Method for extracting DNA

Abstract

The invention relates to a method for extracting DNA, comprising the steps of: providing (400) a lysate (11) in a sample vessel (1), feeding (500) an DNA absorbing substance (15) to the sample vessel (1), closing (700) the sample vessel (1) by means of a wash filter element (3), feeding (600) a wash fluid (12) to the sample vessel (1) through the wash filter element (3), and discharging (700) the wash fluid (12) from the sample vessel (1) through the wash filter element (3). The DNA absorbing substance can be retained from the wash filter element (3).

Claims

1. A method for extracting DNA, comprising the steps: lysing cells in a first sample vessel (2) to form a lysate, filtering the lysate through a lysate filter element (4) into a second sample vessel (1) to form a filtered lysate (11) comprising DNA released from the cells, adding a DNA-adsorbing substance (15) to the second sample vessel (1) comprising the DNA released from the cells, wherein the DNA-adsorbing substance binds to and forms a complex with at least some of the DNA released from the cells, closing the second sample vessel (1) with a wash filter element (3) formed of the same material as the lysate filter element (4), delivering a wash fluid (12) into the second sample vessel (1) through the wash filter element (3), and discharging the wash fluid (12) from the second sample vessel (1) through the wash filter element (3), wherein the complex is retained within the second sample vessel (1) by the wash filter element (3), and wherein any DNA that is not bound to the DNA-adsorbing substance is not retained by the wash filter element (3), introducing an additional substance into the second sample vessel (1) in order to separate the DNA-adsorbing substance (15) from the DNA, producing free DNA, and separating the DNA-adsorbing substance from the free DNA in the second sample vessel (1), leaving the free DNA in the second sample vessel (1).

2. The method as claimed in claim 1, characterized in that the step of lysing cells to form a lysate comprises the following steps: introducing cellular material (13) and a grinding element (14) into the first sample vessel (2), and breaking down the cellular material (13) by moving the first sample vessel (2).

3. The method as claimed in claim 1, characterized in that the wash filter element (3) or the lysate filter element (4) is arranged in a plunger (5), the plunger (5) being insertable into the second sample vessel (1) or the first sample vessel (2), and, through the plunger (5), the wash fluid (12) being sucked into the second sample vessel (1) and pressed out of the second sample vessel (1), or the lysate (11) being pressed out from the first sample vessel (2) into the second sample vessel (1).

4. The method as claimed in claim 3, characterized in that the plunger (5) and the second sample vessel (1) or the first sample vessel (2) form a syringe.

5. The method as claimed in claim 1, characterized in that the second sample vessel (1) is held in a holding device (6) throughout the entire method, and/or the first sample vessel (2) is held in an additional holding device at least during the step of filtering the lysate (11).

6. The method as claimed in claim 5, characterized in that the holding element (6) simultaneously holds a multiplicity of second sample vessels (1), and/or the additional holding element simultaneously holds a multiplicity of first sample vessels (2), so that the method is applied simultaneously to the multiplicity of second sample vessels (1) and/or first sample vessels (2).

7. The method as claimed in claim 4, characterized in that a multiplicity of plungers (5) are fixed on a carrier element (7), so that, at the same time, the multiplicity of plungers (5) are inserted simultaneously into the multiplicity of second sample vessels (1) and/or first sample vessels (2).

8. The method as claimed in claim 7, characterized in that the step of delivering the wash fluid (12) and/or the step of discharging the wash fluid (12), and/or the step of filtering the lysate (11), are carried out by moving the carrier element (7) and the holding element (6) and/or the additional holding element relative to one another.

9. The method as claimed in claim 7, characterized in that the step of delivering the wash fluid (12) is carried out by simultaneous suction of wash fluid (12) through the multiplicity of plungers (5) from a common reservoir (8) or from an individual reservoir (9) provided for each plunger (5).

10. The method as claimed in claim 9, characterized in that the reservoir (8) is subdivided by at least one subdivision element (10) into individual subreservoirs (11) for each plunger (5).

11. The method as claimed in claim 1, characterized in that the steps of delivering the wash fluid (12) and of discharging the wash fluid (12) are carried out repeatedly.

12. The method as claimed in claim 1, characterized in that after the step of delivering the wash fluid (12) and before the step of discharging the wash fluid (12), air is delivered to the second sample vessel (1).

13. The method as claimed in claim 1, characterized in that after the step of delivering the wash fluid (12) and before the step of discharging the wash fluid (12), the second sample vessel (1) is moved circularly or alternatingly.

14. The method as claimed in claim 1, characterized in that the wash filter element (3) and the lysate filter element (4) are identical, and/or the second sample vessel (1) and the first sample vessel (2) are constructed identically.

15. The method as claimed in claim 1, characterized in that the step of lysing cells to form a lysate (11) comprises the following steps: introducing cellular material (13) and a grinding ball (14) into the first sample vessel (2), and breaking down the cellular material (13) by moving the first sample vessel (2) with frequency in an alternating movement.

16. The method as claimed in claim 9, characterized in that the reservoir (8) is subdivided by at least one retractable subdivision element (10) into individual subreservoirs (11) for each plunger (5).

17. The method as claimed in claim 1, characterized in that after the step of delivering the wash fluid (12) and before the step of discharging the wash fluid (12), air is delivered to the second sample vessel (1), the air being delivered to the second sample vessel (1) in the same way as the wash fluid (12).

18. The method as claimed in claim 1, characterized in that after the step of delivering the wash fluid (12) and before the step of discharging the wash fluid (12), the second sample vessel (1) is moved with a frequency, circularly or alternatingly.

19. The method as claimed in claim 1, characterized in that the additional substance is a chaotropic salt disrupts binding between the DNA and DNA-adsorbing substance to form a mixture of free DNA and the DNA-adsorbing substance.

20. The method as claimed in claim 19, characterized in that the DNA-adsorbing substance is coated on magnetic beads, and wherein a magnet is used to separate the DNA-adsorbing substance from the free DNA in the second sample vessel (1).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Exemplary embodiments of the invention will be described in detail below with reference to the appended drawings. In the drawings:

(2) FIG. 1 shows a schematic view of a sequence of a method according to an exemplary embodiment of the invention,

(3) FIG. 2 shows a schematic view of a first alternative of a washing process of the method according to the exemplary embodiment of the invention,

(4) FIG. 3 shows a schematic view of the components used for carrying out the washing process according to the first alternative,

(5) FIG. 4 shows a schematic view of a second alternative of a washing process of the method according to the exemplary embodiment of the invention,

(6) FIG. 5 shows a schematic view of the components used for carrying out the washing process according to the second alternative,

(7) FIG. 6 shows a schematic view of a third alternative of a washing process of the method according to the exemplary embodiment of the invention,

(8) FIG. 7 shows a schematic view of the components used for carrying out the washing process according to the third alternative,

(9) FIG. 8 shows a schematic view of a fourth alternative of a washing process of the method according to the exemplary embodiment of the invention,

(10) FIG. 9 shows a schematic view of the components used for carrying out the washing process according to the fourth alternative,

(11) FIG. 10 shows a schematic view of a fifth alternative of a washing process of the method according to the exemplary embodiment of the invention,

(12) FIG. 11 shows a first schematic view of the components used for carrying out the washing process according to the fifth alternative,

(13) FIG. 12 shows a second schematic view of the components used for carrying out the washing process according to the fifth alternative,

(14) FIG. 13 shows a schematic view of a sixth alternative of a washing process of the method according to the exemplary embodiment of the invention,

(15) FIG. 14 shows a first schematic view of the components used for carrying out the washing process according to the sixth alternative,

(16) FIG. 15 shows a second schematic view of the components used for carrying out the washing process according to the sixth alternative,

(17) FIG. 16 shows a schematic view of a first mixing process during the method according to the exemplary embodiment of the invention,

(18) FIG. 17 shows a schematic view of a second mixing process during the method according to the exemplary embodiment of the invention, and

(19) FIG. 18 shows a schematic view of a third mixing process during the method according to the exemplary embodiment of the invention.

DETAILED DESCRIPTION

(20) FIG. 1 schematically shows a sequence of a method according to an exemplary embodiment of the invention. The method is subdivided in particular into two steps, a step of providing 400 lysate 11 in a sample vessel 1, and a washing process 900 in order to purify the DNA inside the lysate 11.

(21) The step of providing 400 the lysate 11 comprises, in particular, a plurality of substeps. Thus, introduction 100 of cellular material 13 and of a grinding element 14 into an additional sample vessel 2 is initially carried out. The grinding element 14 is, in particular, a grinding ball. Subsequently, closure of the additional sample vessel 2 is carried out by means of a stopper 16.

(22) After the introduction step 100, breakdown 200 of the cellular material 13 is carried out. To this end, the additional sample vessel 2 is moved alternatingly, in particular alternatingly with a high frequency. Particularly advantageously, the additional sample vessel 2 is configured cylindrically, the alternating movement of the additional sample vessel 2 being carried out parallel to a midaxis of the cylindrical shape. The effect achieved by such a movement of the additional sample vessel 2 is that cells inside the cellular material 13 are broken down by the grinding element 14, so that the DNA can emerge from the cells.

(23) After the breakdown step 200 has been carried out, the cellular material 13 is a mixture of cells not broken down, cells broken down and cell contents, which are located inside the additional sample vessel 2. In a further step, filtering 300 of the lysate 11 out into a sample vessel 1 is therefore carried out. For the filtering out 300, removal 310 of the stopper 16 is carried out. Instead of the stopper 16, a plunger 5 is used, so that after the removal 310, application 320 of the plunger 5 is carried out. The plunger 5, and preferably likewise the additional sample vessel 2, is configured cylindrically. The plunger 5 can therefore advantageously be introduced into the additional sample vessel 2. In this case, a fluid-tight seal is formed between an outer wall of the plunger 5 and an inner wall of the additional sample vessel 2.

(24) The plunger 5 preferably comprises an opening 17. The opening 17 therefore constitutes a connection of the interior of the additional sample vessel 2 to an environment. The opening constitutes, in particular, a channel extending lengthwise along a midaxis of the cylindrical shape of the plunger 5, so that the plunger 5 has the shape of a hollow cylinder. A lysate filter element 4 is arranged in the opening 17, so that all fluid which is intended to pass through the plunger 5 into the additional sample vessel 2, or is intended to be removed from the additional sample vessel 2, must pass through the opening 17 and the lysate filter element 4.

(25) The combination of the additional sample vessel 2 and the plunger 5 forms, in particular, a syringe. By relative movement of the additional sample vessel 2 and the plunger 5 with respect to one another, it therefore becomes possible to suck fluid into the additional sample vessel 2 or press it out from the additional sample vessel 2. This simplifies the delivery and discharge of fluid into or out from the additional sample vessel 2.

(26) After the application 320 of the plunger 5, the additional sample vessel 2 is closed in a fluid-tight fashion, so long as no relative movement occurs between the plunger 5 and the additional sample vessel 2. Rotation 330 of the combination of the additional sample vessel 2 and the plunger 5 therefore takes place without the risk arising that the content of the additional sample vessel 2 will flow out. Lastly, pressing 340 of the lysate 11 out from the additional sample vessel 2 into the sample vessel 1 takes place. The pressing out 340 is initiated by the plunger 5 and the additional sample vessel 2 being moved relative to one another. Because of the lysate filter element 4, only the lysate 11 passes out of the additional sample vessel 2 through the plunger 5 into the sample vessel 1. The rest of the cellular material 13, i.e. in particular still intact cells as well as coarse cellular waste, are, just like the grinding element 14, retained by the lysate filter element 4. All undesired components therefore remain inside the additional sample vessel 2, while only the lysate 11 enters the sample vessel 1.

(27) After the provision 400 of the lysate 11, the process 900 of washing the lysate 11 is carried out. The washing process 900 in turn comprises a multiplicity of substeps. In this case, provision is made that, at the start of the washing process 900, the lysate 11 is provided in a fresh sample vessel 1, the DNA contained in the lysate 11 no longer leaving the sample vessel 1 until the washing process 900 has ended.

(28) First, supply 500 of a DNA-adsorbing substance 15 into the sample vessel 1 takes place. The DNA-adsorbing substance 15 is, in particular, silicon dioxide. The DNA-adsorbing substance 15 is, in particular, in a suspension. In this way, the dosing of the DNA-adsorbing substance 15 is simplified. Subsequently, closure 600 of the sample vessel 1 is carried out by means of a plunger 5, which comprises a wash filter element 3. The plunger 5 is, in particular, constructed identically to the plunger 5 previously used. Particularly advantageously, provision is furthermore made that the wash filter element 3 is identical to the wash filter element 4. Therefore, only a single type of plunger 5 is used for the entire method, that is to say in particular for the provision 400 and for the washing process 900. With the combination of the plunger 5 and the sample vessel 1, the sample vessel 1 is closed in a fluid-tight fashion. Introduction and discharge of fluid is possible only by moving the plunger 5 and the sample vessel 1 relative to one another, the fluid being conveyed through the plunger 5 and through the wash filter element 3.

(29) After these preparation measures, in particular repeated delivery 700 of a wash fluid 12 and discharge 800 of the wash fluid 12 take place. In particular, a delivery 700 of different wash fluids 12 takes place, each wash fluid 12 being delivered only when the previous wash fluid 12 has been removed fully from the sample vessel 1 by the discharge step 800. Use of the different wash fluids 12 ensures that all the remaining cellular waste is removed from the lysate 11, in order to obtain DNA which is as pure as possible. During each discharge 800 of wash fluid 12, because of the DNA-adsorbing material 15 the DNA is retained by the wash filter element 12 and therefore remains inside the sample vessel 1 throughout the entire method.

(30) In order finally to provide only DNA, reception of an additional substance into the sample vessel 1 is carried out. The additional substance is used to release the DNA-adsorbing substance 15 from the DNA. Maximally pure DNA is therefore available inside the sample vessel 1.

(31) The method described above is, in particular, carried out in an automated fashion, a multiplicity of additional sample vessels 2 and/or sample vessels 1 being processed at the same time. To this end, a holding element 6 (cf. FIG. 2) is used, which can receive a multiplicity of sample vessels 1. Likewise, an additional holding element (not shown) preferably configured identically to the holding element 6 is used, which can receive a multiplicity of additional sample vessels 2. In this way, a multiplicity of sample vessels 1 and/or additional sample vessels 2 can be handled.

(32) Furthermore, a carrier element 7 (cf. FIG. 2) is used in order to hold a multiplicity of plungers 5. In this way, a multiplicity of plungers 5 can be inserted simultaneously into a multiplicity of additional sample vessels 2, so as to simultaneously filter lysate 11 out from a multiplicity of additional sample vessels 2 into a multiplicity of sample vessels 1. The carrier element 7 may likewise be used in order to simultaneously insert a multiplicity of plungers 5 into the multiplicity of sample vessels 1. The effect achieved in this way is that the washing process 900 can be carried out simultaneously for the multiplicity of sample vessels 1. This is shown below in FIGS. 2 to 15.

(33) FIGS. 2 and 3 show a first alternative of the washing process 900. In the first alternative, suction of wash fluid 12 out from a common reservoir 8 takes place. The multiplicity of plungers 5 are immersed into the common reservoir 8. To this end, in particular, the carrier element 7 is placed onto the reservoir 8. Subsequently, a relative movement is carried out between the holding element 6 and the carrier element 7, during which the holding element 6 is moved away from the carrier element 7. In this way, a reduced pressure is generated inside the sample vessels 1, so that the wash fluid 12 is sucked through the plungers 5 into the sample vessels 1. There, the wash fluid 12 is mixed with the lysate 11 and subsequently pressed out from the sample vessel 1 by a new relative movement between the holding element 6 and the carrier element 7. To this end, the holding element 6 is moved relatively toward the carrier element 7. Particularly advantageously, provision is in this case made that an air cushion remains inside the sample vessel 1, so that the wash fluid 12 can be fully pressed out together with the foreign substances to be washed out.

(34) In the first alternative, the reservoir 8 is arranged in a pan, so that the multiplicity of plungers 5 can engage into the reservoir 8 from above in order to be immersed in the wash fluid 12. The plungers 5 are in this case immersed in the same wash fluid 12 and therefore take the wash fluid 12 from the same reservoir 8.

(35) FIGS. 4 and 5 show a second alternative of the washing process 900. In this case, the second alternative corresponds essentially to the first alternative, with the only difference that the reservoir 8 is delimited into individual subreservoirs 11 by at least one, in particular retractable, subdivision element 10. In this way, in particular, the reservoir 8 is simply too early, in which only a single bowl of the wash fluid 12 is to be provided. Subsequently, the subdivision element 10 may be inserted. By the subdivision element, the reservoir 8 is divided into a multiplicity of subreservoirs 11, a separate subreservoir 11 being available for each plunger 5. As an alternative, it is likewise possible for each subreservoir 11 to be filled separately, so as to adjust a predetermined quantity of wash fluid 12 individually for each individual plunger 5.

(36) FIGS. 6 and 7 show a third alternative of the washing process 900. In contrast to the first alternative, a single reservoir 8 is not provided, rather there are a multiplicity of individual reservoirs 9 so that each plunger 5 is immersed in a separate individual reservoir 9. The amount of wash fluid 12 to be supplied to the respective sample vessel 1 can therefore be adjusted individually.

(37) FIGS. 8 and 9 show a fourth alternative of the washing process 900. Again, provision is made for there to be a single reservoir 8, which is filled with the wash fluid 12. From this common reservoir 8, the wash fluid 12 is send in for each individual sample vessel, in which all the plungers 5 are immersed into the common reservoir 8. In contrast to the first alternative described above, however, immersion does not take place from above into the reservoir 8, but rather in a bottom of the reservoir 8 an opening 18 is provided, through which the multiplicity of plungers 5 can penetrate. Each plunger 5 is therefore assigned its own opening 18. The multiplicity of openings 18 are, in particular, fluid-tight so long as no plunger 5 penetrates through the opening 18. Filling of the reservoir 8 is advantageously carried out from above, so that a removal direction and a filling direction of the reservoir 8 are separate. Apart from the different removal direction, the removal of wash fluid 12 is carried out in a similar way to the first alternative.

(38) FIGS. 10, 11 and 12 show a fifth alternative of the washing process 900. In this case, provision is again made that the common reservoir 8 is subdivided by at least one subdivision element 10 into a multiplicity of subreservoirs 11. Each of the subreservoirs 11 comprises an opening 18 described above. Removal of wash fluid 12 therefore again takes place from below through a bottom of the reservoir 8. In other regards, the washing process 900 according to the fifth alternative does not differ from the second alternative.

(39) FIGS. 13, 14 and 15 show a sixth alternative of the washing process 900. In this case, provision is again made for there to be a multiplicity of individual reservoirs 9, each individual reservoir 9 comprising an above-described opening 18. Each plunger 5 can therefore take wash fluid 12 out from the associated individual reservoir 9 through the corresponding opening 18. In contrast to the third alternative, removal of wash fluid 12 though the bottom of the multiplicity of individual reservoirs 9 is therefore again provided. In other regards, the washing process 900 according to the sixth alternative does not differ from the washing process 900 according to the third alternative.

(40) If the wash fluid 12 is taken from above, as in the first alternative, the second alternative and the third alternative, precautions are to be taken against contamination of the wash fluid 12. At the same time, however, very accurate quantity assignment of wash fluid 12 to the respective samples vessel 1 is made possible. If, however, the wash fluid is taken from below, as in the fourth alternative, the fifth alternative and sixth alternative, the reservoir 8 or the individual reservoir 9 can be fully closed and therefore hermetically sealed from the environment. Wash fluid 12 can only be taken by the multiplicity of plungers penetrating through the openings 18. The reservoir 8 and/or the subdivision element 10 and/or the individual reservoirs 9 may preferably be used several times. A rinsing process and/or a cleaning process can, in particular, be carried out between the individual uses.

(41) In all alternatives, provision is preferably made that the discharge 800 of the wash fluid 12 by pressing out the wash fluid 12 takes place into a separate waste container. In this way, the contamination risk is reduced.

(42) The multiplicity of sample vessels 1 advantageously remain inside the holding element 6 during the entire washing process 900. To this end, the sample vessels 1 are capable of being fixed on the holding element 6. In particular, form-fit fixing is carried out, the form-fit fixing being releasable. In this way, the multiplicity of sample vessels 1, which are held by the holding element 6, can be handled simply and with little outlay. In particular, simultaneous conduct of a washing process 900 for a multiplicity of sample vessels 1 can be carried out. The effect therefore achieved is, in particular, that a throughput during the extraction of DNA is maximized. Particularly advantageously, each holding element 6 can receive a number of 96 sample vessels 1.

(43) As for the washing process 900, provision is preferably made that the step of providing 400 the lysate 11 for a multiplicity of additional sample vessels 2 is also carried out simultaneously. The additional sample vessels 2 are held on an additional holding element (not shown) structurally equivalent to the holding element 6, the additional holding element also preferably being capable of receiving 96 additional sample vessels 2. In this way, in particular, the process of breaking down 200 the cellular material 13 is simplified, since a multiplicity of additional sample vessels 2 can be moved simultaneously by moving the additional holding element.

(44) In order to permit efficient mixing of the wash fluid 12 and the lysate 11 to form a purification mixture 19, provision is advantageously made that movement of the sample vessel 1 is carried out. This is represented in FIG. 16. The sample vessel 1 is, in particular, moved circularly at high frequency. In this way, optimal mixing takes place. Likewise, in this way particles adhering to the wash filter element 3 are released and mixed with the purification mixture 19. Such adhering particles are, in particular, DNA which has been retained by the wash filter element 3 because of the DNA-adsorbing substance 15. The mixing with the purification mixture 19 therefore ensures that the DNA is purified with a high quality.

(45) FIG. 17 shows a further possibility of releasing particles from the wash filter element 3. To this end, after the delivery 800 of the wash fluid 12 by suction through the plunger 5, air is sucked in the same way through the plunger 5. This is therefore done by a relative movement between the plunger 5 and the sample vessel 1, particularly advantageously by the relative movement between the holding element 6 and the carrier element 7. By the loosening of particles from the wash filter element 3, the efficiency of the washing process 900 can be increased.

(46) Lastly, FIG. 18 shows a further possibility of releasing particles from the wash filter element 3 and ensuring optimal mixing of the purification mixture 19. To this end, alternating of the tilting of the sample vessel 1 and of the plunger 5 is carried out. Since the sample vessel 1 is closed in a fluid-tight fashion by the plunger 5, escape of fluid is in this case prevented.

(47) Particularly advantageously, the DNA is present exclusively inside the sample vessel 1 during the entire washing process 900. Handling of the DNA is therefore possible in a very simple way and therefore optimally automatable. By the simple conduct of the washing process 900 by repeated relative movement of the holding element 6 and the carrier element 7, the washing process 900 can also be automated very simply and with little outlay. The same applies for the provision 400 of the lysate 4. Because of the simple automatability, a very large multiplicity of extractions of DNA can therefore be carried out simultaneously. In this case, the entire method takes place in sequential steps, and can therefore be controlled and monitored simply and with little outlay. At the same time, a high throughput is ensured because of the parallel conduct of the steps. Lastly, provision is advantageously made that highly pure DNA, which can be made available for further steps, can be extracted because of the described method according to the exemplary embodiment of the invention.