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EP2134840A1 - Procédé de purification de biomolécules - Google Patents

Procédé de purification de biomolécules

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Publication number
EP2134840A1
EP2134840A1 EP08718091A EP08718091A EP2134840A1 EP 2134840 A1 EP2134840 A1 EP 2134840A1 EP 08718091 A EP08718091 A EP 08718091A EP 08718091 A EP08718091 A EP 08718091A EP 2134840 A1 EP2134840 A1 EP 2134840A1
Authority
EP
European Patent Office
Prior art keywords
biomolecules
centrifugation
sample
reaction vessel
acceleration value
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Withdrawn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
EP08718091A
Other languages
German (de)
English (en)
Inventor
Friederike Wilmer
Anja Schultz
Claudia Fritz
Claudia Dienemann
Andreas Schäfer
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.)
Qiagen GmbH
Original Assignee
Qiagen GmbH
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 Qiagen GmbH filed Critical Qiagen GmbH
Publication of EP2134840A1 publication Critical patent/EP2134840A1/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Withdrawn legal-status Critical Current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

Definitions

  • the present invention relates to a method for the purification of biomolecules, in particular of nucleic acids, such as DNA and RNA molecules.
  • biomolecules from biological samples plays an increasingly important role in basic bio-medical research, clinical research and diagnostics, forensic analysis, population genetic research, epidemiological analytics and related fields. This applies in particular to nucleic acids such as DNA and RNA molecules, but also to amino acids, oligopeptides, polypeptides, monosaccharides, oligosaccharides, polysaccharides, fats, fatty acids and / or lipids.
  • the analysis of the genome ie the whole of the cellular DNA, by molecular biological methods, such. As PCR, NASBA, RFLP, AFLP or sequencing allows z. As the detection of genetic defects or the determination of the HLA type and other genetic markers. Moreover, DNA fingerprinting for forensic, population genetic or food law analysis fall under this generic term.
  • the analysis of genomic DNA and RNA is also used for the direct detection of infectious agents such as viruses, bacteria, etc.
  • biomolecules e.g. Amino acids, oligopeptides, polypeptides, monosaccharides, oligosaccharides, polysaccharides, fats, fatty acids and / or lipids
  • Amino acids, oligopeptides, polypeptides, monosaccharides, oligosaccharides, polysaccharides, fats, fatty acids and / or lipids can be used e.g. Provide information about certain physiological states, about contamination in food, about the content of certain nutrients and so on.
  • a method for purifying nucleic acids following this principle is e.g. the so-called "boom principle" method disclosed in EP389063.
  • a sample containing nucleic acids in the vicinity of a chaotropic salt is placed in a vessel with a silicate matrix. Subsequently, the vessel is centrifuged or a vacuum is applied. This results in the nucleic acids binding to the silicate matrix while all other components of the sample (especially cell debris, organelles, proteins and the like) pass through the silicate matrix and are discarded. Subsequently, the bound nucleic acids are eluted with a suitable agent and supplied for further analysis.
  • binding mechanisms relevant to binding are e.g. in Melzak et al. (1996), Driving Forces for DNA Adsorption to Silica in Perchlorate Solutions, Journal of Colloid and Interface Science 181 (2), 635-644.
  • spin columns are often used for this process.
  • These are microreaction vessels which have a disk-shaped silicate matrix, are open at the bottom, and are placed in another microreaction vessel closed at the bottom.
  • the nucleic acid-containing sample is pipetted into the microreaction vessel together with a chaotropic salt.
  • the combination of both microreaction vessels is introduced into a centrifuge and centrifuged at an acceleration value of about 10,000 xg.
  • the nucleic acids bind to the silicate matrix, while all other constituents of the sample pass through the silicate matrix and are transferred to the second, closed micro reaction vessel. The latter is subsequently discarded, while the bound nucleic acids are eluted with a suitable agent and supplied for further analysis.
  • the present invention has for its object to overcome the described, resulting from the prior art disadvantages.
  • step c) it may be a binding step, a washing step and / or an elution step in step b).
  • the multistage step b) is a binding step in which the biomolecules are bound to the binding matrix by centrifugation.
  • this step may also be a washing step.
  • the method comprises at least one binding step, a washing step and an elution step, which consistently comprise at least one optionally multi-stage centrifugation step.
  • biomolecules are substances selected from the group consisting of nucleic acids, amino acids, oligopeptides, polypeptides, monosaccharides, oligosaccharides, polysaccharides, fats, fatty acids and / or lipids.
  • nucleic acids is understood in particular as meaning RNA and DNA.
  • plasmidic, genomic, viral and mitochondrial DNA are suitable as DNA, while mRNA, siRNA, miRNA, rRNA, snRNA, tRNA, hnRNA and total RNA are particularly suitable as RNA.
  • the nucleic acids introduced herein may be any type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine base.
  • the nucleic acid can be single, double or multi-stranded, linear, branched or circular. It may correspond to a molecule occurring in a cell, such as genomic DNA or messenger RNA (mRNA), or be generated in vitro, such as Complementary DNA (cDNA), counterstrand RNA (aRNA), or synthetic nucleic acids.
  • the nucleic acid can consist of a few nucleotides or even of many thousands of nucleotides.
  • reaction vessel with a binding matrix is to be understood in the following as a biochemical separation principle in which a binding matrix which associates selectively with certain substances is arranged in a reaction vessel or a miniaturized column.
  • the acceleration value is also referred to as “spin speed” and does not correspond to the rotational speed of the centrifuge, which is usually measured in revolutions per minute (9.81 ms "2) .
  • the acceleration value is determined constructively by the centrifuge drum diameter (effective diameter) and the speed.
  • centrifugation step is understood below to mean a method step which is characterized by a definable duration and a definable acceleration value.
  • This bonding matrix preferably comprises an anion exchanger, a silicate substrate, a plastic substrate, or a chitosan-containing substrate.
  • silicate substrate is to be understood below as a membrane, a pellet, a packing or a disk of porous silicate, which has a large inner surface and is arranged in the reaction vessel so that a solution which is added to the reaction vessel is driven through the membrane, pellet, packing or disc upon application of a vacuum or centrifugation, such that the components in the solution contact the constituents of the matrix.
  • the silicate substrate is preferably a matrix of silica gel.
  • the silicate substrate may be made of pressed glass fibers or "glass beads.” Silicate substrates are used, for example, in the purification kits sold by the assignee under the trademarks QIAprep and RNeasy.
  • Anionenleyer are well known from the prior art.
  • a resin is used here, which, for example, interacts with the negatively charged phosphate residues of the nucleic acid backbone.
  • the salt concentration and pH of the buffers used determine whether the nucleic acid binds to the resin or is eluted from the column.
  • anion exchangers for example, by the applicant under the trademarks! QIAGEN Genomic-tip and Plasmid-tip distributed.
  • Chitosan has only recently come into contact as a binding agent for biomolecules. It is a copolymer of ß-l, 4-glycosidically linked N-acetyl-glucosamine residues and glucosamine residues. Under physiological conditions, chitosan carries net positive charges and is therefore able to bind many negatively charged biomolecules, especially nucleic acids, amino acids, oligo- and polypeptides, fats and fatty acids.
  • the binding matrix prefferably has a silicate substrate and for the sample containing the biomolecule also to be mixed with at least one chaotropic salt prior to centrifugation.
  • This embodiment is particularly suitable for nucleic acids.
  • the separation principle used is based on the "boom principle" method already discussed. In this case, a sample containing nucleic acids is placed in a vessel with a silicate matrix in the presence of a chaotropic salt. Subsequently, the vessel is centrifuged or a vacuum is applied. This results in the nucleic acids binding to the silicate matrix while all other components of the sample (especially cell debris, organelles, proteins and the like) pass through the silicate matrix and are discarded. Subsequently, the bound nucleic acids are eluted with a suitable agent and supplied for further analysis.
  • a columnar reaction vessel having a binding matrix comprising a silicate substrate in a centrifuge, wherein before or after this step, a solution or suspension of a sample containing nucleic acids and at least one chaotropic salt prepared in the reaction vessel or filled into the reaction vessel becomes; b) inserting a first centrifugation step at a first acceleration value;
  • step d) optionally introducing further centrifugation steps between step c) and step d) or after step d);
  • the multi-step centrifugation step is a binding step in which the nucleic acids are bound to the silicate matrix.
  • This embodiment leads to a considerably improved yield of nucleic acids to be purified compared to "one-step processes with silicate matrices" known from the prior art.
  • the washing and / or the elution step are designed in a multi-step manner in the sense of the above protocol.
  • washing or washing steps are preferably carried out with a washing buffer.
  • a washing buffer This may in particular contain ethanol and / or acetone.
  • the elution solution used to elute the biomolecules bound to the binding matrix, in particular nucleic acids, may be e.g. to water (including distilled water) or a low molar solution act.
  • a weakly concentrated saline solution in question.
  • the chaotropic salt is preferably already in solution.
  • the sample containing nucleic acids may be in solution or suspension and closing with a chaotropic salt.
  • sample and chaotropic salt may be present as a solid and be solubilized together.
  • reaction vessel is to be understood in the following to mean a vessel which is optionally closable at the top and optionally open at the bottom "as they eg manufactured and distributed by the applicant.
  • the reaction vessels may preferably be designed so that they fit accurately in a commercially available, slightly larger reaction vessel, as e.g. can be arranged by the company Eppendorf.
  • the larger reaction vessel serves as a collecting vessel for the liquid passing through the binding matrix.
  • the abovementioned process according to the invention has in common that the yield of the biomolecule purification is increased by up to 20% as a result of the first combination of a centrifugation step at a low acceleration value and a centrifugation step at a high acceleration value, as investigations by the Applicant have shown (see examples).
  • analytical investigations are considerably facilitated and in many cases even made possible; these are cases where e.g. the amount of nucleic acid in the sample is so low that with the conventional purification methods, the yield is insufficient to amplify and / or detect the nucleic acids.
  • the method according to the invention is carried out in an automatic and / or programmable centrifuge.
  • the centrifuge already has one or more internally stored centrifugation protocols with at least two centrifugation steps at different acceleration values.
  • Such a centrifuge is expressly within the scope of the present invention.
  • the biological sample is a material selected from the group consisting of sample material, plasma, body fluids, blood, serum, cells, leukocyte fractions, crusta phlogistica, sputum, urine, sperm, faeces, forensic specimens, smears, punctates, biopsies , Tissue samples, tissues and organs, food samples, environmental samples, plants and parts of plants, bacteria, viruses, viroids, prions, yeasts and fungi, and fragments or constituents of the aforementioned materials, and / or isolated, synthetic or modified proteins, nucleic acids, lipids, Carbohydrates, metabolites and / or metabolites.
  • all methods of analysis known to those skilled in the art may be used, preferably methods selected from the group consisting of light microscopy, electron microscopy, confocal laser scanning microscopy, laser microdissection, scanning electrochemistry - Nuclear microscopy, Western blotting, Southern blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, affinity chromatography, mutation analysis, polyacrylamide gel electrophoresis (PAGE), especially two-dimensional PAGE, HPLC, polymerase chain reaction (PCR), RFLP (Restriction Fragment Length Poly- morphism) analysis, Serial Analysis of Gene Expression (SAGE) analysis, Fast Protein Liquid Chromatography (FPLC) analysis, mass spectrometry such as MALDI -TOFF mass spectrometry or SELDI mass spectrometry, microarray analysis, LiquiChip analysis, analysis of the activity of enzymes, HLA typing, sequencing, WGA ("whole genome amplification
  • the method is preceded by a step for the lysis of cells or tissues containing biomolecules.
  • This lysis step may be e.g. to act a physical or a chemical lysis.
  • a physical or a chemical lysis In particular, the use of ultrasound, successive freezing / thawing, the use of rotating knives, the use of oscillating microbeads, the action of a hypotonic shock, the so-called “French Press - Method "or the so-called” cell-bombing "used.
  • a special form is the alkaline lysis. This is used in particular to isolate plasmid DNA from already lysed bacteria.
  • the hydrogen bonds between the complementary DNA strands of both the chromosomal and the plasmid DNA dissolve, the plasmid DNA is due to their conformation capable of completely renaturing.
  • the chromosomal DNA After neutralization of the pH with potassium acetate and glacial acetic acid, the chromosomal DNA, which has been fragmented by the individual preparation steps, can not be renatured, DNA double strands with only short complementary regions and through the undirected connection of many individual DNA strands are formed it comes to the formation of a confused DNA mass.
  • the chaotropic salt used according to the invention is a salt or a mixture of salts selected from the group comprising guanidinium hydrochloride, guanidinium umrhodanide, guanidinium iodide, urea, ammonium sulfate, sodium iodide, potassium iodide, sodium perchlorate, sodium (iso) thiocyanate and guanidium thiocyanate.
  • Chaotropic salts are salts that have a high affinity for water and therefore form a hydration shell. In the presence of these salts, hydrophobic interactions in proteins are destabilized because the solubility of the hydrophobic side chains increases and the protein denatures. On the other hand, nucleic acids such as DNA and RNA are not impaired because no hydrophobic interactions are required to stabilize them. In addition, the cations of the chaotropic salts in high concentrations saturate the negative charges on the surface of silicates, especially in silicate matrices, and generate a net positive charge, which significantly promotes binding of the nucleic acids to the silicate matrices.
  • the first centrifugation step of the process is preferably carried out at an acceleration value in the range between 5 and 2000 g.
  • Particularly suitable acceleration values are 10 ⁇ g, 27 ⁇ g, 50 ⁇ g, 15 ⁇ g, 300 ⁇ g, 500 ⁇ g, 800 ⁇ g, 1000 ⁇ g and 1500 ⁇ g.
  • This centrifugation step may, for example, have a duration of 5 s-20 min. Particularly preferred is a duration of 10 s - 10 min. Particularly preferred is a duration of 30 s - 5 min.
  • the second centrifugation step of the process is preferably carried out at an acceleration value in the range between 100-25,000 ⁇ g.
  • Particularly suitable acceleration values are 180 ⁇ g, 610 ⁇ g, 1000 ⁇ g, 2500 ⁇ g, 8000 ⁇ g, 12000 ⁇ g and / or 17000 g.
  • This centrifugation step may also have a duration of 5 seconds to 20 minutes, for example. Particularly preferred is a duration of 10 s - 10 min. Very particular preference is a duration of 30 s - 5 min.
  • the value ranges for the acceleration values of the first and the second centrifugation step overlap.
  • it must be ensured that the acceleration value of the first centrifuging step is always below the acceleration value of the second centrifuging step.
  • the reaction vessels are centrifuged in a centrifuge rotor of the "swing-out type."
  • the required centrifugation angle only sets in when the rotor is set in motion fixed angle rotors, the above-mentioned improvements in the yield, however, centrifugal rotors of the "swing-out type" are preferably used when substances in already arranged in the rotor reactor ons or Zentrifugiergefäße to be given, for example by pipetting or using a pipetting robot.
  • the individual steps of the method are automated.
  • the applicant u.A. developed its own device that combines the functions of a pipetting robot and a programmable centrifuge. With the help of such an automated process, the laboratory throughput can be significantly increased and at the same time largely avoid allocation errors. Both factors play an important role in clinical, forensic, epidemiological and population genetic studies.
  • a reaction vessel comprising a binding matrix is provided for use in a method of purifying biomolecules, preferably nucleic acids, from a sample.
  • a reaction vessel is e.g. shown in Fig. 3.
  • a composition for use in a method for the purification of biomolecules, preferably nucleic acids is provided from a sample, the composition having at least one constituent selected from the group consisting of alkaline agents, phenol, lytic enzymes, isoamyl alcohol, chloroform, chaotropic salts , Alcohols, water, inorganic or organic salts.
  • This composition may be e.g. a lysis buffer (phenol, lytic enzymes, isoamyl alcohol, chloroform), a binding buffer (chaotropic salts), a washing buffer (alcohols, inorganic or organic salts) or an elution buffer (inorganic or organic salts).
  • a lysis buffer phenol, lytic enzymes, isoamyl alcohol, chloroform
  • a binding buffer chaotropic salts
  • washing buffer alcohols, inorganic or organic salts
  • elution buffer inorganic or organic salts
  • kit of parts comprising at least one such composition.
  • This kit particularly preferably has at least one reaction vessel as mentioned above and also reagents for analysis of biomolecules in or from a biological sample or for analysis of the morphology of a biological sample.
  • Reagents used for the analysis of biomolecules include in particular reagents for the detection and quantification of nucleic acids, amino acids, oligopeptides, polypeptides, monosaccharides, oligosaccharides, polysaccharides, fats, fatty acids and / or lipids.
  • reagents for the detection and quantification of nucleic acids, amino acids, oligopeptides, polypeptides, monosaccharides, oligosaccharides, polysaccharides, fats, fatty acids and / or lipids are examples of the reagents from the literature without their own inventive performance. Frequently, such reagents are already readily available as kits for the respective biomolecules to be analyzed.
  • reagents include in particular dyes for staining cells or cell components, antibodies optionally labeled with fluorescent dyes or enzymes, an absorption matrix such as DEAE cellulose or a silica membrane, substrates for enzymes, agarose gels, polyacrylamide gels, solvents such as ethanol or phenol, aqueous buffer solutions, RNase-free water, lysis reagents, alcoholic solutions and the like.
  • the composition may already be filled in the vessel.
  • the kit comprises as a further constituent a metering device which is filled with the composition and by means of which defined portions of the composition, preferably under sterile conditions, can be introduced into the vessel.
  • a dosing device can be designed, for example, in the form of a soap dispenser.
  • a device for purifying biomolecules, preferably nucleic acids, from a sample comprising a centrifuge, which is characterized in that the device has means which make it possible for at least two centrifugation steps with different heights during a centrifugation without user intervention Acceleration values are automatically inserted.
  • a microprocessor control is usually required, which has a memory device in which Multi-step centrifugation protocols are stored and / or stored.
  • a centrifuging device which comprises means for carrying out the method described above for the purification of biomolecules from a sample according to.
  • a microprocessor control is particularly conceived which makes it possible to automatically insert at least two centrifugation steps with differently high acceleration values during a centrifugation without user intervention.
  • Such a centrifuging device has means for the automated implementation of the method according to the invention. This includes u.A. in addition to the mentioned microprocessor control e.g. a pipetting robot.
  • a purified nucleic acid which can be prepared by a method, a composition, a kit and / or a device according to the present invention.
  • this nucleic acid is plasmid, genomic, viral and mitochondrial DNA or mRNA, siRNA, miRNA, rRNA, snRNA, t-RNA, and hnRNA.
  • Example 1 Basic Procedure (One-Step Method According to the Prior Art)
  • Bacterial colonies grown on an agar plate containing a plasmid to be isolated are picked, suspended in 3 ml of LB liquid culture medium and incubated overnight at 37 ° C. for multiplication.
  • the saturated 3 ml bacterial overnight cultures are pelleted in a bench centrifuge at 13000 rpm.
  • the isolation of the plasmid DNA is carried out according to a modified standard protocol of Qiagen according to the methodology of Birnboim: The supernatant of the bacterial culture is removed and discarded.
  • the pellet is mixed with 250 ⁇ l buffer Pl (Qiagen) and resuspended.
  • Example 2A Comparison of DNA yield between one-step and two-step centrifugation (binding step)
  • the main differences in the centrifugation protocol are highlighted in gray.
  • the buffers Pl, P2, N2, PE and EB are components of the QIAprep kit.
  • the yield of plasmid DNA was examined. In each case 8 parallel experiments were carried out, the results were evaluated statistically and are shown in Fig. 2A. While a DNA yield of 8454 ng was achieved with the one-step process, a yield of 9540 ng was achieved with the two-step process. The differences are significant. It can be seen throughout that the DNA yield was about 13% higher with the two-step procedure.
  • Example 2B Comparison of the DNA yield between one-step and two-step centrifugation method (washing step)
  • Example 2C Comparison of the RNA yield between one-step and two-step centrifugation method
  • Jurkat cells were lysed by a standard method (Qiagen RNeasy) and transferred to Spin Columns (model RNeasy) followed by a conventional manual standard protocol ("manual 2-step binding") centrifugation procedure.
  • the process parameters were as follows:
  • RPE, RWl and RLT are components of the RNeasy kit. Subsequently, the Yield of RNA examined. In each case 8 parallel experiments were carried out, the results were statistically evaluated and are shown in FIG. 2C.
  • RNA yield 1836 ng was achieved with the one-step procedure
  • a yield of 2011 ng was achieved with the two-step procedure. The differences are significant. It can be seen throughout that the RNA yield was about 9% higher with the two-step procedure.
  • the multi-step centrifugation step is a binding step in which the biomolecules are bound to the binding matrix by centrifugation.
  • the sample to be purified is mixed with the binding buffer and then centrifuged initially for 1 min at 500 x g. The centrifuge then accelerates until an acceleration value of 8000 x g is reached, and the sample is centrifuged at this value for a further 75 sec. During this procedure, the nucleic acids bind to the silicate matrix while all remaining components pass through the silicate matrix and can be discarded. It is then washed with a washing buffer and the nucleic acids are washed from the column with an elution buffer and collected.
  • Fig. 2 shows the results of the experiments described in Examples 2A, 2B and 2C. On the one hand, the absolute yields of nucleic acid in ng are shown, and on the other hand, the performance advantage of each two-step process is shown in%.
  • Fig. 3 shows a reaction vessel 30 comprising a silicate matrix 31 for use in a process according to the invention.
  • the reaction vessel 30 was charged with a solution or suspension of a nucleic acid-containing sample and at least one chaotropic salt or such a solution or suspension was prepared in the reaction vessel, the reaction vessel is placed in a larger sized collecting vessel 32.
  • the combination of the two vessels is then subjected to the centrifugation protocol according to the invention with a first centrifuging step at a first acceleration value and a second centrifuging step at a second acceleration value, which is higher than the first acceleration value, in a centrifuge, not shown.
  • the nucleic acids bind to the silicate matrix while all remaining components pass through the silicate matrix and can be discarded. It is then washed with a washing buffer and the nucleic acids are washed from the column with an elution buffer and collected.
  • FIG. 4 shows as a time diagram the exemplary sequence of two further centrifugation protocols according to the method according to the invention.
  • the centrifuge is briefly stopped between the individual centrifugation steps at different acceleration values.

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Abstract

L'invention concerne un procédé de purification de biomolécules à partir d'un échantillon, présentant les étapes suivantes : a) mise en place d'un récipient de réaction avec une matrice de liaison dans une centrifugeuse, une solution ou suspension d'un échantillon contenant des biomolécules étant préparée dans le récipient de réaction ou introduite dans ce dernier, avant ou après cette étape; et b) exécution d'au moins une étape de centrifugation à plusieurs phases, constituée d'au moins une première phase de centrifugation à une première valeur d'accélération et d'au moins une deuxième phase de centrifugation à une deuxième valeur d'accélération supérieure à la première valeur d'accélération, c) la phase b) pouvant être une phase de liaison, une phase de lavage et / ou une phase d'élution.
EP08718091A 2007-04-04 2008-03-20 Procédé de purification de biomolécules Withdrawn EP2134840A1 (fr)

Applications Claiming Priority (2)

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DE102007016707A DE102007016707A1 (de) 2007-04-04 2007-04-04 Verfahren zur Aufreinigung von Biomolekülen
PCT/EP2008/053375 WO2008122500A1 (fr) 2007-04-04 2008-03-20 Procédé de purification de biomolécules

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EP2134840A1 true EP2134840A1 (fr) 2009-12-23

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CN (1) CN101675163A (fr)
AU (1) AU2008235605A1 (fr)
DE (1) DE102007016707A1 (fr)
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CN111868260B (zh) 2017-08-07 2025-02-21 约翰斯霍普金斯大学 用于评估和治疗癌症的方法和材料
CN115516108A (zh) 2020-02-14 2022-12-23 约翰斯霍普金斯大学 评估核酸的方法和材料

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JP2010523094A (ja) 2010-07-15
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US20100113758A1 (en) 2010-05-06
CN101675163A (zh) 2010-03-17

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