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
• • 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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/02—Burettes; Pipettes
  • B01L3/0275—Interchangeable or disposable dispensing tips
• • 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
  • C12N15/1013—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 using magnetic beads
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/405—Concentrating samples by adsorption or absorption
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/02—Adapting objects or devices to another
  • B01L2200/023—Adapting objects or devices to another adapted for different sizes of tubes, tips or container
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0848—Specific forms of parts of containers
  • B01L2300/0854—Double walls
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/16—Surface properties and coatings
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
  • Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction

Definitions

• the invention relates to a device and a method with which
• Nucleic acids can be rapidly, highly efficiently and quantitatively automated, isolated and purified.
• the next generation of processes for the isolation of nucleic acids is based on a method developed by Vogelstein and Gillespie (Proc Natl Acad., USA, 1979, 76, 615-619) for the preparative and analytical purification of DNA fragments from agarose gels.
• the method combines the dissolution of the agarose containing the DNA band to be isolated in a saturated solution of a chaotropic salt (NaJ) with a binding of the DNA to glass particles.
• the DNA fixed to the glass particles is then washed with a washing solution (20 mM Tris HCl [pH 7.2], 200 mM NaCl, 2 mM
• Support materials as well as the type of buffer used for the binding of nucleic acids. Examples include the binding of nucleic acids to mineral carriers in the presence of solutions of different chaotropic salts, in which as a carrier material
• nucleic acids to the mineral materials known and used in the art and so-called antichaotropic salts can be used very efficiently and successfully as a component of lysis / binding buffer systems (EP 1135479).
• the state of the art can thus be described in such a way that nucleic acids to mineral materials in the presence of buffers containing chaotropic or antichaotropic salts or in the presence of buffers, the mixtures chaotropic and
• the mineral carriers used are present in the form of loose beds, in the form of filter membranes or in the form of suspensions.
• Paramagnetic or magnetic particles are often used to perform automated extraction operations. These are e.g. silicatic materials that possess or also have a magnetic or paramagnetic core
• Iron oxide particles whose surface is modified so that they carry the necessary for the attachment of nucleic acids functionalities.
• modified pipette tips were used. These are characterized in that they contain the carrier materials necessary for the binding of nucleic acids (porous mineral carrier materials or porous
• Nucleic acids The patent EP 1951904 discloses a pipette tip consisting of a top and bottom part, between which there is also a porous chromatographic support material and which is to be used for the automated isolation of nucleic acids.
• a modified pipette tip for extraction of nucleic acids also disclosed in the patent US2013 / 0078619. Also in this pipette tip is a porous mineral carrier material (porous glass) for direct binding of nucleic acids. All these modified pipette tips have in common is that it is a porous chromatographic material (loose bed or solid porous body). These substrates are always located horizontally within the pipette tip. The liquids to be processed flow through the porous material used.
• porous chromatographic material loose bed or solid porous body
• Extraction procedure is based on that after lysis of the sample and adjustment of the necessary binding conditions for the adsorption of the nucleic acids to the support material, this approach is drawn by means of a pipetting process through the porous support material.
• the nucleic acids bind to the carrier material.
• wash buffers are pipetted through the carrier material.
• a drying step (frequent on and
• the invention was therefore based on the object to solve the known problems and thus to perform the automated extraction of nucleic acids much easier and faster than before.
• a broad object of the invention is to enable existing liquid handling device platforms for the automated
• Claim 1 describes a device for the automated extraction of nucleic acids, comprising a body which can partially or completely submerge in a reaction cavity, wherein at least the part immersed in the reaction cavity has a rough or structured surface.
• a corresponding pipette tip is used, which was either roughened or a rough or structured object is infected outside.
• the precipitation of the nucleic acids takes place when the polarity of the solution is lowered. This is either done preferably either by addition of an organic solvent (alcohols) or by a binding buffer known in the art. It has been found that the attachment of the nucleic acids to this article does not take place if this article has a smooth surface.
• Claims 2 to 6 represent preferred embodiments of the invention.
• the invention also provides a device according to the walk-away principle, comprising the device according to the invention, e.g. an automatic pipetter or a
• the invention is based on the observation that nucleic acids adsorb to the surface of patterned or rough materials (e.g., polymer materials). For this purpose, it is only necessary to lyse a nucleic acid-containing biological sample to the
• nucleic acid This can be done with buffers known to those skilled in the art.
• the sample is mixed with a substance which lowers the polarity of the aqueous solution, preferably organic solvents such as alcohols or ketones.
• a substance which lowers the polarity of the aqueous solution preferably organic solvents such as alcohols or ketones.
• This approach is now brought into contact with a material which is characterized by a non-smooth surface. Under these conditions, nucleic acids adsorb to the surface of the material used. Subsequently, if necessary, washing steps with known alcoholic washing solutions. After drying, the adsorbed nucleic acid is removed from the material by the addition of water or a low salt buffer (eg 10 mM Tris HCl) and can be used for downstream applications.
• a low salt buffer eg 10 mM Tris HCl
• the device according to the invention and the method according to the invention make use of this possibility for a simple and automatable extraction process.
• the device consists of a body that can partially dip into a reaction cavity, wherein the part that dips into this reaction cavity, has a non-smooth surface.
• a hollow body is used which can absorb and deliver liquids.
• a pipette tip is used.
• the pipette tip is constructed so that there is a structured or rough material on its outer surface in the last lower third. This can be done, for example, by attaching a suitable ring (eg, such a ring can be placed on common pipette tips, which underscores the universality).
• the hollow body itself may also have such a structural feature (roughness) and thus consist of one part and be produced in an injection mold.
• the inventive device is exemplary of a
• the invention also includes a method for the automated extraction of
• Nucleic acids characterized by the following steps: a) A lysed biological sample is placed in a reaction cavity and mixed with at least one substance which lowers the polarity of the aqueous solution or with a means for binding nucleic acids to a solid phase
• rough surface should be understood to mean that it is not smooth by touching or by viewing the surface, but it may also be a surface having a structure (eg grooves).
• This structure eliminates the smoothness of the surface, even though the structure, ie, the grooves, itself may be smooth, and according to the invention such surfaces are referred to as "structured surfaces". If, by viewing or touching the surface, it is not possible to detect whether a surface is smooth or rough, a test may be performed by directing a laser beam at that surface. With a smooth surface, the laser is only in
• the inventive device is for the inventive method for automated extraction of nucleic acids is used as follows: Preferably, a classic walk-away principle is used, ie the solutions required for the extraction are presented and successively included in the extraction process. According to the
• the sample is placed in a reaction cavity and mixed with lysis buffer and possibly proteolytic enzymes. Thereafter, the sample lysis takes place. After lysing the sample with known lysis buffers, an organic component is added to the lysate.
• the device according to the invention immersed and is in the solution several times vertically moved up and down. Now the nucleic acids are on this device. Thereafter, the device is removed from the solution and moved to a new reaction cavity. In this there is an alcoholic wash buffer (this well known wash buffer can be used) or only one alcohol.
• the agent according to the invention is immersed in this solution and is moved up and down vertically several times in the solution.
• washing steps can be repeated several times. After the last washing step, the device is removed from the solution and briefly dried outside the cavity to remove the residual alcohol. In the last step, the device according to the invention is immersed in a further cavity in which water or another low-salt buffer is located. The device according to the invention also dips into this cavity and is moved up and down vertically several times in the solution. This leads to detachment of the bound nucleic acid. From this general flowchart it becomes clear how easy the automated extraction is now. There is no separation of magnetic particles to be made, as in the automated extraction using
• Magnetic particles is the case.
• the process does not require any vacuum filtration steps, as is necessary with the use of filter plates. It only requires the device according to the invention and a pipetting platform.
• the advantage is the universality and simplicity of the device according to the invention, since it is commercially available
• Standard pipette tips can take. These pipette tips are attached by plugging e.g. a ring with the specific necessary for the isolation of nucleic acids
• the reagents required for the extraction in corresponding reaction cavities already be submitted, so that the extraction process can proceed on the walk-away principle.
• a further particular advantage is revealed by the fact that the agent according to the invention not only enables the binding of the nucleic acid but, moreover, can also move liquids in a separate manner. In this combined function, the extraction process can be further optimized.
• the lysis of the sample can already be done on a machine. The necessary during the lysis continuous movement of the sample is achieved by the pipetting function of the device according to the invention.
• the eluate can also be removed from the elution cavity by means of the pipetting function and transferred to a storage vessel.
• Variant A semi-autoclave extraction procedure (sample lysis is done separately)
• NIH 3T3 cells For the nucleic acid extraction different amounts of NIH 3T3 cells were used.
• the extraction chemistry used for the isolation of the nucleic acids became partly taken from the commercial extraction kit innuPREP Blood DNA KÜ7IPC16X (Analytik Jena AG).
• a lysis buffer Lisis Solution CBV
• proteinase K the cells were lysed at 60 ° C for 15 min in a 2.0 ml reaction vessel. The lysis was not carried out in the extraction machine. This was followed by the
• the ring was dried on the hollow body outside the cavity for 10 minutes, thereby removing the residual ethanol.
• the elution of the nucleic acids was carried out by 30 repetitions of a single and
• the method is extremely simple to carry out and thus extremely fast. Compared to the standard method of nucleic acid extraction with the InnuPure C16 and the use of magnetic particles for the binding of the nucleic acids, the time saved is over 50%.
• the detection of the isolated nucleic acid was carried out by means of spectrophotometric measurement and geletrophoretischer representation in an agarose gel.
• FIG. 2 shows the gel electrophoretic separation of the isolated nucleic acid. Shown is the electrophoretically separated in a 0.8% agarose gel
• Nucleic acid isolated by the method of the invention were applied from left to right beginning with sample 1.
• the applied volume was 5 ⁇ .
• Variant B fully automatic extraction process (sample lysis takes place in the device)
• the lysis of the sample is also automated.
• the user only has to add the sample and the proteinase K, the further preparation is completely automated with the technique of the InnuPure C16.
• the lysis of the sample this is heated by Innupure C16 to 50 ° C and by 25 Ox and
• FIG. 3 shows the gel electrophoretic separation of the isolated nucleic acid.
• Figure 1 is an exemplary embodiment of the ring, which is attached to a hollow body. The picture is greatly enlarged.
• This non-smooth surface shaped body can be plugged onto any commercially available pipette tips so that it is located in the last lower third of the tip.

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• 2016
  • 2016-02-26 EP EP16711780.3A patent/EP3286313A1/de not_active Withdrawn
  • 2016-02-26 JP JP2018506479A patent/JP6797185B2/ja active Active
  • 2016-02-26 CN CN201680036526.2A patent/CN108076644B/zh active Active
  • 2016-02-26 PL PL16711779.5T patent/PL3286312T3/pl unknown
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  • 2016-02-26 WO PCT/EP2016/054180 patent/WO2016169679A1/de not_active Ceased
  • 2016-02-26 JP JP2018506477A patent/JP6979940B2/ja active Active
  • 2016-02-26 CA CA2983619A patent/CA2983619C/en active Active
  • 2016-02-26 CA CA2983626A patent/CA2983626C/en active Active
  • 2016-02-26 US US15/568,471 patent/US10851368B2/en active Active
  • 2016-02-26 EP EP16711779.5A patent/EP3286312B1/de active Active
  • 2016-02-26 WO PCT/EP2016/054178 patent/WO2016169677A1/de not_active Ceased
  • 2016-02-26 CN CN201680036436.3A patent/CN108064262B/zh active Active
  • 2016-02-26 US US15/568,474 patent/US10704039B2/en active Active
  • 2016-02-26 JP JP2018506478A patent/JP2018524017A/ja active Pending
  • 2016-02-26 WO PCT/EP2016/054179 patent/WO2016169678A1/de not_active Ceased
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  • 2016-02-26 CN CN201680036533.2A patent/CN108124454A/zh active Pending
  • 2016-02-26 ES ES16711778T patent/ES2928055T3/es active Active
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• 2022
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