Method and device for purifying nucleic acids

Technical field [0001] The invention relates to a method for isolating and purifying nucleic acids from a sample and a suitable device for it.

Background Art [0002] The introduction of polymerase chain reaction (PCR) and later alternative nucleic acid amplification systems have made it possible to use this genetic material as a sample in diagnostic tests. As a consequence, new analytical methods have now become available, especially in the diagnosis of hereditary diseases, predisposition to certain diseases and infectious diseases, which, among other things, allow early diagnosis of the disease state.

[0003] In order to transform genetic material into a form suitable for enzymatic amplification, it is necessary to release it from the material of the biological sample. In addition, the nucleic acid must be protected from degradation by nucleases derived from biological material or the environment, and against degradation by chemical reaction conditions. The most stringent requirements relate to the release of the biological sample and the nucleic acid isolated from it. The nucleic acid for amplification should be in a buffered aqueous solution, essentially salt-free.

[0004] While the PCR reaction usually uses very small amounts of analyte (in the pg-ng range), particular problems require the processing of a larger amount of sample. For example, to identify circulating tumor cells with the sensitivity of one tumor cell against the background of normal cells, the nucleic acid should be isolated from a 10-20 ml blood sample. After homogenizing the sample, an aliquot of isolated RNA can be tested for expression of tumor associated genes.

In addition to classical methods for the isolation of nucleic acid by enzymatic, mechanical or chemical lysis of a material sample, subsequent extraction of proteins and lipids with phenol and phenol / CHCl3, and precipitation of the nucleic acid from the aqueous phase with ethanol or iso-propanol (Sambrook, J. and et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989, 2nd Edition, 9.16-9.23; Ausubel, FM et al. Current Protocols in Molecular Biology, John Wiley & Sons, 1987, 2.1.1-2.4.5), several commercial kits have been developed in recent years, in particular for the preparation of PCR samples that use the nucleic acid property known since the late 1970s to bind to glass surfaces in the presence of chaotropic salt, (Vogelstein, B. et al., Proc. Natl. Acad. Sci. USA 75 (1979) 615-619). Other components of the biological material, such as proteins, lipids or salts, are not bound and are therefore separated. Centrifugal vessels lined with glass fleece or a silica gel suspension enabling the portioning process are known. In addition, multiple-type devices in the form of strips and 96-well microtiter plates, with a built-in glass fleece, are known that are supported by a vacuum chamber mounted below, as well as by centrifugation. In such methods, the volume of samples is often limited. In addition, large amounts of buffer are required for effective leaching of nucleic acids from the glass fleece, which results in a diluted solution of isolated molecules and requires additional preparation steps for certain applications.

[0006] The modified method (Miller et al., Nucl. Acids Res., 16: 1215) uses a concentrated salt solution to precipitate proteins and other accompanying substances after lysing the sample material. Nucleic acids in

The EP supernatant is then precipitated with ethanol and collected by centrifugation. After suspension, the nucleic acids can be used for amplification.

[0007] WO 93/11221 discloses a method and apparatus for isolating and purifying nucleic acids that uses anion exchangers and mineral carrier substances. US 5104533 discloses a pressure compensation filter unit. US 4,270,921 discloses a combination of microcolumn and centrifuge tube. WO 98/32877 discloses a nucleic acid isolation device that consists of two vessels connected by a closure element that contains material for binding nucleic acids. US 4956298 discloses a separation or reaction column consisting of a centrifuge vessel and a receiving body, the receiving body comprising column material, and the centrifuge vessel collecting leakage from the receiving body. DE 19512361 discloses a method for isolating biological material that uses a compressible porous matrix to bind the material and in which the material is compressed to gel the material. EP 588564 describes an affinity separation device comprising a capture membrane disposed in a pipette tip. WO 96/41810 discloses the removal of DNA from a cell suspension using a hollow membrane filter and an ion exchange step. The production of a device containing material for binding nucleic acids is known from EP 738733. WO 02/053256 discloses a device and purification method comprising a sample container with an insert part for a column, while a column module is mounted in the said insert part for a column. US 6177009 and German utility models DE 298 03 712 U1 and DE 202 18 503 U1 describe a device for treating biomolecules comprising a separation column, which has a separation device and a collecting vessel for leaking liquid.

Summary of the invention [0008] The object of the present invention was to provide a new device and a new method for purifying or isolating nucleic acids from larger volumes.

The object is achieved according to the invention by a device, the individual parts of which are shown in Fig. 1. Furthermore, a method is described in which the device according to the invention is used and whose individual phases are also schematically shown and as an example in Fig. in which the device of the invention is used, it provides nucleic acids with greater efficiency.

[0010] One object of the present invention is a nucleic acid purification or isolation device consisting of a first hollow body 100 with sample inlet 101 and an outlet 102 and a second hollow body 200 with inlet 201 and outlet 202, the inlet the second hollow body 201 is operably connected to the outlet opening of the first hollow body 102, the first and second hollow bodies can be separated from each other, nucleic acid-binding material 203 is placed in front of the outlet opening of the second hollow body 202, the second hollow body 200 has a smaller volume compared to the first hollow body 100, and the first hollow body 100 has a volume greater than 5 ml.

[0011] Another aspect of the invention relates to a closable vessel 400 that comprises a device according to the invention.

[0012] The invention also relates to a method of purifying or isolating nucleic acids from a sample, comprising the steps of:

a) providing the device according to the invention,

b) transferring the sample to the device through the inlet opening of the first hollow body,

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c) passing the sample from the second hollow body through the nucleic acid binding material to the vessel during which the nucleic acids bind to the nucleic acid binding material,

d) optional washing of the nucleic acids associated with the nucleic acid binding material,

e) disconnecting the second hollow body from the first hollow body and transferring the second hollow body to the receiving vessel 900,

f) washing of nucleic acids associated with nucleic acid binding material,

g) elution of the nucleic acids associated with the nucleic acid binding material, whereby the nucleic acids are collected in a second receiving vessel 13 and are thus purified or isolated.

[0013] The invention also relates to a kit for purifying or isolating nucleic acids from a sample, consisting of the device according to the invention or a vessel according to the invention and chaotropic reagents for binding the nucleic acids to the nucleic acid binding material.

[0014] Another object of the present invention is the use of the device according to the invention or the vessel according to the invention for purifying or isolating nucleic acids from a sample.

[0015] Nucleic acid binding material is understood to be material with which nucleic acids bind non-covalently under certain conditions, while other substances from the sample do not bind under these conditions. This binding of nucleic acids is reversible and hence the nucleic acids can later be re-eluted from the material by changing the conditions.

[0016] A matrix within the scope of the present invention is understood as a material in which particles or fibers of a nucleic acid binding material are embedded. The matrix material is liquid permeable so that the sample can pass through the matrix, the nucleic acids may come into contact with the nucleic acid binding material, and other components of the sample may leave the matrix. Solid materials, having a small diameter, are referred to by specialists as particles. Such particles preferably have a substantially spherical surface. Lamellar or filamentary particles made of nucleic acid binding material are referred to as fibers.

[0017] Within the scope of the invention, the hollow body is a hollow structure with an inlet through which the sample can enter the hollow body and with an outlet through which the sample can leave the hollow body. In contrast, the vessel is an empty structure with only one inlet opening through which the sample can enter the vessel. Thus it can be used to collect the sample.

[0018] Within the scope of the invention, the functional connection of the hollow bodies means that the two hollow bodies are connected in such a way that it is possible to carry out the method according to the present invention. To this end, it should be possible to disconnect the connection as required, it should be liquid impermeable and, for specific applications, it should also prevent air exchange with the environment. In addition, it should ensure that the sample passes without loss from the first hollow body to the second hollow body.

[0019] Chaotropic reagents are understood to be substances that alter the secondary, tertiary and / or quaternary structure of proteins or nucleic acids, but at least do not affect their primary structure. Examples are guanidinium thiocyanate, guanidinium hydrochloride, NaI, KI, sodium thiocyanate or combinations of these substances. Within the scope of the invention, chaotropic reagents are understood to be all chemicals that disrupt the orderly structure of liquid water and thereby cause DNA or RNA from such an aqueous solution to bind to the glass surface. Other substances, such as NaCl, KCl or, may be present in the solution to modify the ionic strength

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CaCl2. The ability of DNA and RNA to bind to glass surfaces under chaotropic conditions can be used to isolate them from a solution containing other biological materials, since binding to the glass surface is reversible. If, for example, the concentration of chaotropic reagents is reduced or the chaotropic reagents are completely removed, the DNA or RNA may be re-eluted.

Description of the figures [0020]

Figure 1: Schematic representation of the method according to the invention shown as an example of the use of the device according to the invention.

Figure 2: An alternative embodiment of the device according to the invention, in which means are introduced that allow a pressure difference to be generated and thus ensure the passage of the sample through the nucleic acid binding material.

Detailed description of the invention [0021] One object of the present invention is a nucleic acid purification or isolation device consisting of a first hollow body 100 with sample inlet 101 and an outlet 102 and a second hollow body 200 with inlet 201 and outlet 202, at what the inlet from the second hollow body 201 is operatively connected to the outlet of the first hollow body 102, the first and second hollow bodies can be separated from each other, the nucleic acid binding material 203 is located in front of the outlet opening of the second hollow body 202, the second hollow body 200 has a smaller volume compared to the first hollow body 100 and the first hollow body 100 has a volume greater than 5 ml.

[0022] Materials that bind nucleic acids are known to the person skilled in the art. The material may be in the form of particles as well as of fibers. It has been proven that if the material consists of particles, it is preferable to immobilize these particles, for example by introducing them between liquid-permeable layers, e.g. fabrics or fleece, made of fibrous material such as cellulose or plastics that have such narrow pores, that the particles are held between layers. The nucleic acid binding material preferably consists mainly of silicon dioxide or contains silicon dioxide in the form of fibers or particles. The nucleic acid binding material is particularly preferably a glass fleece or silica gel or consists of zeolite. The nucleic acid binding material also preferably consists of metal oxides or mixed metal oxides in the form of fibers or particles. The nucleic acid binding material particularly preferably consists of alumina, hafnium oxide or zirconia or contains alumina, hafnium oxide or zirconia in the form of fibers or particles.

[0023] The nucleic acid binding material is preferably a fibrous material, e.g. in the form of fabrics or fleece. Suitable materials are, for example, known for methods of isolating nucleic acids using centrifuge tubes (EP 738733) or multiple strip devices (EP 616638). The nucleic acid binding material must have the property that a liquid sample can pass through this material without any additional force or force application, e.g. applying pressure or vacuum. However, because the nucleic acids are not bound in the present method by filtration of the nucleic acids from the sample, but by a method that uses the affinity of the nucleic acids for the surface, it is possible to use material with relatively thick pores. This facilitates the flow of even relatively viscous liquid samples.

4EP 1702066 B1 [0024] The liquid-permeable nucleic acid binding material is capable of binding nucleic acids, but allows the surrounding liquid and other components dissolved in it, such as proteins, etc. to pass. In the first variant, the nucleic acids can be specifically bound sequences using capture probes attached to the surface of the material. Capture probes have a base sequence that can bind to the complementary base sequence in nucleic acids for isolation under hybridization conditions. The use of sequence-specific materials allows the selective isolation of nucleic acids with a specific sequence. A method of binding nucleic acids to peptide nucleic acids on the surface of solids is described, for example, in WO 95/14708.

[0025] Suitable nucleic acid binding material are, for example, zirconia, hafnium oxide or alumina (EP 897978), as well as titanium oxide. The hydrated surfaces of these materials have a sufficient positive charge to bind negative nucleic acids. Oxide surfaces can be hydrated in alkaline solutions. The nucleic acids can then be eluted by washing with low molecular weight alcohols or other washing with low pH solutions.

[0026] In a preferred embodiment, the liquid-permeable nucleic acid-binding material has a surface containing glass. The ability of glass, in the form of particles or fibrous, to bind nucleic acids has long been known. Chaotropic reagents such as guanidinium thiocyanate, guanidinium hydrochloride, NaI, KI, sodium thiocyanate or combinations of these substances are necessary for reversible binding to glass surfaces (US 5,234,809). DE-A-19512369 describes the use of a glass fleece to isolate nucleic acids. EP-B-0389 063 proposes a method in which a sample is mixed with a chaotropic mixture of guanidinium salt and silica particles. Under such conditions, the nucleic acids bind to the silica surface in a sequence-independent manner. Other components of the sample can be washed away and the nucleic acids can then be eluted with an aqueous buffer.

[0027] Nucleic acids within the meaning of the invention are understood to be nucleic acids of any origin, e.g. nucleic acids of viroidal, viral, bacterial or cellular origin. If the nucleic acids are not readily available in the sample, they are preferably made available by the action of appropriate factors. These include pH (alkaline) changes, heating, repeated extreme temperature changes (freezing / thawing), changes in physiological growth conditions (osmotic pressure), the action of detergents, chaotropic salts or enzymes (e.g. proteases and lipases). Specimen materials from which nucleic acids can be released in this way are, in particular, cell-containing media, cell smears and tissue sections. The nucleic acids can be RNA as well as DNA.

[0028] The device according to the invention consists of two 100/200 hollow bodies which are connected by a functional connection 3 and each having an inlet opening 101/201 and an outlet opening 102/202 for the sample. The device according to the invention is preferably of such a shape that it at least partially fits the vessel 400. In this context, operatively connected means that the two hollow bodies are connected in such a way that the method of the present invention can be carried out. To this end, it should be possible to disconnect the connection as required, it should be impermeable to liquids and, for specific applications, should prevent air exchange with the environment. In addition, it should ensure that the sample passes without loss from the first hollow body to the second hollow body. The hollow bodies are preferably substantially cylinders. The holes are essentially round. In addition to the cylindrical portion at the inlet opening, the first hollow body particularly preferably also has a portion

-5EP 1702066 B1 tapering conically towards its outlet. The first hollow body preferably has a volume above 5 ml. Sample 5, which has passed through the outlet opening 102 of the hollow body 100, enters without loss into the second hollow body 200 through the preferably circular inlet opening 201 due to the functional connection 3.

[0029] The liquid-permeable, nucleic acid-binding material 203 is in a second hollow body. The nucleic acid binding material is preferably located at the circular outlet 202 of the hollow body in such a way that the emerging sample liquid must pass through the nucleic acid binding material. In this process, nucleic acids bind to nucleic acid-binding material. [0030] The second hollow body may be similar to the first hollow body in appearance and material. In particular, the hollow bodies should be able to receive at least the volume of the liquid sample 5. The second hollow body preferably has a smaller volume compared to the first hollow body. This hollow body preferably has a shape such that it at least partially fits into the receiving vessel 900. In addition, the second hollow body contains material and has a size such that it can be centrifuged at speeds up to 14,000 rpm.

[0031] Both hollow bodies have means that allow functional and reversible connection between them. The functional connection of the first and second hollow bodies is preferably a screw connection, i.e. for example with an external or internal thread. If the first hollow body, for example, has a thread, the center of the second hollow body is fitted with a counter thread. In another preferred embodiment of the present invention, the first and second hollow bodies are joined together by pressure forces, e.g., by a plug connection.

[0032] The two hollow bodies preferably together have a volume that allows them to receive the entire sample and other reagents, e.g. to facilitate the binding of nucleic acids to the nucleic acid binding material.

The volume is above 500 microliters, preferably in the range of 1 - 1000 ml, particularly preferably in the range of 1 - 100 ml and most preferably in the range of 5 - 50 ml or 10 - 30 ml. A volume of 10, 20 or 30 ml is particularly preferred. The first hollow body preferably has a volume in the range of 1 - 100 ml, particularly preferably in the range of 5 - 50 ml, most preferably in the range of 10 - 30 ml. The second hollow body preferably has a volume below 2 ml, particularly preferably in the range of 1-2 ml.

[0033] A preferred variant of the device according to the present invention is a device characterized in that the two hollow bodies together have a volume in the range of 5 - 50 ml.

[0034] Furthermore, the first hollow body preferably has, near the inlet opening 101, means for supporting 103 in the hollow body vessel 400 such that its position is fixed. The vessel has an inlet 401 which is preferably round. The vessel preferably has a cylindrical portion at the inlet 401 and a conical region at its end. The vessel is used to receive liquid 8 which appears from the second hollow body 200 after passing through the nucleic acid binding material 203. Preferably the vessel can be closed.

[0035] The vessel 400 has a particularly advantageous shape and is provided with the closing means 6 in such a way that it can completely take up the functionally connected hollow bodies 100/200 and the vessel 400 can be closed by the closing element 7. The closing element is preferably a plastic nut plastic. A preferred means for supporting the first hollow body in the vessel 400 is a circular flange 103 which extends beyond the edge of the vessel such that the hollow body rests on it and does not fall further into the vessel. The circular collar preferably has a shape such that it is pressed against the vessel

-6EP 1702066 B1 through the closing element 7 and acts as a closure. In this case, the closing element is preferably a nut that is applied from above and the vessel has a correspondingly opposite thread.

[0036] Alternatively, the vessel may have a tapered portion in its inlet opening 401, such that the first hollow body cannot fall further into the vessel in a particular position even if there is no additional support means. In this case, the vessel can also be closed with a closing element. It cannot be ruled out that the two functionally connected hollow bodies only partially fit into the vessel, and in this case at least the outlet opening of the second hollow body must completely extend into the vessel.

[0037] In a further preferred embodiment of the device according to the present invention, which is shown in Fig. 2, the vessel 400 is provided with means for creating a pressure difference. This pressure difference can force or sustain the passage of the sample through the nucleic acid binding material. For this purpose, a vessel is provided with a connecting element 16 for creating an overpressure or underpressure and a closing element 7 with a pressure equalizing connection 17 is provided. In this device variant, it is necessary that the space connecting the two hollow bodies is also gas impermeable, otherwise the pressure difference will not be generated by the nucleic acid binding material.

[0038] The second hollow body 200 preferably has a shape such that it at least partially fits into the receiving vessel 900. This receiving vessel preferably has a substantially circular inlet opening 901. The receiving vessel 900 is preferably closed and consists of a cylindrical portion at the inlet opening and a conical portion at its end. The receiving vessel is particularly preferably designed such that the second hollow body 200 provided with the support means 204 fits thereto and the vessel can be closed with the closing element 10. Like the first hollow body 100, the second hollow body also preferably has a round flange as the supporting means 204, which extends beyond the edge of the receiving vessel. When a screw cap 10 with thread 11 is used, the circular collar is pressed against the vessel and acts as a closure. Also in this case, the receiving vessel may also be cone-shaped to support the second hollow body in a fixed position in which the hollow body fully or partially enters the receiving vessel.

[0039] In a further embodiment of the present invention, the receiving vessel is preferably provided with means for creating a pressure difference. This pressure difference can force or sustain removal of residual liquid from the fleece and elution of nucleic acids from the material. To this end, a receiving vessel is provided with an overpressure or negative pressure connecting element and a closing element with a pressure equalizing connection is provided.

[0040] The first and second hollow bodies, receiving vessels or vessels of the devices of the invention are made of materials that do not bind nucleic acids. The first and second hollow bodies, vessels or receiving vessels of the devices according to the invention are preferably made of plastic, metal or composite material. Particularly preferred plastics are polypropylene, polystyrene,_® polyethylene or Luran^®. They have the advantage that they can easily be produced in a multiple injection process and have high mechanical durability under the conditions of the insulation method according to the invention.

[0041] Plastics that can be injection molded are particularly preferred as materials for hollow bodies because they immediately allow the introduction of a liquid-permeable nucleic acid binding material during manufacture of the second hollow body. Especially in the case of glass fiber rune, the material can be permanently flooded in the closing element during the process

-7EP 1702066 B1 injection molding. Although, it is also possible to attach the material after production of the second hollow body in the injection molding process e.g. by gluing, welding or immobilizing in the clamping ring. The process for producing a hollow body containing nucleic acid binding material is described in patent document EP 0 738 733. Such separation columns containing nucleic acid binding material are also commercially available (High Pure column^TM from Roche Diagnostics GmbH, Mannheim).

[0042] The invention also relates to a closable vessel which comprises a device according to the invention. The closable vessel preferably has a shape such that it is capable of receiving the entire inventive connection of two hollow bodies and can be closed by means of a closing member. The closable vessel is particularly preferably a commercial centrifuge vessel, such as a plastic tube (Falcon tube), 50 ml, which can be closed with a screw cap.

[0043] Another aspect of the present invention is a method of purifying or isolating nucleic acids from a sample, comprising the steps of:

a) providing the device according to the invention,

b) transferring the sample to the device through the inlet opening of the first hollow body,

c) passing the sample from the second hollow body through the nucleic acid binding material to the vessel during which the nucleic acids bind to the nucleic acid binding material,

d) optional washing of the nucleic acids associated with the nucleic acid binding material,

e) disconnecting the second hollow body from the first hollow body and transferring the second hollow body to the receiving vessel 900,

f) washing of nucleic acids associated with nucleic acid binding material,

g) elution of the nucleic acids associated with the nucleic acid binding material, whereby the nucleic acids are collected in a second receiving vessel 13 and are thus purified or isolated.

[0044] A preferred variant of the method according to the present invention uses the device according to the invention with a nucleic acid binding material which consists mainly of silicon dioxide or contains silicon dioxide in the form of particles or fibers, or is particularly preferably a glass fleece or silica gel or consists of zeolite , and a sample to which chaotropic reagents have already been added through the inlet opening of the first empty body before being transferred to the device, the concentration of chaotropic reagents is in the range of 1 M - 8 M.

[0045] A preferred embodiment of the method according to the invention, which is based on the device according to the invention, is described below. First, cells with 5 to 30 ml of whole blood, serum, plasma or other body fluids are lysed, destroyed and additionally required reagents e.g. chaotropic salt or / and protease are added to the liquid sample. In addition, the device according to the invention is placed in a collection vessel for nucleic acid-free liquid (see stages I and II in Fig. 1). After the sample 5 has been transferred to the device through the inlet opening of the first hollow body, the vessel is preferably sealed with a lid (stage III in Fig. 1).

[0046] In the next step, the liquid sample is passed through the nucleic acid binding material (step IV in Fig. 1). The sample can pass due to gravity or also preferably due to the centrifugation of the device. The sample can also advantageously pass through the nucleic acid binding material by applying a pressure difference. In this embodiment of the invention (see Fig. 2) the device is

-8EP 1702066 B1 provided with additional means e.g. two connecting elements 16/17 for creating pressure. One connecting element for creating an overpressure or underpressure is preferably located on the vessel itself, and the other connecting element is placed on the vessel closing element, acting as a pressure equalizing connection.

[0047] As the liquid sample passes through the nucleic acid binding material, the nucleic acids present in the sample are bound to the nucleic acid binding material, while the other components of the sample along with the liquid 8 pass into the vessel. Isolated nucleic acids are now found in the nucleic acid binding material of the second hollow body, which, if necessary, can be separated from the first hollow body and further processed by any desired method.

[0048] Since some amounts of liquid containing impurities usually still adhere to the liquid-permeable material, even after centrifugation, it is possible to remove still adhering substances by an optional rinsing step to isolate particularly pure nucleic acids before removing the device from the vessel and separating the functional connection of the empty bodies . For this purpose, the flushing liquid can, for example, be added through the inlet opening of the first hollow body, so that it flushes the material with which the nucleic acids have bound during passage, and is then collected in the vessel. The rinsing step can preferably be carried out by introducing a pressure difference or by centrifuging the device.

[0049] To re-detach the nucleic acids from the liquid-permeable nucleic acid binding material, the second hollow body can be removed from the device (stage V in Fig. 1). For this purpose, the second hollow body, after separating it from the first hollow body, is preferably connected to a receiving vessel 900 to first remove residual liquid 12 present in the fleece (stage VI in Fig. 1). This rinsing is preferably carried out by centrifuging the device because the second hollow body connected to the receiving vessel can be subjected to strong centrifugation forces due to its small diameter. Then the second hollow body is connected to the second receiving vessel 13, which should collect the eluate (stage VII in Fig. 1). Elution liquid 14 is such a composition of components that abolishes the binding of nucleic acids to the nucleic acid binding material. The conditions under which nucleic acids can be detached again depend on the material used and the process that can again be supported by introducing a pressure difference or by centrifugation (step VIII in Fig. 1).

[0050] The elution is preferably carried out by centrifugation because it allows the nucleic acids to be dissolved in very small volumes of elution liquid, and also allows the reduction of the amount of nucleic acids remaining in the nucleic acid binding material. Centrifugation for elution and rinsing is preferably carried out using higher centrifugation forces than centrifugation in sample passages or optional rinsing. Centrifugation for elution and rinsing is preferably carried out at less than 5000 g, and centrifugation in sample passages or optional rinsing is preferably carried out at less than 5000 g. The second hollow body and receiving vessel are particularly preferably shaped so that they can be spun together, e.g. in an Eppendorf centrifuge (Eppendorf, Hamburg, Germany) at over 10,000 g. This is possible because the collection vessel must have a significantly smaller volume than the first vessel that can be subjected to a significantly smaller centrifugal force (e.g. in a Beckman table top centrifuge (Beckman Coulter, Inc., USA) at about 3000 g) and thus requires a larger volume of elution liquid. Thus, the device of the invention is not only useful for isolating nucleic acids, but also for transferring nucleic acids from a larger volume of 5 to a smaller volume of 15.

-9EP 1702066 B1 [0051] The volume of the nucleic acid purification or isolation device is an essential feature of the present invention. On the one hand, the volume of the device must be large due to the application of diluted, large-volume samples, and on the other hand, the size of the device must be small in order to perform the lysis procedure with small amounts of lysis buffer. The amount of lysis buffer needed for efficient nucleic acid elution depends, among other things, on the size of the nucleic acid binding material and on the forces supporting the lysis procedure. The reversible attachment of the large first hollow body to the small second hollow body, including the nucleic acid binding material, meets both of these requirements. First, the total volume of both hollow bodies provides the possibility of using diluted, bulky samples during the binding process. Secondly, after the binding process, the small second hollow body can be removed from the device and the nucleic acid can be eluted with a much higher centrifugal force, which is possible for a device used in the field with the same initial sample volume.

[0052] Another aspect of the invention is a kit for purifying or isolating nucleic acids from a sample, which consists of a device according to the invention or a vessel according to the invention and chaotropic reagents for binding nucleic acids to the nucleic acid binding material. The kit may additionally contain other plastic parts that are required to carry out the method of the invention, such as microtiter plates or simple reaction vessels, such as Eppendorf reaction tubes (Eppendorf, Hamburg, Germany). In addition, the kit may contain further reagents that are necessary in the process of the invention, e.g. lysing buffer containing chaotropic reagents, detergent, alcohol or mixtures of these substances that lys cells, rinsing buffer containing chaotropic reagents and / or alcohol or acidic buffer for washing the nucleic acid binding material to which the nucleic acids or elution buffer are bound, enabling the detachment of nucleic acids from the nucleic acid binding material. According to the invention, the components of the kit can be provided individually or in storage containers. Reagents are usually offered ready-to-use, although they may be sold as stock solutions, which should be diluted before use.

[0053] The invention also relates to the use of the device according to the invention or a vessel according to the invention for purifying or isolating nucleic acids from a sample.

[0054] Specifically, the invention includes the following aspects:

1. Nucleic acid purification or isolation device consisting of a first hollow body 100 with sample inlet 101 and an outlet 102 and a second hollow body 200 with inlet 201 and outlet 202, the inlet from second hollow body 201 being operatively connected with the outlet opening of the first hollow body 102, the first and second hollow bodies can be separated from each other and the nucleic acid binding material 203 is located in front of the outlet opening 202 of the second hollow body.

2. The device of item 1, wherein the second hollow body has a smaller volume compared to the first hollow body.

3. The device according to one of the items 1 or 2, in which the device has a shape which at least partly fits into the vessel 400.

4. The device according to one of items 1 to 3, in which the second hollow body has a shape which at least partly fits into the receiving vessel 900.

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5. The device according to one of items 1 to 4, in which the first and second hollow bodies are connected together by a bolted connection.

6. The device according to one of items 1 to 4, in which the first and second hollow bodies are joined together by pressure forces.

7. The device according to one of items 1 to 6, in which the first and / or second hollow body are substantially cylinders.

8. The device according to item 7, in which the first hollow body in addition to the cylindrical part at the inlet has a part which tapers conically towards the outlet.

9. The device according to one of items 1 to 8, wherein the holes of the first and / or second hollow body are substantially round.

Ten. The device according to one of items 1 to 9, characterized in that the first hollow body has a volume of more than 5 ml.

11. The device according to one of the items 3 to 10, in which the vessel is closed.

12. The device according to one of items 3 to 11, in which the vessel consists of a cylindrical part at the inlet and a conical part at its end.

13. The device according to one of items 3 to 12, in which the vessel has means for generating a pressure difference.

14. The device according to one of items 4 to 13, in which the receiving vessel is closed.

15. The device according to one of the items 4 to 14, in which the receiving vessel is closed and consists of a cylindrical part at the inlet opening 401 and a conical part at its end.

16. The device according to one of items 4 to 15, in which the receiving vessel has means for generating a pressure difference.

17. The device according to one of items 4 to 16, in which the inlet openings of the vessel 401 and / or the receiving vessel 901 are substantially circular.

18. A device according to one of items 1 to 17, characterized in that the nucleic acid binding material consists mainly of silicon dioxide or contains silicon dioxide in the form of fibers or particles.

19. Device according to one of items 1 to 18, characterized in that the nucleic acid binding material is a glass fleece or silica gel or consists of zeolite.

twenty. A device according to one of items 1 to 19, characterized in that the nucleic acid binding material consists of metal oxides or mixed metal oxides or contains metal oxides or mixed metal oxides in the form of fibers or particles.

21. A device according to one of items 1 to 20, characterized in that the nucleic acid binding material consists of alumina, hafnium oxide or zirconia or contains alumina, hafnium oxide or zirconia in the form of fibers or particles.

22. A device according to one of items 1 to 21, characterized in that the first or second empty body, receiving vessel or vessel is made of a material that does not bind nucleic acids.

23. A device according to one of items 1 to 22, characterized in that the first or second empty body, receiving vessel or vessel is made of plastic, metal or mixed material.

24. The device according to item 23, characterized in that the second hollow body is made of polypropylene.

25. A closable vessel 400 that contains the device according to one of items 1 to 24.

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26. A method for purifying or isolating nucleic acids from a sample by:

a) delivery of the device according to one of items 1 to 25,

b) transfer of the sample to the device through the inlet opening of the first hollow body,

c) passing the sample from the second hollow body through the nucleic acid binding material to the vessel during which the nucleic acids bind to the nucleic acid binding material,

d) optional washing of nucleic acids associated with the nucleic acid binding material,

e) disconnecting the second hollow body from the first hollow body and transferring the second hollow body to the receiving vessel 900,

f) washing of nucleic acids associated with nucleic acid binding material,

g) elution of the nucleic acids associated with the nucleic acid binding material, whereby the nucleic acids are collected in a second receiving vessel 13 and are thus purified or isolated.

27. The method of item 26, characterized in that the device in one of items 18 or 19 is used and additional chaotropic reagents are added to the sample through an inlet in the first empty body prior to transfer to the device such that the concentration of chaotropic reagents is between 1 M and 8 M.

28. The method according to item 26, characterized in that the passage of the sample in step c), optional rinsing in step d), rinsing in step f) or elution in step g) are carried out by applying a pressure difference.

29. The method according to item 26, characterized in that the passage of the sample in step c), optional rinsing in step d), rinsing in step f) or elution in step g) are carried out by centrifugation.

thirty. The method according to item 29, characterized in that the centrifugation in steps f) and g) is performed at a higher centrifugation force compared to the centrifugation in steps c) and d).

31. The method according to item 29, characterized in that the centrifugation in steps c) and d) is carried out at a centrifugation force of less than 5000 g.

32. The method according to item 29, characterized in that the centrifugation in steps f) and g) is carried out at a spin force of more than 5000 g.

33. Kit for purifying or isolating nucleic acids from a sample, consisting of:

a) devices according to one of items 1 to 24 or dishes according to item 25,

b) chaotropic reagents for binding nucleic acids to the nucleic acid binding material.

34. Use of the device according to one of items 1 to 24 or the vessel according to item 25 for purification or isolation of nucleic acids from the sample.

[0055] The invention is further elucidated by means of the following examples, publications and figures, the scope of protection of which is based on the claims. The described methods should be understood as examples that describe the invention even after modifications thereof.

Examples

Example 1 [0056] The present example of DNA isolation from 5 ml serum is intended to further clarify the present invention. The so-called 'UpScale HighPure' method uses a set called 'UpScale HighPure', which consists of commercially available 'HighPure' columns^TM"(Roche Diagnostics GmbH, Mannheim, Germany), by volume insert and reagents that are identical to the kit reagents

-12EP 1702066 B1 _® "MagNA Pure^®LC Total Nucleical Acid Isolation Kit-Large Volume "(Roche Diagnostics GmbH, Mannheim, Germany). The volumetric connection (made of polypropylene) has a cylindrical shape in this example (internal diameter 2.5 cm, length 5 cm) with a filling volume of about 25 ml, a circular inlet and outlet opening in which the HighPure column (volume about 1, 5 ml). Eppendorf centrifuge (Eppendorf, Hamburg, Germany), Beckman centrifuge (Beckman Coulter, Inc., USA) with Falcon tubes, commercial vortex stirrer and Eppendorf reaction tubes (Eppendorf centrifuge) can also be used to isolate DNA by means of the "UpScale HighPure" method. Eppendorf, Hamburg, Germany).

DNA isolation procedure [0057] 250 μl Proteinase K 40 mg / ml solution is placed in a 50 ml Falcon tube. 5 ml of sample (serum or plasma) are added, shaken with vortex and then incubated for 10 min. in room temperature. In the next step, 6.25 ml of lysis / binding buffer are added, shaken with vortex and the solution is incubated for 10 min. at 65 ° C. Then centrifuged at 1900 g (foam removal). In the next step, 3.125 ml of isopropanol are added, mixed and centrifuged at 1900 g, then the mixture is allowed to stand for 10 min. in room temperature. In the next step, the mixture is applied in one portion (about 15 ml) to the volumetric connection of the device, the remaining liquid residue is also applied to the column by pipetting. It is centrifuged first for 2 minutes. at 1900 g (including acceleration) and then for 1 min. at 3300 g. In the next step, the eluate is discarded.

[0058] Various washing steps are followed (the wash buffers used are commercially available _® "MagNA Pure^®LC Total Nucleic Acids Isolation Kit-Large Volume "Cat. No. 3264793, Roche Diagnostics GmbH, Mannheim, Germany): First place the device in a new 50 ml Falcon tube, add 2 ml of wash buffer 1 and centrifuge for 2 min. at 3300 g. Then 2 ml of wash buffer 2 are added and centrifuged for 2 min. at 3300 g. In the next step, add 2 ml of wash buffer 3 and centrifuge for 2 min. at 3300 g (3 rinsing steps can be carried out without changing the Falcon tube). The volumetric attachment is removed from the HighPure column and the HighPure column is placed in an Eppendorf tube, the cap is sealed and centrifuged in an Eppendorf centrifuge for 1 min. at 20,000 g. This removes the residual liquid from the fleece.

[0059] The next step involves DNA elution. 50 - 100 μl of elution buffer are applied to the fleece and zipper HighPure column. Then incubate for 3 minutes. at room temperature and then centrifuged for 1 min. at 20,000 g in Eppendorf centrifuge. The Eppendorf tube contains the eluate and the HighPure column can be discarded.

Example 2 [0060] This example shows a comparison of DNA isolation according to the inventive method and isolation according to the state of the art. A serum sample is prepared analogously to Example 1 and separated equally into two HighPure columns with a volume connection. The experimental procedure for both columns is identical to the liquid residue removal step. In one column, the volume attachment is separated from the HighPure column, liquid residue from the fleece is removed by centrifugation using an Eppendorf centrifuge (about 15 μθ and bound nucleic acid is then eluted in 50 μl using an Eppendorf centrifuge.

[0061] In contrast, in the second column the connection between the HighPure column and the volumetric connection is left, the whole device is put into a Falcon tube and the elution is carried out with a 50 μl solution in a Beckman centrifuge (2 min. 3300 g), without removing liquid residues first fleece.

[0062] The results show that the average efficiency of DNA recovery was about 30% lower in 10 experiments without separation of the device and without centrifugation in the Eppendorf centrifuge.

Example 3 [0063] The following example shows another comparison of DNA isolation according to the inventive method and isolation according to the prior art. A serum sample is prepared analogously to Example 1 and distributed equally between the HighPure column^TM with volumetric connection and column "NucleoSpin^® Funnel ”from Macherey & Nagel, Duren, Germany (catalog number 740959). The experimental procedure for both columns is identical to the liquid residue removal step. In the case of the HighPure column^TM the volumetric attachment is separated from the HighPure column, liquid residue in the fleece is removed by centrifugation with an Eppendorf centrifuge (about 15 μΐ), and the bound nucleic acid is then eluted in 60 μΐ with an Eppendorf centrifuge. [0064] In contrast, the Macherey & Nagel column is inserted into a Falcon tube and the elution is carried out with a 60 μl solution in a Beckman centrifuge (2 min. 3300 g), without first removing liquid residue from the fleece. [0065] The results show that the average efficiency of DNA recovery using a HighPure column^TM with a volumetric attachment was about 3 to 4 times better for 5 different serum samples (3 determinations per sample) than when using the Macherey & Nagel column.

Example 4 [0066] Five serum samples used in Example 3 are also used to compare the performance of the inventive device with the performance of two commercial isolation precipitation kits or _® DNA purification, the "RTP^® DNA / RNA Virus Supersense "from Invitek Gm, Berlin, Germany (NT cat. 10404002, series OC030036) and the" QIAmp^® UltraSens^TM"From Qiagen GmbH, Hilden, Germany (cat. No. 53704, series 11862713). Both kits are used according to the procedure recommended by the manufacturers (Invitek: version TH11 / 03.2 March 2003; Qiagen: 01/2003), while for the Qiagen kit the procedure is adapted to a volume of 5 ml instead of 1 ml.

[0067] Compared to the HighPure column^TMadapted to larger volumes, the average amount of DNA isolated with the Qiagen kit was reduced by about 9 times, and the average amount of DNA isolated with the Invitek kit was reduced up to about 80 times.

List of references [0068]

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