Reststoffen wird durch Anwendung von Mikropartikel aus dem dreidimensionalem Polymer-Matrix mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-N'N'- methylenbisacrylamid-co-Natrium-acrylat) mit LD: von 2 bis 4 mol-96) durchgeführt. 4. Process for the purification of the DNA / RNA from proteins and cellular residues in the cell lysate, characterized in that flocculation of proteins and cellular residues is achieved by using 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-96).
5. Verfahren zur Reinigung der DNA/RNA von Proteinen und zelluläre Reststoffen im Zellenlysat, dadurch gekennzeichnet, dass durch Anwendung der Zusammensetzung aus dem ionischen (Natriumlaurylsulfat) und nichtionischen (Triton X-100) Tensiden, SS Brücken spaltender Substanz (2-Merkaptoethanol) im ersten und der Zusammensetzung aus der Polyanionen (Polly(acrylamid-co-Natrium-acrylat) mit LD: 50-60 mol-%), Pollymeren mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-Natrium-acrylat) mit LD: 2-4 mol-%), Mikropartikel aus dem dreidimensionalen Polymer-Matrix mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-N'N'-methylenbisacrylamid-co- Natrium-acrylat) mit LD: von 2 bis 4 mol-%) im zweiten Schritt, im Puffer mit niedriger pH (3,o<pH<5,o), nur Proteinen und zellulären Reststoffen werden selektiv geflockt, DNA/RNA aber nicht.5. Process for the purification of the DNA / RNA from proteins and cellular residues in the cell lysate, characterized in that by using the composition of the ionic (sodium lauryl sulfate) and nonionic (Triton X-100) surfactants, SS bridging substance (2-mercaptoethanol) in the first and the composition of the polyanions (polly (acrylamide-co-sodium-acrylate) with LD: 50-60 mol%), polymers with negatively charged and hydrophobic groups (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 -%) in the second step, in the buffer with low pH (3, o <pH <5, o), only proteins and cellular residues are selectively flocculated, but not DNA / RNA.
6. Verfahren zur Reinigung der DNA/RNA von Proteinen und zelluläre Reststoffe n im Zellenlysat, dadurch gekennzeichnet, dass durch die Anwendung von Mikropartikel aus dem dreidimensionalem Polymer-Matrix mit negativ geladenen und gydrophoben Gruppen (Polly(acrylamid-co-N'N'-methylenbisacrylamid-co-Natrium-acrylat) mit LD: von 2 bis 4 mol-%) im zweiten und Polykationen ((PoUyCacrilamid-co-NjNjNrtrimetylammonium- ethylacrylat)-chlorid) mit LD: 50-60 mol-%)) im dritten Schritt werden Flocken genug groß und schwer für die sichere Trennung, von der bleibenden im Losung DNA/RNA, bei der abschließender Zentrifugierung. 6. Process for the purification of the DNA / RNA from proteins and cellular residues n in the cell lysate, characterized in that by the use of 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%) in the second and polycations ((PoUyCacrilamid-co-NjNjNrtrimetylammonium-ethyl acrylate) -chloride) with LD: 50-60 mol%)) in the third Step flakes are big enough and heavy for the safe separation, from the DNA / RNA remaining in the solution, during the final centrifugation.
EP05819710A 2005-02-07 2005-12-22 Method for the purification of dna/rna of proteins and cellular residual substances in cell lysate Withdrawn EP1904633A1 (en)

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AT1952005A AT501380B1 (en) 2005-02-07 2005-02-07 METHOD OF CLEANING THE DNA / RNA FROM PROTEINS AND CELLULAR RESIDUAL MATERIALS IN THE CELL LYSATE
PCT/AT2005/000519 WO2006081597A1 (en) 2005-02-07 2005-12-22 Method for the purification of dna/rna of proteins and cellular residual substances in cell lysate

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FR3038616B1 (en) * 2015-07-06 2020-11-06 Gl Biocontrol PROCESS FOR PURIFICATION AND CONCENTRATION OF NUCLEIC ACIDS.
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