WO2007101653A1

WO2007101653A1 - Method of carrying out a reaction for replicating a nucleic acid

Info

Publication number: WO2007101653A1
Application number: PCT/EP2007/001903
Authority: WO
Inventors: Jürgen SCHÜLEIN; Ugur ÜLKER; Jürgen KRAUSE; Dirk Kuhlmeier; Roland Barten; Herbert Argauer
Original assignee: Directif Gmbh; Kloiber, Michael
Priority date: 2006-03-06
Filing date: 2007-03-06
Publication date: 2007-09-13
Also published as: DE102006010958A1; EP1993730A1

Abstract

The invention relates to a method of carrying out a reaction for replicating a nucleic acid (nucleic acid replication reaction; NAR) in a liquid-conveying system (14), where the nucleic acid is present in a first liquid (26), a cylindrical reaction vessel (12) which has an opening at a first end which is connected by a first liquid line (16) to the liquid-conveying system (14) so that liquid can be conveyed and which can be filled and emptied with/of the first liquid (26) by the first liquid line (16) is provided and the reaction vessel (12) has a first piston (18) which can be moved in the reaction vessel (12) between an outer space and the interior space of the reaction vessel (12).

Description

Method for carrying out a reaction for the amplification of a nucleic acid

The invention relates to a method for carrying out a reaction for the amplification of a nucleic acid (nucleic acid amplification reaction, - NSV) in a liquid-conducting system and to a device suitable for carrying out the method. A fluid-conducting system is understood to be an arrangement of fluid-conducting or fluidically interconnected fluid conduits and fluid chambers.

Such a method and apparatus are known in the art. In this case, a liquid which contains a nucleic acid to be amplified and all reagents required for carrying out a polymerase chain reaction (PCR) is passed through a hose. The tube is heated at different intervals in different sections, so that the liquid flowing through it passes through the temperatures required for the PCR. A disadvantage of this method is that the temperature profile, which passes through the liquid, is determined by the course of the tube by predetermined temperature zones. It is not or only with great difficulty possible with such a device to change the temperature profile required for carrying out the PCR and thereby adapt it, for example, to the nucleic acid to be amplified. Furthermore, in such an arrangement there is the problem that at temperatures close to the boiling point of the liquid in the liquid gas bubbles form, which lead to a pulling apart of the sample in the hose. As a result, the temperature profile that passes through the liquid is uneven for different parts of the liquid. Leading to inconsistent, poorly reproducible and often insufficient duplication reactions.

From EP 1 123 980 A2 a method for carrying out a nucleic acid analysis is known. A capillary is connected via a three-way cock with two syringes. Liquid can be sucked through the capillary via the syringes and pumped back and forth between the syringes for mixing. Both binding of nucleic acids and subsequent amplification of the nucleic acids in a PCR takes place in the capillary. To carry out the PCR, the capillary is removed from the syringes and sealed with plugs. As a result, the method can not be automated. Without closing the capillary, however, there would be a loss of fluid when carrying out the PCR.

The object of the present invention is to provide a method and a device which make it possible to carry out a NSI within a fluidic system while avoiding the abovementioned disadvantages. In particular, it should be possible to choose any, favorable for the NSV, temperature conditions. In addition, it should be avoided that liquid, for example in the form of vapor, escapes from the device. This would change the defined concentration or amount of reagents or nucleic acid to be amplified present in the liquid.

This object is solved by the features of claims 1 and 35. Advantageous embodiments of the invention will become apparent from the features of claims 2 to 34 and 36 to 57th

According to the invention, a method for carrying out a reaction for amplifying a nucleic acid (nucleic acid amplification reaction, - NSV) in a liquid-conducting provided the system, wherein the nucleic acid is contained in a first liquid. In this case, a cylindrical reaction vessel is provided which has an opening at a first end, which is connected in fluid-conducting manner via a first liquid line to the liquid-conducting system. Via the first liquid line, the reaction vessel can be filled with the first liquid and emptied. Between an outer space and the interior of the reaction vessel, the reaction vessel has a first piston displaceable in the reaction vessel.

The method comprises the following steps:

a) introducing the first liquid and reagents necessary for the execution of the NSV, as far as the reagents are not present in the reaction vessel (12), through the first liquid line (16) into the reaction vessel (12), so that all for the implementation the NSV required reagents are present in the reaction vessel (12),

b) incubating the first liquid in the reaction vessel under conditions required for the NSV and

c) Detection of the amplified nucleic acid produced in the NSV.

For the purposes of the invention, the reaction vessel is also connected to the liquid-conducting system in a liquid-conducting manner, when the flow of the liquid between the reaction vessel and the remaining liquid-conducting system is reversibly interrupted by a valve or another device. At step lit. a, the first liquid can partially replace the reagents required to carry out the NSV in addition to the nucleic acid to be amplified or completely included. The first fluid may also contain only the nucleic acid. For the purposes of the invention, the first liquid is any liquid containing the first liquid. Accordingly, a first liquid is understood as meaning a mixture of first liquid and other liquids or a solution of reagents in the first liquid. The introduction of the first liquid into the reaction vessel according to step lit. a) can also be done after the first liquid has been moved back and forth between the reaction vessel and another vessel for better mixing or dissolution of reagents in the liquid.

In the method according to the invention, a reaction vessel is provided by the first piston, which has a variable volume by the movable first piston. An advantage achieved thereby is that an escape of the first liquid in the form of vapor or, for example, by the first liquid is forced out by the formation of a vapor bubble from the reaction vessel is prevented. The provision of a cylindrical reaction vessel allows a far-reaching thermal decoupling of the reaction vessel from the remaining liquid-conducting system, so that only the first liquid present in the reaction vessel can be tempered according to the temperatures required for the NSV. Due to the thermal decoupling of the reaction vessel, it is also possible to make faster temperature changes in the reaction vessel. A NSV, which requires multiple temperature changes, such. As a PCR, can be carried out faster. In addition, it is possible by a cylindrical reaction vessel to provide a large surface in relation to the volume of the reaction vessel. This also favors a rapid temperature change in the reaction vessel and a multiple temperature change requiring NSV can be performed faster.

The method according to the invention makes it possible to carry out a NSI in a fluidic system under precisely defined conditions with regard to temperature, concentration of the reagents used and amount of nucleic acid used. Preventing fluid loss through the plunger also helps prevent contamination of the infectious specimen. Furthermore, contamination of the first liquid by contaminants from the environment can also be avoided by the first piston. This is particularly important in carrying out an NSV which causes an exponential nucleic acid amplification since even the smallest impurities, for example due to dander falling into the reaction vessel, can falsify the result.

In addition, the first piston allows a pressure equalization between the interior of the reaction vessel and the

External space, so that in the system no or only a predetermined by the position of the piston pressure builds up. As a result, a pressure load of the liquid-conducting system and in particular valves contained therein can be limited or even prevented. By limiting the pressure load can be further prevented that liquid is pressed by the pressure on the liquid-conducting system in areas of the liquid-conducting system, in which the liquid is undesirable. Overall, the method according to the invention makes possible an automated, contamination-free NSI, which takes place under precisely defined conditions. The fluidic system is preferably a microfluidic system. A microfluidic system is understood to mean a fluid-conducting system in which the fluid conduits have a diameter or a diagonal in a predominant part of the length of the fluid conduits, which is shorter than 2 mm, in particular shorter than 1 mm, preferably shorter than 0.5 mm , is.

In a preferred embodiment of the method according to the invention, the first piston is located at the first end of the cylindrical reaction vessel before the first liquid is introduced. As a result, the volume of the interior of the reaction vessel is minimized, so that after the introduction of the first liquid no or almost no gas such. B. air, located in the reaction vessel. A gas in the reaction vessel is compressible and thereby makes it more difficult to discharge a precisely defined volume from the reaction vessel by pressing the first piston.

Preferably, the first piston is displaced by the introduction of the first liquid. The NSV is preferably a polymerase chain reaction (PCR) or another reaction in which the temperature of the first liquid is changed. If the first liquid changes its volume or forms gas by a temperature change, either by outgassing air dissolved therein or by vaporization, the first piston can be moved by the first liquid, thereby changing the volume of the interior of the reaction vessel.

It is particularly preferred if the first piston is only exposed during the entire process by the first liquid and / or by gas formed from the first liquid and / or by pressing the first piston from outside the reaction vessel. vessel is moved. As a result, a particularly simple design actuator for automated implementation of the method can be used, because no constructive measures must be taken to move the first piston by train.

It is advantageous if the first piston lit. a) and / or lit. b) is moved to a predetermined stop. The predetermined stop makes it possible to fill a defined volume in the reaction vessel. Given a concentration of the nucleic acid to be amplified in the first liquid, a precisely defined amount of nucleic acid to be amplified can be filled into the reaction vessel. By the stop can be ensured that enters the reaction vessel a defined volume, without the amount of liquid which is forced through the opening of the reaction vessel, must be regulated on the part of the remaining liquid-conducting system.

It is advantageous if the stop between the steps lit. a) and lit. b) is displaced or removed in the opposite direction to the opening. The attack can be z. B. be formed by a displaceable or removable rod. As a result, a respective desired volume of liquid can be adjusted by the stop when filling the reaction vessel. After filling, the stop may be removed or withdrawn to carry out the reaction, thereby allowing pressure equalization.

It is also advantageous if such a counter force is exerted by the first piston with respect to the first liquid which expands upon heating in such a way that a pressure is built up thereby which prevents the formation of gas bubbles in the process. immediately reduced to a pressure-free system. The build-up of pressure is less than the pressure that would build up in a closed with a immovable closure vessel. For example, this can be achieved by retracting the rod forming the stop when the liquid is being filled in after the liquid has been introduced so that the piston can move in a region which corresponds to the temperature-induced change in volume of the liquid. The counterforce can be generated by a force acting on the first piston by an expansion of the first liquid against gravity to moving weight or by a frictional resistance of the first piston relative to the reaction vessel. The weight can be z. B. act on the piston by the piston itself has this weight. Due to the frictional resistance of the piston moves only when a certain minimum pressure is reached and it comes to a stop before complete pressure in the reaction vessel is reached.

In the implementation of the method, the reaction vessel is preferably arranged in such a way that bubbles forming in the first liquid can rise to the first piston. This can be achieved, in particular, by arranging the longitudinal axis of the reaction vessel essentially vertically. As a result, it can be avoided that the bubbles enter the liquid-conducting system during the subsequent emptying of the reaction vessel. This can be done in particular by the fact that the first piston for pushing out the first liquid in the direction of the first end but not pressed to the first end. It can be a defined

Volume remain in the cylinder in which the bubbles are contained. In a preferred embodiment of the invention. In the case of the cylindrical reaction vessel, the method is the ratio of height to inner diameter greater than 2, preferably greater than 4, in particular greater than 6. The cylindrical reaction vessel can in particular also run slightly conically towards the first end. In the case of a first piston made of an elastic material, this is achieved in that the first piston can easily be displaced by the introduction of the first fluid and a lower pressure is required for the introduction.

Furthermore, it is advantageous if the wall thickness of the cylindrical reaction vessel is less than 1 mm, preferably less than 0.5 mm, in particular less than 0.3 mm. As a result, good heat transfer to the interior of the reaction vessel is possible. The heat transfer is better, the thinner the wall thickness. Most preferably, the wall thickness is as low as it can just be made by an injection molding process.

It is preferred if the liquid-conducting system or the opening or the first liquid line between the steps lit. a) and lit. b) is sealed liquid-tight. It is understood that the opening or the first liquid line after step lit. b) is opened again when the first liquid is then to be passed out of the reaction vessel again. By closing the liquid-conducting system, after the introduction of the first liquid any contamination of the environment can be avoided. This is especially important for infectious samples. It is very particularly preferred if the nucleic acid, in particular in the form of a biological sample, before step lit. a) is introduced into the liquid-conducting system and the liquid-conducting system after and before performing the step lit. a) is sealed liquid-tight. Both the NSV and the detection can be done within the liquid-tight liquid-conducting system. Thereafter, the liquid-conducting system, including any liquids contained therein, can be disposed of completely. As a result, the inventive method is very safe. By closing the opening or the first liquid line, it can be achieved that a pressure building up in the reaction vessel can not affect the remaining liquid-conducting system.

Preferably, the reaction vessel is lit. b) to set the conditions required by the NSA, in particular by blowing with an air stream, heated or cooled. Due to the cylindrical shape of the reaction vessel, the reaction vessel can be isolated and exposed so that substantially only the reaction vessel is heated or cooled. By blowing with a heated or cooled air flow, a particularly rapid temperature change within the reaction vessel and thereby a quick execution of the NSV is possible. Preferably, the reaction vessel in step lit. b) a cyclic temperature profile. This means that several times the same temperature levels are passed through within a given time sequence. It is particularly preferred if the

Air flow through an air duct, in particular an air deflector, at least partially, is guided around the reaction vessel. As a result, a particularly intensive and therefore faster heat exchange between the air stream and the reaction vessel is possible.

It is particularly preferred if the proof according to step lit. c) takes place outside the reaction vessel and the first liquid between the steps lit. b) and lit. c) by the opening and the first liquid line is passed, in particular by advancing the first piston, out of the reaction vessel. In the liquid-conducting system, the detection can be effected, for example, by means of an electrode with capture molecules immobilized thereon, with which the amplified nucleic acids hybridize, by means of an electrochemical reaction. The first liquid is preferably conducted via the liquid-conducting system into a detection chamber in fluid communication with the liquid-conducting system. Preferably, in step lit. c) a hybridization of the amplified nucleic acids with, in particular immobilized, further nucleic acids. The further nucleic acids can be immobilized on a chip, in particular arranged in the detection chamber. The proof according to step lit. c) can be done on the chip by means of an optical or an electrical detection method. In the optical detection method, the presence of nucleic acids which have bound to other nucleic acids immobilized on the chip can be detected, for example, by measuring the fluorescence of fluorophores incorporated into the amplified nucleic acids. The electrochemical detection can be carried out, for example, by means of graphic electrodes arranged on the chip, to which the further nucleic acids are immobilized as probes. After binding of the specific amplified nucleic acids, electrochemical detection of bound nucleic acids can then be performed.

In an advantageous embodiment of the method, the reaction vessel and the liquid-conducting system or the reaction vessel, at least part of the detection chamber and the liquid-conducting system, preferably in one piece, of the same material, in particular of an injection molded Plastic, preferably polycarbonate, produced. By one-piece production, the dead volume between the detection chamber and the remaining liquid-conducting system and the reaction vessel and the rest of the liquid-conducting system can be minimized. This allows the procedure to be performed with less fluid, fewer nucleic acids and fewer reagents for the NSV as a whole. The process is thereby more sensitive and cheaper to perform. Furthermore, the one-piece production by an injection molding process is particularly cost. It does not conflict with the one-piece configuration if the liquid-conducting system comprises a line plate containing at least partially open lines and a closure means connected thereto, which closes the open lines. The closing means can z. Example, be a plastic plate which is fixedly connected to the conduit plate by laser welding along the open lines in the conduit plate. Such a liquid-conducting system is simple and inexpensive to manufacture and has a high density and pressure stability of the lines.

Alternatively, the reaction vessel can also be provided in the form of a cartridge, which before the step lit. a) is connected in liquid-tight manner by plugging onto a receiving unit provided for this purpose and fluid-tightly to the outside with the liquid-conducting system. The receiving unit can be provided for example by a connecting piece for inserting a connecting portion of the cartridge. By forming the reaction vessel as a cartridge, for example, the nucleic acid or a reagent required for the NSV can be introduced into the reaction vessel. In a preferred embodiment of the method, at least one cylindrical vessel is provided, which contains a second or further liquid and is connected fluid-conducting to the liquid-conducting system, the vessel having between an outer space and an inner space of the vessel a second or further piston displaceable in the vessel, wherein the second or further liquid is pressed into the reaction vessel by actuating the second or further piston via the liquid-conducting system. It is preferred if three cylindrical

Containers are provided, each having a liquid and a flask, each containing one of the liquids required for performing a PCR reagent, in particular polymerase, primer and buffer.

It is particularly preferred if the cylindrical vessel is provided in the form of a cartridge, which before the step lit. a) by connecting to a dedicated receiving unit with the liquid-conducting system liquid-conducting and liquid-tightly connected to the outside. As a result, it is possible, for example, to provide uniform cartridges with polymerase and buffer and, for the particular nucleic acid to be detected, individual cartridges with a respectively required primer pair. It is preferred if the cylindrical vessel or, in the case of the provision of a plurality of cylindrical vessels, the cylindrical vessels contain or contain all the reagents required for carrying out the NSV in addition to the nucleic acid, in particular polymerase, primer and buffer.

In an embodiment of the method, the liquid-conducting system comprises a plurality of second liquid lines arranged in a plane in a plate-like conduit element and the receiving unit / receiving units for attaching the cartridge / cartridges. The implementation of the method with such a liquid-conducting system is particularly advantageous because the specificity of the method for certain nucleic acids can be chosen arbitrarily by attaching appropriate cartridges. In the second fluid lines can be provided, in particular provided by a membrane, valves. The membrane is arranged and designed so that by applying pressure, a passage through the second liquid lines can be shut off. The pressure may, for example, be exerted by a plunger actuated by an automated actuator on the diaphragm.

Furthermore, the liquid-conducting system may have a respective sample preparation chamber and waste chamber in fluid communication with the liquid-conducting system, as well as a connection pipe in fluid communication with the liquid-conducting system, in particular with a profile for producing a bayonet closure. For example, a tube containing a biological sample, such as a so-called Monovette ™, can be connected to the connecting piece. Preferably, the sample preparation chambers, the waste chamber, the connecting piece and the liquid-conducting system are integrally made of the same material, in particular of injection-molded plastic, preferably polycarbonate. It does not conflict with the one-piece construction if the conduit element comprises a conduit plate containing at least partially open conduits and a closure means connected thereto and closing off the open conduits.

Preferably, in the method according to the invention, the first and the second piston or, in the case of providing a plurality of cylindrical vessels, the first, the second and the further piston (s) is actuated by feed from the same side of the plate-like conduit element. In a preferred embodiment of the method, the first, second, an optional further liquid and / or; a mixture thereof before step lit. b) reciprocated between the cylindrical vessel and an optionally present further vessel or the reaction vessel and the cylindrical vessel. As a result, a particularly good mixing of all the reagents required for the NSV and the nucleic acids to be detected can be achieved.

The invention further relates to an apparatus for carrying out the method according to the invention, consisting of a cylindrical reaction vessel, which is connected in liquid-conducting manner via a provided at a first end of the reaction vessel opening and opening into the opening first liquid line to a liquid-conducting system, so that the reaction vessel via the liquid-conducting system can be filled and emptied with a first, second or further liquid. The reaction vessel has, between an outer space and an inner space of the reaction vessel, a first piston which is displaceable in the reaction vessel, wherein the first piston is designed so that it can be moved through the first, second or further liquid as it flows through the reaction vessel Opening is introduced or changes its volume or gas forms at a temperature change, thereby changing the volume of the interior of the reaction vessel is changed. At least one vessel is provided, which contains the second or further liquid and is connected in a fluid-conducting manner to the liquid-conducting system. The vessel or, in the case of the presence of multiple vessels, the vessels contain / contain reagents necessary for the performance of the NSV in addition to the nucleic acid. All can be included for performing the NSV in addition to the nucleic acid to be amplified reagents. If some of the reagents required for NSA are already introduced into the reaction vessel or contained in the first fluid, only the remaining part of the reagents necessary for the NSV can also be present.

The reaction vessel preferably has a rigid vessel wall, in particular consisting of polycarbonate. A rigid vessel wall improves the tightness between the piston and the vessel wall. Polycarbonate allows good heat transfer from the outside to the reaction vessel and is also compatible with performing a PCR. In addition, the rigid vessel wall ensures a precise change in the volume of the interior of the reaction vessel and a precise

Fluid movement through a defined movement of the first piston.

Further advantageous embodiment of the device according to the invention can be found in the claims.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the respective combinations indicated, but also in other combinations or in isolation, without departing from the scope of the present invention.

Hereinafter, an advantageous embodiment of the invention will be explained in more detail with reference to an embodiment. Show it:

1 is a schematic representation of a device according to the invention prior to carrying out the method according to the invention, 2 is a schematic representation of a device according to the invention at the beginning of filling the reaction vessel with a liquid,

3 is a schematic representation of a device according to the invention after filling with the liquid,

Fig. 4 is a schematic representation of a device according to the invention in carrying out the method according to the invention and

Fig. 5 is an exploded view of a device according to the invention contained liquid-conducting system.

The device 10 according to the invention consists of the reaction vessel 12 and a liquid-conducting system 14, to which the reaction vessel 12 via at least a first

Liquid line 16 is connected. The device 10 has a movable closure in the form of a first piston 18. Furthermore, the device 10 has a stop 21 formed by a removable rod 20 for the first piston 18. The liquid-conducting system 14 has a closable or self-closing, designed here as a valve 22, inlet opening. Instead of the valve 22, a septum or a rubber stopper could also be provided here, which can be pierced by means of a cannula. Furthermore, the device 10 has a further valve 24.

In Fig. 2, the beginning of the filling of the reaction vessel 12 with the first liquid 26 is shown schematically. The The first liquid 26 is initially in the syringe 28. The first liquid 26 is injected by means of a cannula through the open valve 22 into the liquid-conducting system 14. Since the further valve 24 is closed, the first liquid 26 is pressed into the reaction vessel 12 via the first liquid line 16. In this case, the first piston 18 is raised. Into the reaction vessel 12 passes a precisely defined volume, which is predetermined by the stop 21 formed by the rod 20 for the first piston 18. After filling the reaction vessel 12, the syringe 28 is removed and the valve 22 is brought into the closed position. This condition is shown in FIG.

Fig. 4 shows the device 10 schematically in carrying out the method according to the invention. The rod 20 has been removed to allow the first piston 18 to move freely. The first liquid 26 is, as usual in performing a PCR, heated and cooled again. In this case, the volume of the first liquid changes 26. This change in volume causes a movement of the first piston 18. As a result, the volume change of the first liquid 26 is compensated by a corresponding change in volume of the interior of the reaction vessel 12. It is also possible to withdraw the rod 20 only slightly, so that by a movement of the first

Piston 18 Although the volume change of the first liquid 26 is indeed compensated, the formation of gas bubbles by a still existing formed by the rod 20 stop 21 for the first piston 18 is avoided or at least weakened.

FIG. 5 shows a fluidic system comprising a carrier 30. Liquid lines run on the underside of the carrier 30, which is not shown in the drawing. Finally, the first fluid line 16, which have a downwardly at least partially open side and are closed by the closure plate 32. On the support 30, the reaction vessel 12 is provided. For connecting cartridges 34 containing reagents further

Liquids or sample container 36, a connection element 38 is provided. The connecting element 38 is connected to the carrier 30 and the chip 40, which serves to detect nucleic acids amplified in the reaction vessel 12, is latched to the latching hooks 42. The liquid-conducting system

14 has a sample preparation chamber 44, a waste chamber 46, various connecting pieces for attaching cartridges 34 and sample container 36 and a reaction chamber 12. The liquid-conducting system 14 shown in Fig. 5 may form a unit after plugging the cartridges 34, in the complex processes such. For example, an opening of cells, a purification of biomolecules, a nucleic acid amplification and a detection of biomolecules can take place by means of the chip 40.

LIST OF REFERENCE NUMBERS

10 device

12 reaction vessel 14 liquid-conducting system

16 first fluid line

18 first piston

20 bar

21 stop 22 valve

24 additional valve

26 first liquid

28 injection syringe

30 carrier 32 closure plate

34 cartridge

36 sample containers

38 connection element

40 chip 42 snap hook

44 sample preparation chamber

46 Waste Chamber

Claims

claims

1. A method for carrying out a reaction for amplifying a nucleic acid (nucleic acid amplification reaction, NSV) in a liquid-conducting system (14), wherein the nucleic acid in a first liquid (26) is provided, wherein a cylindrical reaction vessel (12) is provided, which at a first end has an opening which is fluidly connected via a first liquid line (16) to the liquid-conducting system (14) and which can be filled and emptied via the first liquid line (16) with the first liquid (26), wherein the Reaction vessel (12) between an outer space and the interior of the reaction vessel (12) has a in the reaction vessel (12) displaceable first piston (18), comprising the following steps:

a) introducing the first liquid (26) and required for carrying out the NSV reagents, as far as the reagents are not presented in the reaction vessel (12), through the first liquid line (16) in the reaction vessel (12), so that all for the Carrying out the NSV required reagents in the reaction vessel (12) are present,

b) incubating the first liquid (26) in the reaction vessel (12) under conditions required for the NSV and

c) Detection of the amplified nucleic acid produced in the NSV.

2. The method of claim 1, wherein the first piston (18) is displaced by the introduction of the first liquid (26).

3. The method of claim 1 or 2, wherein the NSV is a polymerase chain reaction (PCR) or other reaction in which the temperature of the first liquid (26) is changed.

4. The method of claim 3, wherein the first piston (18) is moved by the first liquid (26) when the first liquid (26) changes its volume or gas by a temperature change, thereby increasing the volume of the interior of the reaction vessel (12) is changed.

5. The method according to any one of the preceding claims, wherein the first piston (18) during the entire process only by the first liquid (26) and / or from the first liquid (26) gas formed and / or by pressing the first piston ( 18) is displaced from outside the reaction vessel (12).

6. The method according to any one of the preceding claims, wherein the first piston (18) in the steps lit. a) and / or b) is moved to a predetermined stop (21).

7. The method according to claim 6, wherein the stop (21) between the steps lit. a) and b) is displaced or removed in the opposite direction to the opening.

8. The method according to any one of the preceding claims, wherein by the first piston (18) opposite to the heating when expanding first liquid (26) such a counter force is applied, thereby establishing a pressure which reduces formation of gas bubbles compared to a non-pressure system.

9. The method of claim 8, wherein the counterforce by a force acting on the first piston (18) by an expansion of the first liquid (26) against gravity or by a frictional resistance of the first piston (18) relative to the reaction vessel (12 ) is produced.

10. The method according to any one of the preceding claims, wherein the reaction vessel (12) in the implementation of the method is arranged so that in the first liquid (26) forming bubbles can rise to the first piston (18).

11. The method of claim 10, wherein the longitudinal axis of the reaction vessel (12) is arranged substantially vertically.

12. The method according to any one of the preceding claims, wherein in the cylindrical reaction vessel (12) the ratio of height to inner diameter is greater than 2, preferably greater than 4, in particular greater than 6, is.

13. The method according to any one of the preceding claims, wherein the wall thickness of the cylindrical reaction vessel (12) is less than 1 mm, preferably less than 0.5 mm, more preferably less than 0.3 mm.

14. The method according to any one of the preceding claims, wherein the liquid-conducting system (14) or the opening or the first liquid line (16) between the steps lit. a) and lit. b) is sealed liquid-tight.

15. The method according to any one of the preceding claims, wherein the nucleic acid, in particular in the form of a biological sample, before step lit. a) is introduced into the liquid-conducting system (14) and wherein the liquid-conducting system (14) after and before the implementation of step lit. a) is sealed liquid-tight.

16. The method according to any one of the preceding claims, wherein the reaction vessel (12) in step lit. b) for adjusting the conditions which are required for the NSV, in particular by blowing with a Luftström, heated or cooled.

17. The method according to claim 16, wherein the reaction vessel (12) in step lit. b) passes through a cyclic temperature profile.

18. The method of claim 16 or 17, wherein the air flow through an air guide, in particular an air guide, at least partially, around the reaction vessel (12) is guided around.

19. The method according to any one of the preceding claims, wherein the proof according to step lit. c) takes place outside the reaction vessel (12) and the first liquid (26) between the steps lit. b) and lit. c) is passed through the opening and the first liquid line (16), in particular by advancing the first piston (18), from the reaction vessel (12).

20. The method of claim 19, wherein the first liquid (26) via the liquid-conducting system (14) in a liquid-conducting with the liquid-conducting system (14) communicating detection chamber is passed.

21. The method according to any one of the preceding claims, wherein at step lit. c) a hybridization of the amplified nucleic acids with, in particular immobilized, further nucleic acids takes place.

22. The method of claim 21, wherein the further nucleic acids are immobilized on a, in particular in the detection chamber, chip (40).

23. The method of claim 22, wherein the proof according to step lit. c) takes place on the chip (40) by means of an optical or an electrochemical detection method.

24. The method according to any one of the preceding claims, wherein the reaction vessel (12) and the liquid-conducting system (14) or the reaction vessel (12), at least a portion of the detection chamber and the liquid-conducting system (14), preferably in one piece, of the same material, in particular from an injection-molded plastic, preferably polycarbonate, are produced.

25. The method according to any one of claims 1 to 23, wherein the reaction vessel (12) is provided in the form of a cartridge (34), which before the step lit. a) by connecting to a designated Aufnähmeeinheit liquid-conducting and liquid-tight to the outside with the liquid-conducting system (14) is connected.

26. The method according to any one of the preceding claims, wherein at least one cylindrical vessel is provided which contains a second or further liquid and is connected to the liquid-conducting system (14) liquid-conducting, wherein the vessel between an outer space and the interior of the vessel has a second or further piston displaceable in the vessel, wherein the second or more liquid is pressed into the reaction vessel (12) by actuating the second or further piston via the liquid-conducting system (14).

27. The method of claim 26, wherein the cylindrical vessel is provided in the form of a cartridge (34), which before the step lit. a) by connecting it to a designated Aufnähmeeinheit with the liquid-conducting system (14) liquid-conducting and liquid-tightly connected to the outside.

28. Method according to claim 27, wherein the cylindrical vessel or, in the case of providing a plurality of cylindrical vessels, the cylindrical vessels contain / contain all the reagents required for performing the NSV in addition to the nucleic acid, in particular polymerase, primer and buffer.

29. The method of claim 27 or 28, wherein the liquid-conducting system (14) comprises a plurality of arranged in a plane in a plate-like conduit member second fluid lines and the receiving unit / receiving units for attaching the cartridge / cartridges (34).

30. Method according to claim 29, wherein valves (22, 24) provided in the second liquid lines, in particular by a membrane which can block a passage through the second liquid lines by exerting pressure, are provided.

31. The method according to any one of the preceding claims, wherein the device (10) further comprises a respective liquid having in connection with the liquid-conducting system (14) in connection sample processing chamber (44) and waste chamber (46) and a liquid-conducting with the liquid-conducting system (14) communicating connection piece, in particular with a profile for producing a bayonet closure comprises.

32. The method of claim 31, wherein the sample preparation chamber (44), the waste chamber (46), the connecting piece and the liquid-conducting system (14) are integrally made of the same material, in particular of an injection-molded plastic, preferably polycarbonate.

33. The method according to any one of claims 29 to 32, wherein the first (18) and the second piston or, in the case of providing a plurality of cylindrical vessels, the first (18), the second and the / the further (s) of the same piston Side of the plate-like conduit element forth are operated by feed.

34. The method according to any one of claims 26 to 33, wherein the first (26), second, an optional further liquid and / or a mixture thereof before step lit. b) is moved back and forth between the cylindrical vessel and any other further vessel or the reaction vessel (12) and the cylindrical vessel.

35. Device (10) for carrying out a method according to one of claims 1 to 34, consisting of a cylindrical reaction vessel (12) which has an opening provided at a first end of the reaction vessel (12) and opening into the opening first Liquid line (16) is liquid-conductively connected to a liquid-conducting system (14), so that the reaction vessel (12) via the liquid-conducting system (14) with a first (26), second or further liquid can be filled and emptied, wherein the reaction vessel (12) between an outer space and the interior of the reaction vessel (12) in the reaction onsgefäß (12) displaceable first piston (18), wherein the first piston (18) is adapted to be moved by the first (26), second or further liquid as it is introduced into the reaction vessel (12) through the opening or upon a temperature change their volume changes or gas forms so that thereby the volume of the interior of the reaction vessel (12) is changed, wherein at least one vessel is provided which contains the second or further liquid and is connected to the liquid-conducting system (14) liquid-conducting, at the vessel or, in the case of the presence of several vessels, the vessels for carrying out NSV in addition to the nucleus ure required reagents contains / contain.

36. Device (10) according to claim 35, wherein the reaction vessel (12) comprises a rigid, preferably made of polycarbonate,

Vascular wall has.

37. Device (10) according to claim 35 or 36, wherein in the cylindrical reaction vessel (12) the ratio of height to inner diameter is greater than 2, preferably greater than 4, in particular greater than 6 ,.

38. Device (10) according to one of claims 35 to 37, wherein the wall thickness of the cylindrical reaction vessel (12) is less than 1 mm, preferably less than 0.5 mm, in particular less than 0.3 mm.

39. Device (10) according to one of claims 35 to 38, wherein the first piston (18) is formed so that it has a Counterforce generated when the volume of the interior of the reaction vessel (12) is increased by a first liquid extending therein (26).

40. Device (10) according to claim 39, wherein the counterforce by a force acting on the first piston (18) by an expansion of the first liquid (26) against gravity or by a frictional resistance of the first piston (18) relative to the Reaction vessel (12) is generated.

41. Device (10) according to one of claims 35 to 40, wherein the first piston (18) is formed and arranged in the device (10) such that by means of the first piston (18) in the reaction vessel (12) contained first Liquid (26) from the reaction vessel (12) through the opening and the first liquid line (16) can be pressed.

42. Device (10) according to one of claims 35 to 41, wherein the liquid-conducting system (14) is a microfluidic

System is.

43. Device (10) according to any one of claims 35 to 42, wherein the reaction vessel (12) is freestanding.

44. Device (10) according to one of claims 35 to 43, wherein an air guide, in particular an air guide, is provided, which is arranged so that thereby an air flow, at least partially, to the reaction vessel (12) is guided around.

45. Device (10) according to any one of claims 35 to 44, wherein the device (10) is formed liquid-tight lockable.

46. Device (10) according to any one of claims 35 to 45, wherein a liquid-conducting with the liquid-conducting system (14) in communication standing detection chamber is hen provided.

47. Device (10) according to any one of claims 35 to 46, wherein in the detection chamber a, in particular immobilized nucleic acids exhibiting, chip (40) is provided.

48. Apparatus (10) according to any one of claims 35 to 47, wherein the reaction vessel (12) and the liquid-conducting system (14) or the reaction vessel (12), the detection chamber and the liquid-conducting system (14), preferably einstük- kig from the same material, in particular of an injection-molded plastic, preferably polycarbonate, are produced.

49. Apparatus (10) according to any one of claims 35 to 47, wherein the reaction vessel (12) in the form of a cartridge (34) is formed, which is attached to a dedicated receiving unit and thereby liquid-conducting connected to the liquid-conducting system (14) is.

50. Device (10) according to any one of claims 35 to 49, wherein the vessel is cylindrical and has between an outer space and the interior of the vessel a displaceable in the vessel second or further piston.

51. Device (10) according to claim 50, wherein the cylindrical vessel has the shape of a cartridge (34) which is liquid-connected by attaching to a dedicated receiving unit with the liquid-conducting system (14) and fluid-tight to the outside.

52. The device (10) according to any one of claims 35 to 51, wherein the reagents are polymerase, primer and buffer.

53. Device (10) according to one of claims 35 to 52, wherein the fluid-conducting system (14) comprises a plurality of second fluid conduits arranged in a plane in a plate-like conduit element and the cartridge / cartridge mounting / receiving units (34). includes.

54. Device (10) according to claim 53, wherein in the second liquid lines valves (22, 24) are provided, which in particular are provided by a membrane, which can shut off a passage through the second liquid lines by applying pressure.

55. Apparatus (10) according to any one of claims 35 to 54, further comprising a respective sample preparation chamber (44) and waste chamber (46) in fluid communication with the liquid-conducting system (14) and a liquid-conducting system with the liquid-conducting system (14). associated connection piece, in particular with a profile for producing a bayonet closure, are provided.

56. Device (10) according to any one of claims 35 to 55, wherein the sample preparation chamber (44), the waste chamber (46), the connecting piece and the liquid-conducting system (14) integrally from the same material, in particular from an injection-molded plastic, preferred Polycarbonate, consist.

57. Device (10) according to one of claims 53 to 56, wherein the reaction vessel (12) and the cylindrical vessel or the cylindrical vessels are arranged so that the first (18) and the second piston or the first (18), the second and the / the other (s) piston from the same side of the plate-like conduit member can be actuated by advance.