DEVICE FOR PURIFYING NUCLEIC ACIDS

Abstract

The invention relates to a device (1) for purifying nucleic acids composed of a one-piece hollow body (2) comprising an upper portion (3) having an inlet port (5) and a lower portion (4) having an outlet port (6), wherein within the hollow body (2) at the least one nucleic acid-binding matrix (7) is arranged, wherein the device (1) is characterized in that between the upper portion (3) and the lower portion (4) a predetermined breaking point (10) is provided and the nucleic acid-binding matrix (7) is arranged in the lower portion (4). The invention further relates to a method for producing such a device, a method for purifying nucleic acids by means of a device according to the invention, and a kit comprising a device according to the invention.

Claims

1.-15. (canceled)

16. A method for purifying nucleic acids from a liquid nucleic acid-containing sample comprising a) providing a device composed of a one-piece hollow body comprising an upper portion having an inlet port and a lower portion having an outlet port, wherein at least one nucleic acid-binding matrix is arranged within the hollow body, wherein between the upper portion and the lower portion a predetermined breaking point is provided and the nucleic acid-binding matrix is arranged in the lower portion; b) providing the liquid nucleic acid-containing sample and adjusting the binding conditions so as to achieve binding of the nucleic acids to the nucleic acid-binding matrix; c) transferring the sample into the device through the inlet opening of the device; d) passing the sample through the nucleic acid-binding matrix, wherein the nucleic acids bind to the nucleic acid-binding matrix; e) optionally washing the nucleic acid-binding matrix; f) disconnecting the upper portion from the lower portion along the predetermined breaking point; g) optionally washing the nucleic acid-binding matrix; h) eluting the nucleic acids from the nucleic acid-binding matrix and collecting the eluted nucleic acids in a separate collecting vessel.

17. The method according to claim 16, wherein the volume of upper portion corresponds to a volume that is at least 5-fold the volume of lower portion.

18. The method according to claim 16, wherein the upper portion and lower portion independently of one another have a round or rectangular cross section.

19. The method according to claim 16, wherein the upper portion and lower portion independently of one another have a cylindrical or conical form.

20. The method according to claim 16, wherein the hollow body is manufactured of a plastic.

21. The method according to claim 16, wherein the plastic is selected from the groups consisting of polyolefins, bio-based plastics, polylactates, polyamides, polyimides, acetals, polyvinyl chloride, polytetrafluoroethylene, polyesters, polycarbonates, polymethyl(meth)acrylates, acrylonitrile butadiene styrene terpolymer (ABS), polystyrene, copolymers thereof, and mixtures thereof.

22. The method according to claim 16, wherein the hollow body is manufactured by an injection molding process, blow molding, casting technique with silicone forms, methods of rapid prototyping, fused deposition modeling, laser sintering or electron beam melting, or by means of forming machining methods.

23. The method according to claim 16, wherein the nucleic acid-binding matrix is a membrane, a fiber filter, frit and/or a particulate filter.

24. The method according to claim 16, wherein the nucleic acid-binding matrix is a silica membrane having a mean retention of 0.1 to 5μm measured according to DIN EN 1822-3.

25. The method according to claim 16, wherein the nucleic acid-binding matrix is immobilized on a carrier.

26. The method according to claim 16, wherein the predetermined breaking point is formed by a weakening line that substantially encircles the hollow body in a continuous form.

27. The method according to claim 16, wherein the predetermined breaking point is designed such that it enables a separation of the upper portion from the lower portion without the aid of tools.

28. The method according to claim 16, wherein the outlet opening is designed such that it is suitable for attachment to a vacuum chamber.

29. The method according to claim 16, wherein the nucleic acid yield is between 72 and 99% relative to the total amount of nucleic acids in the sample.

30. The method according to claim 29, wherein the total amount of nucleic acids in the sample is determined via a photometric measurement or quantitative real-time PCR.

31. The method according to claim 29, wherein the purification of nucleic acids is carried out in a clean-up procedure.

32. The method according to claim 16, wherein the nucleic acid yield is sufficient for subsequent PCR-Analysis.

33. A kit for purifying nucleic acids from a liquid nucleic acid-containing sample comprising a device for purifying nucleic acids composed of a one-piece hollow body comprising an upper portion having an inlet port and a lower portion having an outlet port, wherein at least one nucleic acid-binding matrix is arranged within the hollow body, wherein between the upper portion and the lower portion a predetermined breaking point is provided and the nucleic acid-binding matrix is arranged in the lower portion, and an operating manual for performing the method according to claim 29, and/or agents suitable for purifying nucleic acids.

Description

[0067] The present invention will be discussed in more detail below with reference to two drawings and embodiments. It is shown in

[0068] FIG. 1 an embodiment of an inventive device,

[0069] FIG. 2 a sectional enlargement of a portion B of the device of FIG. 1, and

[0070] FIG. 3 a precise embodiment of the device of FIG. 1 with dimensional data.

[0071] In FIG. 1 a device 1 for isolation of nucleic acids is shown in a lateral sectional view. The device comprises an integrally formed hollow body 2 with a round cross section and an upper portion 3, as well as a lower portion 4. Hollow body 2 is constructed of polypropylene as a molded part. At upper portion 3 an inlet opening 5 is provided for introducing a liquid sample containing nucleic acids. In lower portion 4, a nucleic acid-binding matrix in the form of a silica membrane 7 (glass fiber filter) with a retention of 1.4 μm according to DIN EN 1822-3 (January 2011) is fixed on a carrier frit 8 by means of a clamping ring 9. In flow direction of the device 1, an outlet opening 6 is provided below silica membrane 7 that is configured as a luer male adapter.

[0072] Between upper portion 3 and lower portion 4 a predetermined breaking point 10 is formed in form of a circumferential key groove, which allows manual separation of upper portion 3 and lower portion 4 along the dashed line A-A. Upper portion 3 has a void volume of 38.5 mL, lower portion 4 has a void volume of approximately 1 mL of which typically 0.7 mL is usable so as to prevent liquids from spilling over the rim.

[0073] Upper portion 3, which is also referred to as a reservoir, comprises a circumferential lip 11 at inlet opening 5. At its opposite end, i.e., at the side facing predetermined breaking point 10, top portion 3 is provided with a conical tapering 12. By means of conical tapering 12, the outer diameter of upper portion 3 is reduced up to predetermined breaking point 10 to about the outer diameter of the lower section 4.

[0074] As is shown in FIG. 2, an enlarged circular detail B of FIG. 1, lower portion 4 is provided immediately below predetermined breaking point 10 with a two-step tapering 13 which forms latching surfaces 14 on the outer side of lower portion 4 that, after removal of upper portion 3, allows lower portion 4 to be inserted in a centrifuge. The inner wall of lower portion 4 in the area of the outer side of the stepwise tapering 13 is thereby preferably not provided stepwise, but with an inclined tapering 15.

[0075] FIG. 3 shows a specific embodiment of device of 1 according to FIG. 1 with dimensional data. The respective dimensions are summarized in the following table:

TABLE-US-00001 Length to a position x dx Position [mm] Diameter [mm] Angle [°] a — da 32 α  1° b 1 db 30.6 β 45° c 56.4 dc 29.6 γ 1.1°  d 65 dd 12.4 δ 55° e 66.4 de 11.1 ε 1.7°  f 69.3 df 8.8 g 88.4 dg 8.4 h 89.8 dh 4.3 i 93.2

[0076] The length values in the table indicate the distance of the respective position to the top edge of device 1, the position a. The length of the sections can be calculated from the distance between the respective end section and the end of the previous section. For example, the section a-b extends from the beginning of rim lip 11—position a—to the end of rim lip 11—position b. The diameters values are based on the position shown in FIG. 3. The angles refer to the angle between the wall at the designated position and the imaginary geometric center line extending continuously through the device 1 in flow direction. The hollow volumes of upper and lower portions 3, 4 correspond to those shown in FIG. 1.

[0077] In the following, several examples of embodiments are described for separating nucleic acids using inventive device 1 and with the method according to the invention.

EXAMPLE 1

Protocol for Purification of Plasmid DNA from E. Coli

[0078] 1. 200 mL of an E. coli XL1 Blue culture with low-copy vector and an OD of 1.8 was pelleted and the cells resuspended in 7 mL A1 with RNase A. (A1: NucleoSpin plasmid, REF 740855, MACHEREY-NAGEL, Düren, Germany—commercial Tris/EDTA resuspension buffer containing RNase A).

[0079] 2. After alkaline lysis with 7 mL A2 and neutralization with 8.4 mL of A3, the precipitate was removed by centrifugation (10 min, 10,000×g; NucleoSpin plasmid, REF 740588, MACHEREY-NAGEL, Düren, Germany—A2 commercial buffer for alkaline lysis of bacteria containing NaOH/SDS; A3 commercial neutralization- and binding buffer containing potassium acetate and guanidine hydrochloride).

[0080] 3. The clear lysate was loaded by vacuum onto the column according to the invention and washed with 5 mL Wash Buffer AW, and 2×5 mL Wash Buffer A4 under vacuum (NucleoSpin plasmid, REF 740855, MACHEREY-NAGEL, Düren, Germany—AW commercial high salt wash buffer containing guanidine hydrochloride and ethanol; A4 commercial alcohol wash buffer containing 80% ethanol). The column according to the invention was designed in accordance with FIG. 1 with a predetermined breaking point in circumferential direction. Six layers of silica membrane were placed in the lower part of the column and immobilized in the column by means of a plastic clamping ring.

[0081] 4. Then, the reservoir part was removed (broken off), and the lower part of the column was transferred to a collection tube (2 mL).

[0082] 5. The column was dried in a benchtop centrifuge for 2 min at 11,000×g.

[0083] 6. The DNA was eluted in 50 μL of elution buffer AE (5 mM Tris/HCl) by centrifugation for 1 min at 11,000×g.

Result

[0084] The photometric measurement of the eluate determined a yield of 11.8 μg and purity of A260/A280=1.84 or A260/A230=2.23.

[0085] Without the column according to the invention the lysate would have needed to be loaded onto a commercially available mini spin column in approx. 30 individual steps of 700 μL each.

EXAMPLE 2

Protocol for Purification of PCR Fragments

[0086] 1. A band weighing 5 g containing 20 μg of plasmid DNA was excised from a 1% TAE agarose gel (30 minutes, 90V) and incubated in 10 mL NTl buffer (NucleoSpin Gel and PCR Clean-up, REF 740609, MACHEREY-NAGEL, Düren, Germany—commercial binding buffer containing guanidine thiocyanate) at 55° C. until it was completely dissolved.

[0087] 2. The sample was loaded under vacuum onto the column according to the invention (2 layers of silica membrane, polyethylene frit as a carrier) and washed with 5 mL Wash Buffer NT3 under vacuum (NT3, NucleoSpin Gel and PCR Clean-up, REF 740609, MACHEREY-NAGEL, Düren, Germany—commercial washing buffer containing 80% ethanol).

[0088] 3. Then, the reservoir part was removed (broken off), and the lower part of the column transferred to a collection tube (2 mL).

[0089] 4. The column was dried in a benchtop centrifuge for 2 min at 11,000×g.

[0090] 5. The DNA was eluted in 100 μL of elution buffer NE (5 mM Tris/HCl) by centrifugation for 1 min at 11,000×g.

Result

[0091] The photometric measurement of the eluate determined a yield of 12.8 μg (64%) and purity of A260/A280=1.85 or A260/A230=2.06.

[0092] Without the column according to the invention, the lysate would have needed to be loaded onto a commercially available mini spin column in approx. 20 individual steps of 700 μL each.

EXAMPLE 3

Protocol for Purification of Genomic DNA from Complex Biological Samples

[0093] 1. 1 g of cooked ham was incubated in 2750 μL lysis buffer CF and 50 μL proteinase K for 3 h at 65° C. (lysis buffer CF: NucleoSpin Food, REF 740945, MACHEREY-NAGEL, Düren, Germany—commercial SDS-lysis buffer).

[0094] 2. Undigested sample material was pelleted at 10,000×g for 10 min. To the clear supernatant 1 volume of binding buffer C4 and one volume of ethanol was added (C4: NucleoSpin Food, REF 7409454, MACHEREY-NAGEL, Düren, Germany—commercial binding buffer containing guanidine hydrochloride).

[0095] 3. The sample was loaded under vacuum onto the column according to the invention (3 layers of silica membrane, polyethylene frit as carrier) and washed with 5 mL Wash Buffer CQW, 2×5 mL Wash Buffer C5 under vacuum (NucleoSpin Food, REF 740945, MACHEREY-NAGEL, Düren, Germany—CQW: commercial wash buffer containing guanidine hydrochloride and ethanol; C5 commercial alcohol wash buffer containing 80% ethanol).

[0096] 4. Then, the reservoir part was removed (broken off), and the lower part of the column transferred to a collection tube (2 mL).

[0097] 5. The column was dried in a benchtop centrifuge for 2 min at 11,000×g.

[0098] 6. The DNA was eluted in 2×100 μL of elution buffer CE (5 mM Tris/HCl) by centrifugation for 1 min at 11,000×g.

Result

[0099] The photometric measurement of the eluate determined a yield of 120 μg and purity of A260/A280=1.91 or A260/A230=2.19.

[0100] Without the column according to the invention, the lysate would have needed to be loaded onto a commercially available mini spin column in approx. 15 individual steps of 700 μL each.

EXAMPLE 4

Protocol for Purification of Genomic DNA from Human Plasma

[0101] 1. 2.4 ml of plasma was mixed with 3.6 ml of buffer BB (binding buffer BB: NucleoSpin Plasma, REF 740900, MACHEREY-NAGEL, Düren, Germany—commercial binding buffer containing guanidine thiocynate and ethanol).

[0102] 2. The sample was loaded under vacuum onto the column according to the invention (3 layers of silica membrane, polyethylene frit as a carrier). The following washing steps were carried out in a benchtop centrifuge.

[0103] 3. The reservoir of the column was removed (broken off), the lower part of the column inserted into a 2 mL collection tube, 500 μL wash buffer WB added (commercial wash buffer, Tris/HCl, >60% ethanol) and centrifuged for 30 seconds at 11.000×g.

[0104] 4. The collection tube containing the flow-through was discarded and washing performed a second time with 250 μL wash buffer WB.

[0105] 5. The column was then dried by centrifugation at 11,000×g for 3 min.

[0106] 6. The DNA was eluted in 200 μL elution buffer BE (5 mM Tris/HCl) by centrifugation for 30 sec. at 11,000×g.

[0107] In parallel, 2 further samples were processed. To one sample was added 240 μL plasma and 360 μL binding buffer BB following the NucleoSpin Plasma XS High Sens protocol (MN, REF 740900). This corresponds to 1/10 of the volume of the example in the above-mentioned embodiment. As a binding column, the conventional NucleoSpin Plasma XS mini spin column was used. The loading and all other steps were carried out in a tabletop centrifuge under identical conditions.

[0108] Another sample was prepared as described in the example of the above embodiment by adding 3.6 mL binding buffer BB to 2.4 mL plasma. Here, however the column used was a large funnel column (MACHEREY-NAGEL Funnel Column). The samples were processed using a standing floor centrifuge. The individual steps were as follows:

[0109] 1. Wash step 5 mL WB (1,000×g, 3 min)

[0110] 2. Wash step 2.5 mL WB and drying (3,000×g, 3 min)

[0111] 3. Elution: 200 μL BE, 3,000×g 3 min.

[0112] The DNA was quantified by quantitative rtPCR (CyNamo Capillary SYBR Green qPCR Kit).

Result

[0113] DNA was isolated from plasma using all three formats. The DNA yield using the NucleoSpin Plasma XS was 13 ng, 108 ng using the large funnel column (Funnel Column), and 194 ng using the column according to the invention. Compared to the mini spin column the 10-fold amount of plasma could be processed with the column according to the invention, (240 μL vs. 2.4 mL, 13 ng vs. 194 ng DNA yield). The inventive method is also superior to the large funnel column using the same sample volume (in both cases, 2.4 mL), in terms of DNA yield (194 vs. 108 ng), and DNA concentration (0.97 vs. 0.54 ng/μL).

EXAMPLE 5

Protocol for purification of RNA

[0114] 1. RNA was purified by a clean-up protocol. The RNA was present in pre-cleaned form in water at a concentration of 1 ng/μL. 3 ml of the RNA-solution was mixed with 3.0 ml of buffer RCU (binding buffer RCU: NucleoSpin RNA Clean-up XS Kit, REF 740903, MACHEREY-NAGEL, Düren, Germany—commercial binding buffer containing guanidine thiocynate and ethanol).

[0115] 2. The sample was vacuum-loaded onto the column according to the invention (3 layers of silica membrane, polyethylene frit as a carrier). The following washing steps were carried out in a benchtop centrifuge.

[0116] 3. The reservoir of the column was removed (broken off), the lower part of the column inserted into a 2 mL collection tube, 400 μL wash buffer RA3 added (commercial wash buffer, Tris/HCl,>70% ethanol) and centrifuged for 30 seconds at 11,000×g.

[0117] 4. The collection tube containing the flow-through was discarded and washing performed a second time with 200 μL wash buffer RA3.

[0118] 5. The RNA was eluted in 100 μL water by centrifugation for 30 sec. at 11,000×g,

[0119] In parallel, 300 μL of the RNA solution was mixed together with 300 μL RCU and processed with conventional mini spin columns from the NucleoSpin RNA kit ((MACHEREY-NAGEL, Düren, Germany, REF 740955) by centrifugation. All remaining steps were carried out according to the protocol described above.

[0120] RNA was quantified using RiboGreen.

Result

[0121] The RNA yield using the columns according to the invention was 5.5 μg while the yield using the mini spin columns was 0.7 μg. To some extent, the scale-up by the factor of 10 with respect to the starting material (300 μL with the mini spin column, 3 mL with the inventive column) is thus also reflected in the RNA yield.

EXAMPLE 6

Protocol for the Precipitation and Purification of Plasmid DNA

[0122] 1. To 6 μg pcDNA3.1 in 5 mM Tris/HCl water was added to obtain 1, 2, 4, 8, 16 mL.

[0123] 2. The DNA of the samples is precipitated by addition of 1 volume of polyethylene glycol (20% PEG 8000, 2.5 M NaCl) and incubation for 2 h at 4° C.

[0124] 3. The reaction mixture was applied to the column according to the invention and drawn through by applying a vacuum (−300 mbar).

[0125] 4. The reservoir of the column was removed (broken off) and the column was washed under vacuum by addition of 2×700 μL wash buffer A4 (commercially available alcohol wash buffer containing 80% ethanol).

[0126] 5. The lower part of the column was inserted into a 2 mL collection tube and dried by centrifugation for 30 s at 11,000×g in a benchtop centrifuge.

[0127] 6. The DNA was eluted in 2×200 μL of 5 mM Tris/HCL by centrifugation for 30 sec. at 11,000×g.

Result

[0128] The DNA yield for 1, 2, 4, 8, and 16 mL was 5.8, 5.4, 4.8 and 4.5 μg DNA. Thus, yields between 99-72% were obtained.