Classifications

• • 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
• • 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
  • B01L3/56—Labware specially adapted for transferring fluids
  • B01L3/563—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
• • 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
  • B01L3/56—Labware specially adapted for transferring fluids
  • B01L3/563—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
  • B01L3/5635—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors connecting two containers face to face, e.g. comprising a filter
• • 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
• • 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
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0681—Filter
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
  • B01L2400/049—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics vacuum
• • 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
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
  • B01L3/5082—Test tubes per se

Definitions

• the invention relates to a method for isolating nucleic acids from a sample and a device suitable therefor.
• the release from the biological sample material is always necessary.
• the nucleic acid must be protected against degradation by nucleases from the biological material or the environment and degradation by chemical reaction conditions (Fe ++ / DTT or ß-mercaptoethanol; NaOH; heat).
• chemical reaction conditions Fe ++ / DTT or ß-mercaptoethanol; NaOH; heat.
• the greatest demands are placed on the contamination-free nature of the biological sample and the nucleic acid isolated from it. In particular, an entry from the environment, by laboratory personnel and cross-contamination of the samples with one another must be prevented.
• the nucleic acid should be in a buffered, aqueous, salt-free solution.
• PCR generally uses very small amounts of analyte (pg / ng range)
• special questions require the processing of a larger amount of sample.
• the nucleic acid from 10 - 20 ml be isolated from a blood sample. After homogenization of the sample, an aliquot of the isolated RNA can then be examined for expression of a tumor-associated gene.
• a modified method uses a concentrated salt solution after the lysis of the sample material to precipitate proteins and other accompanying substances.
• the nucleic acids in the supernatant are then precipitated by ethanol and collected by centrifugation. After the nucleic acids have been dissolved, they can be used for the amplification.
• the object of the present invention was therefore to provide a simple method for isolating nucleic acids from larger sample volumes.
• the invention therefore relates to a method for isolating nucleic acids from a sample by taking the sample into a first vessel through an opening, closing the opening of the first vessel with a closure element which contains a nucleic acid-binding, liquid-permeable material and which faces the opening on its side
• Side means for attaching the element to the first vessel and on the other side of the material includes means for attaching the element to a second vessel, and transferring the sample through the material into the vessel attached on the other side.
• the invention also relates to a device for carrying out this method.
• a method for isolating nucleic acids is understood to mean a method in which nucleic acids of a sample are separated from other sample components. This is done by binding the nucleic acids to a nucleic acid binding material. After binding, the liquid can be separated from the material with the nucleic acids. For the isolation of particularly pure nucleic acids, any adhering substances can be removed by washing the material with a liquid. If desired, the nucleic acids can be detached from the nucleic acid-binding material. The binding or detachment of the nucleic acids from the material takes place under conditions which depend on the material used.
• Nucleic acid binding materials are known to the person skilled in the art.
• the material can be both particulate and fibrous.
• the material consists of particles, it has proven to be advantageous to immobilize these particles, e.g. B. by insertion between liquid-permeable platelets, for. B. woven or non-woven from fibrous Material such as cellulose or plastics that have such tight pores that the particles are held between the platelets.
• the nucleic acid binding material is a fibrous material, e.g. B. in the form of fabrics or nonwovens themselves. Suitable materials are, for. B. from methods for the isolation of nucleic acids using centrifuge tubes or multiple devices in strip format known.
• the nucleic acid binding material must have the property that the sample liquid without additional force or with force, e.g. B. by exerting pressure or negative pressure through the material.
• the nucleic acids in the present method are not bound by filtration of the nucleic acids from the sample, but rather by a method in which the affinity of nucleic acids for surfaces is exploited, it is possible to use a relatively large-pore material. This facilitates the flow through even relatively viscous sample liquids.
• the liquid-permeable material is able to bind nucleic acids, but to allow the surrounding liquid and other constituents, such as proteins, etc., to pass through.
• the nucleic acids can be bound in a sequence-specific manner by capture probes attached to the surface of the material.
• the capture probes have a base sequence that can bind to a complementary base sequence in the nucleic acids to be isolated under hybridization conditions.
• sequence-specific materials allows the selective isolation of nucleic acids of a certain sequence.
• a method for binding nucleic acids to peptide nucleic acids on the surface of solids is described, for example, in WO 95/14708.
• the liquid-permeable material has a glass-containing surface.
• the ability to bind nucleic acids has long been known for particulate and fibrous materials. For example, DE-A-19512369 describes the use of glass nonwovens for the isolation of nucleic acids.
• nucleic acids are to be understood as meaning nucleic acids of any origin, e.g. B. nucleic acids of viroid, viral, bacterial or cellular origin. If the nucleic acids in the sample are not freely accessible, they are preferably made available with appropriate reagents. This includes both changes in pHs (alkaline), heat, repetition of extreme temperature changes (freeze / thaw), change in the physiological growth conditions (osmotic pressure), exposure to detergents, chaotropic salts or enzymes (e.g. proteases and lipases). Specimen material from which nucleic acids can be released are, in particular, cell-containing media, cell smears and tissue sections. The nucleic acids can be both RNA and DNA.
• Suitable vessels are in particular plastic vessels.
• Such vessels are made of polystyrene, polyethylene or luran, for example. These have the advantage of being easy to manufacture in the multi-injection molding process with simultaneous high mechanical stability under the conditions of the insulation process according to the invention.
• this vessel has a volume which contains the entire sample and possibly other reagents, e.g. B. to facilitate the binding of the nucleic acids to the nucleic acid binding material allowed.
• the volume is preferably between 1 and 100 ml, preferably between 5 and 50 ml.
• This vessel preferably has an essentially cylindrical basic shape which is closed on one side. On the other side of the vessel there is an opening that is suitable for receiving the sample.
• this vessel preferably has means for fastening the vessel to a closure element in the region near the opening. This means can be, for example, an outside or inside screw thread.
• the vessel preferably has a further opening (for example as a pressure compensation opening).
• Another essential element of the invention is a closure element which contains the nucleic acid-binding, liquid-permeable material.
• the nucleic acid-binding material is arranged in the closure element such that sample liquid passing from the first vessel into the second vessel must pass through the nucleic acid-binding material.
• the nucleic acids are bound to the material.
• the closure element therefore connects the opening of the first vessel to the opening of a second vessel, the nucleic acid-binding material being introduced between these openings.
• the closure element On one side of the nucleic acid binding material, the closure element has means for fastening the element on the first vessel. These means depend on the geometry of the first vessel in the area of the opening. If the first vessel has a thread, for example, the middle of the closure element is a matching counter thread. On the side of the closure element facing away from the opening of the first vessel, this means has means for fastening the closure element to a second vessel. It has proven to be advantageous to provide a screw closure here as well.
• the second vessel can be similar in shape and material to the first vessel. In particular, however, the second vessel should be able to hold at least the volume of the sample liquid.
• the second vessel is preferably the same size as the first vessel or has a volume which is so much larger than that of the first vessel that it can also take up washing liquids in addition to the sample liquid.
• the closure element can be made of the same material as the vessels, here the use of injection-moldable plastics is particularly preferred because this allows the introduction of the liquid-permeable material during the manufacture of the closure element.
• the material especially in the case of glass fiber nonwovens, can be firmly poured into the closure element during the injection molding process. However, it is also possible to introduce the material later and to fasten it in the closure element, e.g. B. by gluing or welding.
• the closure element may include other advantageous elements, e.g. B. a neck for applying negative pressure.
• This attachment piece is preferably located on the side of the closure element facing the second vessel.
• the closure element can also contain a pressure compensation connection. This is preferably located in the part of the closure element facing the first vessel or on the side facing away from the intake manifold. It is preferably designed in such a way that under normal conditions no sample liquid can escape through it into the environment. It can e.g. B. is an opening closed with a dense fleece in the area just next to the nucleic acid binding material. Furthermore, the closure element still contain internals, which ensure the controlled and even flow through the nucleic acid binding material. This includes, in particular, a distribution of the liquid flow over the entire available area of the material and the collection of the liquid flow as it enters the second vessel, thereby preventing the liquid from splashing and possible loss through the suction nozzle.
• a system which is already very suitable for carrying out the method according to the invention is based on the components of the commercially available Steriflip TM vacuum filtration system from Millipore, but a glass fiber fleece is used instead of the sterile filtration membrane.
• a preferred embodiment of the method according to the invention based on this device is described below. 5 to 30 ml of whole blood are pipetted into the first tube. In a first step, the cells should be lysed and disrupted. For this purpose, the necessary reagents, e.g. B. a chaotropic salt and / or protease added.
• the first vessel is preferably tightly closed and mixed with a lid. The cover is then removed and the closure element according to the invention is screwed on.
• the second vessel is already attached to the closure element or it is then attached to the closure element on the side of the nucleic acid-binding material facing away from the opening of the first vessel.
• the sample liquid is then caused to pass through the nucleic acid binding material. This can be done on the one hand by tipping over the assembled device (first vessel, closure element, second vessel) or also in a supportive manner by additional application of negative pressure when connecting piece of the closure element.
• the sample liquid passes through the nucleic acid binding material, the now accessible nucleic acids present in the sample are bound to the nucleic acid binding material, while other sample components pass together with the liquid into the second vessel. Air can penetrate into the first vessel from the outside through the second nozzle, and thus compensate for the negative pressure which has arisen in the first vessel.
• the isolated nucleic acids are now in the closure element, bound to the liquid-permeable material. They can now be further processed in any way. Since certain liquid amounts of the sample usually still adhere to the liquid-permeable material even after air has been sucked through, it is preferred to wash off these adhering residues.
• the first vessel can be separated from the first closure element, e.g. B. unscrewed, and the washing liquid to the material to which the nucleic acids are bound, z. B. be dropped. If a larger amount of washing liquid is to be used, it is possible to attach a filler neck to the closure element using the means for attaching the first vessel. The washing liquid can be sucked through the material by applying negative pressure into the second vessel.
• the second vessel can be removed from the closure element and replaced by a fresh, third vessel.
• the third vessel can be attached to the closure element using the means for attaching the second vessel. Then, preferably on the side to which the first vessel was attached, an elution liquid is added and, if desired, sucked through the material into the third vessel under reduced pressure.
• the elution liquid is such that the binding of the nucleic acids to the liquid-permeable material is released. It is now possible to dissolve the nucleic acids in very small volumes of elution liquid.
• the third vessel can therefore also have a smaller volume than the second and the first vessel.
• the device according to the invention is also suitable for converting nucleic acids from a larger volume to a smaller volume.
• FIG. 1A shows a closure element (1) which has both a first screw thread (2) and a second screw thread (7).
• a pressure compensation connection (8) which is sealed with a dense fleece that is air-permeable but liquid-impermeable.
• On the side of the second screw thread there is an exhaust port for negative pressure (3) and a splash guard (5).
• the nucleic acid binding material is located between the screw threads (4)
• FIG. 1B shows a first vessel (10) with a counter thread for the screw thread (2) (11) and a sample liquid (12) containing nucleic acid.
• the closure element (1) is screwed onto the first vessel containing the sample liquid in such a way that the suction nozzle (3) is removed from the sample liquid by the Material (4) is separated.
• the second vessel (20) with a counter thread for the screw thread (7) (21) is indicated.
• FIG. 1D shows the assembled device in a state in which negative pressure has already been applied and part of the sample liquid has passed through the nucleic acid-binding material (4).
• the liquid (22) that has passed collects in the second vessel (20). It can be seen that the device was turned over after screwing on the second vessel (see FIG. IC) (FIG. 1D).
• the invention also relates to a device for isolating nucleic acids from a sample containing a first vessel with an opening capable of receiving the sample and a closure element with a nucleic acid-binding liquid-permeable material, the closure element on a side facing away from the first vessel, means for fastening the element on a second vessel.
• the connection of the first vessel to the closure element should be so tight that no liquid and no gas can penetrate inwards between the closure element and the first vessel.
• the invention also relates to the use of a device with a first and a second vessel and a closure element for firmly connecting these vessels, which contains a nucleic acid-binding material, to avoid contamination during sample preparation for nucleic acid detection.
• the nucleic acids eluted from the liquid-permeable material after isolation can namely advantageously be used for the detection of one or more of these nucleic acids from the sample.
• Appropriate detection methods are known to the person skilled in the art. In particular, they include the amplification of the isolated nucleic acids and their detection using labeled nucleic acid probes.
• the described method allows the simple isolation of nucleic acids from larger sample volumes (> 10 ml) with a reduced risk of contamination of the sample, the operator and the workplace.
• the arrangement of the sample vessels and the filtration unit prevents the spread of aerosols.
• the lysis and further processing the sample can be conveniently staggered in time and space without the need to change vessels (e.g. decentralized sampling and lysis / stabilization).
• Fig. 1 serves as an illustration.
• the vessel is cooled, briefly centrifuged (Heraeus Sepatech Varifuge 3.0R), opened and 5 ml of i-propanol are added and mixed well.
• the filtration unit carries a further 50 ml sample vessel, which can be placed under negative pressure via a connection which is located on the filtration unit.
• a sample vessel can be found, for example, in the Steriflip TM Filter Unit (Millipore, Cat. No. SCGP 005 25).
• the assembled unit is now oriented (e.g. rotated) so that the lysed sample material is in the upper container, and vacuum is applied (e.g. water jet vacuum).
• the vacuum line is usually protected against contamination by an appropriate filter (e.g. Millipore Cat. No. SLFG 050 10).
• the fleece can also be dried by sucking in sterile air. Now the tube with the passage is removed and replaced with a fresh, sterile tube. 1-2 ml of an elution buffer preheated to 70 ° C. (10 mM Tris-HCl, pH 8.5) are applied to the fleece and sucked through with a vacuum. In order to obtain an optimal yield, the unit is finally centrifuged at approx. 8000 xg. The isolated nucleic acid is in the eluate and contains both DNA and RNA and is now suitable for use in PCR or RT-PCR.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Analytical Chemistry (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Wood Science & Technology (AREA)
• Immunology (AREA)
• Zoology (AREA)
• Clinical Laboratory Science (AREA)
• Physics & Mathematics (AREA)
• Biochemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=7818496

Family Applications (1)

Country Status (6)

Families Citing this family (35)

Family Cites Families (11)

• 1997
  • 1997-01-28 DE DE19702907A patent/DE19702907A1/de not_active Withdrawn
• 1998
  • 1998-01-26 EP EP98904143A patent/EP0964934A1/de not_active Withdrawn
  • 1998-01-26 WO PCT/EP1998/000408 patent/WO1998032877A1/de not_active Ceased
  • 1998-01-26 US US09/355,365 patent/US6720417B1/en not_active Expired - Fee Related
  • 1998-01-26 AU AU62132/98A patent/AU6213298A/en not_active Abandoned
  • 1998-01-26 JP JP53160798A patent/JP2001511644A/ja not_active Withdrawn
• 2004
  • 2004-02-25 US US10/786,835 patent/US20040166524A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events