WO2015188994A1 - Procédé d'enrichissement et/ou de purification d'acides nucléiques - Google Patents

Procédé d'enrichissement et/ou de purification d'acides nucléiques Download PDF

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Publication number
WO2015188994A1
WO2015188994A1 PCT/EP2015/060246 EP2015060246W WO2015188994A1 WO 2015188994 A1 WO2015188994 A1 WO 2015188994A1 EP 2015060246 W EP2015060246 W EP 2015060246W WO 2015188994 A1 WO2015188994 A1 WO 2015188994A1
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WO
WIPO (PCT)
Prior art keywords
washing
solid phase
nucleic acids
dna
polyethylene glycol
Prior art date
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PCT/EP2015/060246
Other languages
German (de)
English (en)
Inventor
Martina Reinhardt
Christian Dorrer
Bernd Faltin
Original Assignee
Robert Bosch 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 Robert Bosch Gmbh filed Critical Robert Bosch Gmbh
Publication of WO2015188994A1 publication Critical patent/WO2015188994A1/fr

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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
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • 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 present invention relates to a method for enrichment and / or purification of nucleic acids, wherein the nucleic acids are bound to a solid phase and eluted after one or more washing steps.
  • the invention relates to a kit for carrying out such
  • Microorganisms or viruses are Microorganisms or viruses.
  • RNA from a sample is often provided to, for example, target a particular pathogenic organism, e.g. to detect a bacterium or a virus.
  • a sample is typically a liquid or liquefied
  • Patient sample e.g. Blood, urine, stool, sputum, cerebrospinal fluid, lavage, a flushed out smear or a liquefied tissue sample.
  • the nucleic acid in particular the DNA or RNA, is purified and analyzed.
  • a sequencing, a polymerase chain reaction (PCR), a real-time PCR and / or a detection by means of a microarray can be performed so that a particular gene or a specific sequence can be detected.
  • nucleic acid is adsorbed to a solid phase (binding). Following Usually, one or more washes are performed to
  • elution of the adsorbed nucleic acid occurs under suitable conditions, for example at low salt concentration and / or elevated temperature of an elution buffer. In this way, the
  • Nucleic acid in concentrated form for subsequent steps for example, an amplification and / or detection of certain sequences are provided.
  • An important step in this process is washing after binding
  • washing buffers or washing solutions generally receive an alcoholic component, in particular isopropanol and / or ethanol. Often they are still
  • Components may also contain polyethylene glycol with 2 to 200 ethylene glycol units.
  • WO 2010/149532 describes a method of DNA preparation and amplification in which the DNA is immobilized on a surface and the surface is washed with a washing solution containing tetraethylene glycol before the DNA is recovered by elution.
  • WO 2006/085104 A1 describes a method for the isolation of nucleic acids, wherein the sample in the presence of a Ethylene glycol multimer is brought into contact with a solid phase, and bind the nucleic acids from the solution to the surface of the solid phase.
  • the ethylene glycol multimers used have a molecular weight between 100 and 3,100.
  • DE 199 43 374 A1 describes a process for binding nucleic acids to a solid phase in the presence of polyethylene glycol, the solid phase having hydrophobic and hydrophilic groups on the surface.
  • the inventive method is used for enrichment and / or purification of nucleic acids, in particular of DNA or RNA, wherein the nucleic acids are bound to a solid phase and eluted after one or more washing steps.
  • the washing step (s) are carried out with one or more washing solutions containing polyethylene glycol.
  • the inventors have been able to show that nucleic acids, in particular DNA and in principle RNA, can be effectively purified by this process without the need for an alcoholic component and / or other detergents for washing the reversibly immobilized nucleic acids. With particular advantage in the inventive method therefore to an alcoholic component in the or
  • washing solutions are dispensed with, so that in a preferred embodiment of the method, the washing solution (s) contain no alcohol.
  • the absence of alcohol during the purification of the nucleic acids has several advantages. Alcohols, such as ethanol or isopropanol, interfere with the following
  • polyethylene glycol is not used for a precipitation step
  • Binding step but for washing the reversibly immobilized nucleic acids, wherein the nucleic acids are immobilized, for example, on a porous silica surface, which is integrated into a centrifuge tube.
  • the inventors were able to show for the first time that the present in the washing solution, high molecular weight polyethylene glycol in a very effective manner
  • Cell fragments, proteins and other impurities can be removed from the solid phase in order then to be able to elute the purified nucleic acid.
  • the existing in the wash solution polyethylene glycol interferes
  • polyethylene glycol is understood as meaning polymers with the basic unit ethylene oxide, which generally have an average molar mass of from 200 to 35 000 g / mol. Preference is given for the purposes of the invention Polyethylene glycols used at more than 600 g / mol. Particularly preferred are high molecular weight polyethylene glycols, for example, have an average molecular weight of 5000 g / mol or more. With particular advantage is
  • Polyethylene glycol was a very effective nucleic acid purification can be performed.
  • the concentration of polyethylene glycol in the washing solution (s) is in a concentration range of 10-60% (w / v), more preferably in a range of 30-50% (w / v).
  • the washing solutions with polyethylene glycol concentrations in this area have proven to be particularly effective.
  • the washing solutions may be, for example, aqueous washing solutions or washing buffers.
  • the wash solution (s) contain one or more salts.
  • the use of one or more salts and optionally other ingredients enables the adjustment of chaotropic
  • Guanidinium thiocyanate and / or sodium chloride and / or cesium chloride and / or tris hydrochloride also other salts.
  • Particularly preferred are high molar concentrations, for example 1 - 3 M or optionally higher, preferably 2 M.
  • the suitable concentrations are also dependent on the particular salt. For example, 2 M guanidine hydrochloride in the wash solution has been found to be particularly suitable.
  • substances may be contained in the washing solution which have a stabilizing effect, for example, on the pH or on others
  • Exercise properties of the solution may be, for example, conventional buffer substances such as, for example, tris-ethylenediaminetetraacetic acid (TE) or tris-hydrochloride (HCl) or other buffering substances.
  • TE tris-ethylenediaminetetraacetic acid
  • HCl tris-hydrochloride
  • washing solutions are used successively, for example two different washing solutions or two identical ones
  • a first wash buffer or wash solution may contain polyethylene glycol, preferably PEG 6000, and 1-3 M guanidinium hydrochloride, especially 2 M guanidinium hydrochloride.
  • a second wash solution can be
  • Polyethylene glycol in particular PEG 6000, and 5 to 250 mM NaCl, for example, 100 mM NaCl, and 5 to 50 mM Tris-HCl, preferably 5 to 25 mM Tris-HCl, for example, 10 mM Tris-HCl.
  • concentration of PEG may for example be between 10 and 60% (w / v) and preferably between 30 and 50%.
  • the order of the various wash buffers or wash solutions can be varied.
  • the solid phase to which the nucleic acids are immobilized has substantially hydrophilic surface properties.
  • hydrophilic hydroxy groups are advantageous. The hydrophilic groups are responsible for the binding of the nucleic acids to the solid phase
  • the solid phase may be based on silica.
  • the solid phase may be a silica filter and / or silica beads.
  • corresponding magnetic beads can be used. The magnetic properties of the beads can be used for the
  • the method according to the invention is carried out in a microfluidic device.
  • Microfluidic devices require only a small sample volume and are particularly suitable for performing rapid tests. Since the inventive method dispenses with alcoholic components in the washing solution (s), this method is particularly suitable for microfluidic Devices, because it dispenses with reagents that could attack the materials of the microfluidic device.
  • Polyethylene glycols do not interfere with subsequent analysis steps or at least significantly less than, for example, ethanol or isopropanol. It is therefore not absolutely necessary to preferably completely remove components of the washing solution prior to further analysis, as is the case in conventional processes in which, for example, ethanol and / or isopropanol originates from a
  • Wash solution must be removed as completely as possible in a separate step before subsequent enzymatic reactions can be performed. Depending on the application and subsequent analysis steps, it may also be advantageous in the method according to the invention to remove the washing solution preferably completely. Residues of the washing solution, however, are much less critical than residues of, for example, ethanol or isopropanol.
  • the invention further comprises a kit for carrying out a method for enrichment and / or purification of nucleic acids.
  • This kit includes one
  • Solid phase for binding nucleic acids and optionally at least one binding buffer which is provided for setting suitable conditions, so that the nucleic acids, in particular DNA or RNA, can be reversibly immobilized on the solid phase.
  • a suitable binding buffer may also be provided by the user himself.
  • the kit comprises at least one washing solution which
  • the kit may also contain other reagents suitable for the
  • the kit may further contain an eluant or elution buffer.
  • the kit may contain a lysis buffer containing reagents for chemical lysis or Enzymes for enzymatic lysis, for example proteases and / or lysozymes contains.
  • the kit may contain other reagents suitable for subsequent analysis of the enriched or purified DNA or RNA, for example, reagents for amplification and detection of particular nucleic acid sequences.
  • the invention comprises a method for the detection of pathogenic microorganisms or viruses.
  • the genetic material in particular the nucleic acids, especially DNA and / or RNA
  • the contained biological material especially cells or viruses
  • the sample may be a liquid or liquefied patient sample.
  • the nucleic acids are enriched and / or purified according to the method described.
  • Microorganism or for the virus to be detected can in particular on the basis of a nucleic acid amplification with
  • PCR Polymerase chain reaction
  • the drawing illustrates a quantitative detection of Staphylococcus aureus from the detectable DNA for various washing protocols according to the invention.
  • PEG 6000 is not a hazardous substance, so that no further hazardous substances regulations have to be taken into account during storage, transport and handling.
  • PEG 6000 does not substantially inhibit enzymatic reactions or at least significantly less than, for example, ethanol or isopropanol. Therefore, subsequent enzyme-based analysis reactions, such as a polymerase chain reaction, may be without further
  • wash solutions are compatible with materials typically used in microfluidic devices (lab-on-chip systems) and do not create structural weakenings and / or leaks.
  • wash solutions containing PEG 6000 have a higher contact angle than, for example, ethanol, thereby achieving improved fluidic performance, especially in microfluidic devices.
  • ethanol there is often an uncontrolled progression in microfluidic structures, with channels or surfaces wetted undesirably or at an undesirable time during performance. This problem essentially does not occur with PEG 6000 wash solutions.
  • the invention is based on the fact that in a solid-phase-based enrichment and / or purification of nucleic acids washing solutions with polyethylene glycol are performed waiving alcoholic components in the washing solution.
  • the inventors were able to show that when PEG 6000 is used in particular, chemical conditions are set which make it possible to flow through or flow around a solid phase with reversibly immobilized DNA or in principle also RNA, whereby cellular fragments are removed from the lysate and other contaminations can be without eluting the immobilized nucleic acid.
  • the DNA or RNA released during the elution in the subsequent step is suitable directly for a downstream analysis, for example for amplification by means of PC R.
  • washing solutions for example the kit MolYsis Complete 5 from Molzym GmbH, Bremen.
  • one or more washing solutions according to the invention are used which contain PEG 6000 in different concentrations or comparative substances, with no alcohol, ie in particular no ethanol or isopropanol, being contained in the washing solution.
  • the kit is based on the use of a silica membrane in a special
  • Centrifuge tube wherein the DNA to be isolated is selectively purified by means of the silica membrane.
  • the application of the method according to the invention is not limited to the use of this kit, but rather can generally for the purification and / or enrichment of
  • Nucleic acids in particular of DNA or RNA can be used.
  • washing solution performed. As a reference washing solutions were used, the high percentage of ethanol and isopropanol and 2-10%
  • Ethylenediaminetetraacetic acid (EDTA). There were each
  • Step 1 (Lysis of cells and release of DNA)
  • the cells whose DNA is to be detected can be different
  • Lyzed for example, enzymatically (e.g., with lysozyme), chemically (e.g., with strong alkaline lysis buffers), or physically (e.g., by coupling ultrasound or by milling with beads).
  • the resulting solution is called a lysate.
  • the lysate can be further treated, for example using the enzyme proteinase K, whereby degraded cell fragments and the DNA is optionally freed of associated proteins.
  • Step 2 binding the lysate to a solid phase
  • the lysate is spiked with a binding buffer before passing over a
  • Silica membrane is passed.
  • a high-alcohol buffer e.g. 30 - 100% ethanol or isopropanol or a
  • the lysate was mixed with a mixture of a denaturing solution containing a high proportion of a chaotropic salt (> 50% guanidine thiocyanate) and a buffered binding solution with a high percentage of isopropanol (> 50% isopropanol) and 2-10% tris hydrochloride.
  • a denaturing solution containing a high proportion of a chaotropic salt (> 50% guanidine thiocyanate) and a buffered binding solution with a high percentage of isopropanol (> 50% isopropanol) and 2-10% tris hydrochloride.
  • the centrifuge tube in which the silica membrane is integrated can be centrifuged.
  • magnetic Silica beads are used, the separation of the solid phase can be done by a magnetic field in a conventional manner.
  • binding buffer described here is suitable for purification of DNA. If RNA is to be purified in a comparable manner, a different binding buffer is usually required.
  • the solid phase is washed with one or more washing solutions according to the invention one or more times in succession.
  • the washing solutions contain polyethylene glycol, in particular PEG 6000. Washing solutions having a concentration in a range between 10 and 60% (w / v) are particularly suitable. Details of each washing solution are shown below for individual variants of the process.
  • the washing step is performed by placing the wash solution in the centrifuge tube and then centrifuging.
  • a magnetic field can be applied to separate the solid phase from the wash solution.
  • the elution of the DNA is carried out by contacting the solid phase with a suitable elution buffer. Suitable examples are low or very low salt concentrations in the buffer and / or an elevated temperature of the buffer. This sets conditions in which the DNA dissolves from the solid phase.
  • the liquid phase is separated from the solid phase, for example, in the manner already described above.
  • the resulting solution is generally referred to as eluate and contains the enriched or purified nucleic acid.
  • Step 5 Subsequent Procedures
  • the nucleic acid present in the eluate in particular DNA or optionally RNA
  • the washing of the immobilized DNA is carried out with a first and a second washing solution, the first washing solution containing PEG 6000 and 2 M guanidinium hydrochloride.
  • the second wash contains PEG 6000, 100 mM NaCl, 10 mM Tris-HCl.
  • the PEG concentrations are varied between 0 and 60%.
  • the figure illustrates the quantitative detection of 10 6 cells (Staphylococcus aureus) on the basis of the detectable DNA according to this purification and washing protocol. On the ordinate the recalculated absolute recovery of the cell number for the various washing protocols (abscissa) is shown, the cells in a centrifuge tube with a
  • Washing solutions was performed. The other samples were treated with the washing solutions according to variant 1 in the manner described.
  • the PEG 6000 concentration in the individual experiments was 0%, 10%, 20%, 30%, 40%, 50% and 60%.
  • the first and second wash solutions each contained the same PEG 6000 concentration.
  • the quantification of the DNA was carried out by means of quantitative real-time PCR. It was found that in a washing solution with 10% or 20% in about the same recalculated initial concentration of the cells as in the reference was achieved. At the concentrations of 30 to 60%, an increased recovery rate was Detect cells. At these PEG concentrations, improved enrichment or purity of the eluted DNA could be achieved compared to the reference.
  • the washing of the bound DNA is carried out with a first and a second washing solution, the first washing solution containing PEG 6000, 100 mM NaCl, 10 mM Tris-HCl and the second washing solution PEG 6000 and 2 M
  • the washing of the bound DNA is carried out with only one washing solution containing 100 mM NaCl, 10 mM Tris-HCl and in each case different concentrations of PEG 6000.
  • the PEG concentrations are between 10 and 60%. In comparison with a reference could at the
  • the process according to the invention is reacted in a microfluidic system.
  • the process according to the invention is reacted in a microfluidic system.
  • An exemplary microfluidic system has a lysis chamber for lysing the cells or for receiving the lysate. Furthermore, a filter chamber containing the solid phase, such as a membrane, and reservoirs for receiving the binding and
  • washing solutions provided.
  • valves and pumps for controlling the fluids may be present.
  • Reservoirs are fluidly connected via channels to the filter chamber.
  • the inventive method can be in a particularly advantageous manner in Connection with a microfluidic device for various laboratory routines are used, for example for the diagnosis of infectious diseases.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
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  • Wood Science & Technology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
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  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

L'invention concerne un procédé d'enrichissement et/ou de purification d'acides nucléiques dans lequel les acides nucléiques sont liés à une phase solide et sont élués après une ou plusieurs étapes de lavage. La ou les étapes de lavage sont effectuées avec une ou plusieurs solutions de lavage qui contiennent du polyéthylène glycol (PEG), en particulier du PEG 6000.
PCT/EP2015/060246 2014-06-12 2015-05-08 Procédé d'enrichissement et/ou de purification d'acides nucléiques WO2015188994A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102014211221.4A DE102014211221A1 (de) 2014-06-12 2014-06-12 Verfahren zur Anreicherung und/oder Aufreinigung von Nukleinsäuren
DE102014211221.4 2014-06-12

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WO2015188994A1 true WO2015188994A1 (fr) 2015-12-17

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018189025A1 (fr) * 2017-04-11 2018-10-18 Robert Bosch Gmbh Désorption d'acides nucléiques
CN114196669A (zh) * 2021-12-24 2022-03-18 南京诺唯赞生物科技股份有限公司 一种用于核酸提取的漂洗液
WO2022101304A1 (fr) * 2020-11-10 2022-05-19 Life Technologies As Compositions et procédés d'isolement d'acides nucléiques

Families Citing this family (3)

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Publication number Priority date Publication date Assignee Title
DE102018132710A1 (de) 2018-12-18 2020-06-18 Analytik Jena Ag Filtrierverfahren geeignet zur Isolierung und/oder Quantifizierung zumindest einer zu untersuchenden Substanz aus einer Probe
DE102021204952A1 (de) 2021-05-17 2022-11-17 Robert Bosch Gesellschaft mit beschränkter Haftung Verfahren zur Aufreinigung von Nukleinsäuren, insbesondere in einer mikrofluidischen Vorrichtung
DE102022115445A1 (de) * 2022-06-21 2023-12-21 Ist Innuscreen Gmbh Verfahren und kit zur manuellen und automatisierten probenvorbereitung von long-read-sequenzierungen

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018189025A1 (fr) * 2017-04-11 2018-10-18 Robert Bosch Gmbh Désorption d'acides nucléiques
WO2022101304A1 (fr) * 2020-11-10 2022-05-19 Life Technologies As Compositions et procédés d'isolement d'acides nucléiques
CN114196669A (zh) * 2021-12-24 2022-03-18 南京诺唯赞生物科技股份有限公司 一种用于核酸提取的漂洗液
CN114196669B (zh) * 2021-12-24 2023-02-28 南京诺唯赞生物科技股份有限公司 一种用于核酸提取的漂洗液

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