CA2348054A1 - Processes and means for the isolation and purification of nucleic acids at surfaces - Google Patents

Processes and means for the isolation and purification of nucleic acids at surfaces Download PDF

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Publication number
CA2348054A1
CA2348054A1 CA002348054A CA2348054A CA2348054A1 CA 2348054 A1 CA2348054 A1 CA 2348054A1 CA 002348054 A CA002348054 A CA 002348054A CA 2348054 A CA2348054 A CA 2348054A CA 2348054 A1 CA2348054 A1 CA 2348054A1
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nucleic acids
process according
membrane
employed
isolation container
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French (fr)
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CA2348054C (en
Inventor
Simone Gauch
Helge Bastian
Susanne Ullmann
Uwe Oelmuller
Martin Weber
Guido Fuhrmann
Joachim Schorr
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Qiagen GmbH
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Priority claimed from PCT/EP1998/006756 external-priority patent/WO1999022021A1/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Clinical Laboratory Science (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Detergent Compositions (AREA)
  • Cleaning Or Drying Semiconductors (AREA)
  • Cleaning By Liquid Or Steam (AREA)

Abstract

The invention relates to novel methods and devices for isolating and purifyi ng nucleic acids on surfaces. The invention is directed at methods which use surfaces, e.g. porous membranes, on which the nucleic acids can be immobiliz ed in a simple manner from the sample containing the nucleic acids, and can be detached again using equally simple method steps. The inventive simple proce ss guidance makes it possible to be able to carry out the methods, particularly , in a completely automatic manner. An additional aspect of the invention is directed at binding nucleic acids to an immobile phase, particularly to a membrane, in such a way that they can be easily detached again from said pha se in a successive reaction step, and can optionally be used in additional applications, such as digestion by restriction, RT, PCR or RT-PCR or in ever y other aforementioned suitable analysis or enzyme reaction. Finally, the invention is directed at special isolation vessels with which the inventive methods can be carried out.

Claims (123)

1. A process for isolating nucleic acids comprising the following steps:
- charging a membrane with at least one sample of nucleic acids;
- immobilizing the nucleic acids on the membrane;
- releasing the immobilized nucleic acids from the membrane; and - removing the released nucleic acids through the membrane, whereby said membrane comprises nylon, polysulfone, polyethersulfone, polycarbonate, polyacrylate, acrylate-copolymer, polyurethane, polyamide, polyvinylchloride, polyfluorocarbonate, polytetrafluorethylene, polyvinylidendifluoride, polyethylentetrafluorethylene-copolymerisate, polybenzimidazole, polyethylenechlorotrifluorethylene-copolymerisate, polyimides, polyphenylenesulfide, cellulose, cellulose mixed esters, cellulosenitrate, celluloseacetate, polyacrylnitriles, polyacrylnitrile-copolymers, nitrocellulose, polypropylene and/or polyester.
2. The process according to claim 1, characterized in that the charging takes place from above and the removal takes place downwards.
3. The process according to one of the claims 1 or 2, characterized in that the membrane is placed inside a container with a supply and take-off, positioned in such a manner that the membrane covers the entire cross-section of the container.
4. The process according to one of the claims 1 to 3, characterized in that the membrane is coated.
5. The process according to claim 4, characterized in that the coating renders the membrane hydrophobic.
6. The process according to claim 4, characterized in that the coating renders the membrane hydrophilic.
7. The process according to one of the claims 1 to 6, characterized in that the membrane is less than 1 mm thick.
8. The process according to claim 7, characterized in that the membrane is less than 0.5 mm, preferably less than 0.2 mm thick.
9. A process for isolating nucleic acids comprising the following steps:
- charging a surface with at least one sample of nucleic acids;
- immobilization of the nucleic acids on the surface; and - release of the immobilized nucleic acids from the surface by means of an elution medium, characterized in that the release is carried out at a temperature T, whereby the inequation
10°C > = T > = T S,EM. applies and T S,EM signifies the freezing point of the elution medium.
10. The process according to claim 9, characterized in that the release is carried out at the temperature T , whereby 10°C > = T > = 5°C.
11. The process according to claim 9, characterized in that the release is carried out at the temperature T, whereby 10°C > = T > = 0°C.
12. The process according to claim 9, characterized in that the release is carried out at the temperature T , whereby 10°C > = T > = -5°C.
13. The process according to one of claims 9 to 12, characterized in that the release is carried out at the temperature T, whereby 5°C > = T > =T S,EM.
14. A process for the isolation of nucleic acids comprising the following steps;
- adjustment of a nucleic acid sample to bonding conditions which permit the immobilization of nucleic acids, contained in the sample, at a surface;
- charging of the surface with the sample of nucleic acids; and - immobilization of the nucleic acids at the surface, characterized in that, prior to and/or after the adjustment of the bonding conditions, the sample is pre-purified.
15. The process according to claim 14, characterized in that the pre-purification is effected through demineralization.
16. The process according to claims 14 or 15, characterized in that the pre-purification is carried out by filtration, centrifugation, enzymatic treatment, effect of temperature, precipitation, extraction, homogenization, mechanical particulation and/ or bonding of contaminants at surfaces.
17. The process according to one of the claims 14 to 16, characterized in that the bonding conditions permit an immobilization of RNA.
18. The process according to one of the claims 14 to 17, characterized in that the bonding conditions permit an immobilization of DNA.
19. The process according to one of the claims 14 to 18, characterized in that it contains the following steps:
- release of the immobilized nucleic acids from the surface; and - removal of the released nucleic acids from the surface.
20. The process according to one of the claims 1 to 13 and 19, characterized in that, after the release step, the following step is carried out at least once:
- execution of at least one chemical reaction at the nucleic acids.
21. A process for the carrying out of a nucleic acid amplification reaction with the following steps:
- charging of a surface with at least one sample of nucleic acids;
- immobilization of the nucleic acids at the surface; and - carrying out of an amplification reaction with the nucleic acids.
22. The process according to claim 21, characterized in that the amplification reaction is not isothermal.
23. The process according to claim 21, characterized in that the amplification reaction is isothermal.
24. The process according to one of the claims 21 to 23, characterized in that the amplification reaction is a SDA reaction ("strand displacement amplification), a PCR or a RT-PCR.
25. The process according to one of claims 21 to 24, characterized in that, prior to the execution of the amplification reaction, the nucleic acids are released from the surface by means of a suitable reaction buffer, and the eluate is positioned on or within the membrane.
26. The process according to claim 25, characterized in that it comprises the following further step:

- removal of the released products of the amplification reaction from the surface.
27. The process according to one of the claims 21 to 24, characterized in that the amplification reaction is performed inside a reaction buffer, which does not lead to the release of nucleic acids from the surface.
28. The process according to claim 27, characterized in that it comprises the following further steps:
- release of the amplification reaction products from the surface; and - removal of the released amplification reaction products from the surface.
29. A process for the carrying out of chemical reactions on nucleic acids with the following steps:
- charging of a surface with at least one sample of nucleic acids;
- immobilization of the nucleic acids at the surface;
- release of the immobilized nucleic acids from the surface;
- carrying out of at least one chemical reaction on the nucleic acids; and - removal of the nucleic acids from the surface without additional immobilization.
30. A process for the analysis of nucleic acids inside an isolation container with the following steps:
- providing an isolation container in which a membrane has been placed;
- charging of the isolation container with at least one sample of nucleic acids;
- immobilization of the nucleic acids on the membrane;
- transfer of the liquid parts of the sample through the membrane; and - analysis of at least one property of the nucleic acids on the membrane, contained inside the isolation container.
31. The process according to claim 30, characterized in that, after the transfer of the liquid parts, at least one chemical reaction is carried out on the nucleic acids.
32. The process according to claim 30 or 31, characterized in that the analyzed property is a radioactive marking of the nucleic acids.
33. The process according to claims 30 or 32, characterized in that the analyzed property is the bonding ability of nucleic acids for molecules.
34. The process according to claim 33, characterized in that the molecules are anti-bodies.
35. The process according to claim 33, characterized in that the molecules are nucleic acid-bonding proteins.
36. The process according to claim 33, characterized in that the molecules are colorant molecules.
37. The process according to one of the claims 9 to 36, characterized in that the charging is carried out from above.
38. The process according to one of the claims 1 to 13, 19, 20, 25, 26, 28 and 29, characterized in that washing of the immobilized nucleic acids, with at least one washing buffer, is carried out between the steps of immobilization and release.
39. The process according to claim 38, characterized in that the washing comprises the following steps for each washing buffer:
- application of a predetermined amount of washing buffer onto the surface;
and - transfer of the washing buffer through the surface.
40. The process according to one of the claims 1 to 13, 19, 20, 25, 26, 28, 29 and 38 to 39, characterized in that an aqueous salt or buffer solution is employed for the release of the nucleic acids.
41. The process according to claims 1 to 13, 19, 20, 25, 26, 28, 29 and 38 to 39, characterized in that water is employed for the release of the nucleic acids.
42. The process according to one of the preceding claims, characterized in that the charging and immobilization of the nucleic acids comprises the following steps:
- mixing of the at least one sample of nucleic acid with an immobilization buffer;
- charging of the at least one sample of nucleic acid, together with the immobilization buffer, onto the surface; and - transfer, essentially in the direction of charging, of the liquid parts through the surface.
43. The process according to one of the preceding claims, characterized in that at least one of the steps is carried out fully automatically by a machine.
44. The process according to claim 43, characterized in that all steps of this process are carried out in a controlled sequence by a machine.
45. The process according to one of the preceding claims, characterized in that a multitude of isolations or reactions of nucleic acids are carried out simultaneously.
46. The process according to one of the preceding claims, characterized in that aqueous salt solutions of metal- and/or ammonia cations with mineral acids are employed to immobilize the nucleic acids.
47. The process according to claim 46, characterized in that alkali- and/or earth alkali halogenides and/or -sulfates and/or -phosphates are employed in the immobilization of nucleic acids.
48. The process according to claim 47, characterized in that halogenides of sodium, lithium and/or potassium and/or magnesium sulfate are employed for the immobilization of nucleic acids.
49. The process according to one of the claims 1 to 45, characterized in that aqueous solutions of salts of mono- or polybasic or polyfunctional organic acids, together with alkali- or earth alkali metals, are employed to immobilize nucleic acids.
50. The process according to claim 49, characterized in that aqueous solutions of salts of sodium, potassium or magnesium, together with organic dicarboxylic acids, are employed to immobilize nucleic acids.
51. The process according to claim 50, characterized in that the organic dicarboxylic acid is oxalic acid, malonic acid and/or succinic acid.
52. The process according to claim 49, characterized in that aqueous solutions of salts of sodium or potassium, together with one hydroxy- or polyhydroxycarboxylic acid, are employed to immobilize the nucleic acids.
53. The process according to claim 52, characterized in that the polyhydroxycarboxylic acid is citric acid.
54. The process according to one of the claims 1 to 45, characterized in that hydroxyl derivatives of aliphatic or acyclic saturated or unsaturated hydrocarbons are employed to immobilize the nucleic acids.
55. The process according to claim 54, characterized in that the hydroxyl derivatives employed are C1-C5 alcanoles.
56. The process according to claim 55, characterized in that the C1-C5 alcanoles employed are methanol, ethanol, n-propanol, tert.-butanol and/or pentanols.
57. The process according to claim 54, characterized in that an aldite is employed as a hydroxyl derivative.
58. The process according to one of the claims 1 to 45, characterized in that a phenole or polyphenole is employed to immobilize the nucleic acids.
59. The process according to one of the preceding claims, characterized in that at least one chaotropic agent is employed to immobilize the nucleic acids.
60. The process according to claim 59, characterized in that the chaotropic agent is a salt selected from the group of the trichloracetates, thiocyanates, perchlorates, iodides, or guanidinium-hydrochloride, guanidinium-iso-thiocyanate or urea.
61. The process according to claims 59 or 60, characterized in that 0.01 molar to 10 molar aqueous solutions of the at least one chaotropic agent, alone or in combination with other salts, are employed to immobilize the nucleic acids.
62. The process according to claim 61, characterized in that 0.1 molar to 7 molar aqueous solutions of the at least one chaotropic agent, alone or in combination with other salts, are employed to immobilize the nucleic acids.
63. The process according to claim 62, characterized by the fact that 0.2 molar to 5 molar aqueous solutions of the at least one chaotropic agent, alone or in combination with other salts, are employed to immobilize the nucleic acids.
64. The process according to one of the claims 59 to 63, characterized in that an aqueous solution of sodium perchlorate, guanidinium-hydrochloride, guanidinium-iso-thiocynate, sodium iodide and/or potassium iodide is employed to immobilize the nucleic acids.
65. The process according to one of the claims 38 to 64, characterized in that a salt- or buffer solution, according to one of the claims 46 to 64, is employed to wash the immobilized nucleic acids.
66. The process according to one of the claims 9 to 65, characterized in that the surface is a membrane.
67. The process according to one of the claims 1 to 8 and 66, characterized in that the membrane is a hydrophobic membrane.
68. The process according to claim 67, characterized in that the hydrophobic membrane is formed from a polymer with polar groups.
69. The process according to claim 67 or 68, characterized in that the membrane is a hydrophilic membrane with a hydrophobized surface.
70. The process according to one of the claims 67 to 69, characterized in that the membrane consists of nylon, a polysulfone, polyethersulfone, polycarbonate, polypropylene, polyacrylate, acrylate- copolymers, polyurethane, polyamide, polyvinylchloride, polyfluorocarbonate, polytetrafluorethylene, polyvinylidendifluoride, polyethylenetetrafluorethylene-copolymerisate, one polyethylenechlorotrifluorethylene-copolymerisate, celluloseacetate, nitrocellulose, polybenzimidazole, polyimide, polyacrylnitriles, polyacrylnitrile-copolymers, cellulose mixed esters, cellulosenitrate, or polyphenylenesulfide.
71. The process according to claim 69 or 70, characterized in that the membrane consists of a hydrophobized nylon.
72. The process according to one of the claims 69 to 71, characterized in that the membrane is coated with a hydrophobization agent selected from the group of paraffins, waxes, metallic soaps, with additions of aluminium- or zirconium salts if applicable, quaternary organic compounds, urea derivatives, lipid modified melamine resins, silicones, organic zink compounds and/or glutaric dialdehyde.
73. The process according to one of the claims 1 to 8 and 66, characterized in that the membrane is a hydrophilic or hydrophilized membrane.
74. The process according to claim 73, characterized in that the membrane consists of hydrophilized nylon, polyethersulfone, polycarbonate, polyacrylate, acrylate-copolymers, polyurethane, polyamide, polyvinylchloride, polyfluorocsarbonate, polytetrafluorethylene, polyvinylidendifluoride, polyethylentetrafluorethylene-copolymerisate, one polyethylenechlorotrifluorethylene-copolymerisate, cellulaseacetate, polypropylene, nitrocellulose, polybenzimidazoles, polyimides, polyacrylnitriles, polyacrylnitrile-copolymers, cellulose mixed esters, polyester, polysulfone, cellulosenitrate, or polyphenylenesulfide.
75. The process according to one of the claims 1 to 8 and 66 to 74, characterized in that the membrane has a pore diameter of 0.001 to 50 micrometer, preferably 0.01 to 20 micrometer, particularly preferred 0.05 to 10 micrometer.
76. The process according to claims 9 to 27, characterized in that the surface is a hydrophobic fleece.
77. The process for the precipitation of nucleic acid with the following steps:
- providing an isolation container with at least one membrane placed inside;
- charging of the isolation container with a solution containing nucleic acids;
precipitation of the nucleic acids contained in the solution by means of alcohol, such, that the nucleic acids bond with the at least one membrane, characterized in that the pore size of the at least one membrane is larger or equal 0.2 micrometer.
78. The process according to claim 77, characterized in that alcohol is added to the solution containing nucleic acids prior to charging the isolation container with the solution containing nucleic acids.
79. The process according to claim 77, characterized in that alcohol is added to the solution containing nucleic acids after charging the isolation container with the solution containing nucleic acids.
80. The process according to one of the claims 77 to 79, characterized in that the surface of the membrane is selected such, that all the nucleic acids contained in the solution can bond to the membrane.
81. Use of membranes having a pore size larger or equal to 0.2 micrometer to bind nucleic acids precipitated with alcohol.
82. Use according to claim 81, characterized in that the nucleic acids precipitated with alcohol are DNA and/or RNA.
83. The process according to one of the claims 77 to 80 or the use according to one of the claims 81 and 82, characterized in that the alcohols used preferably are C1-C5 alcanoles, iso-propanol being particularly preferred.
84. The process according to one of the claims 77 to 80 and 83 or the use according to one of the claims 81 and 82, characterized in that the volume ratio of the solution containing nucleic acids to iso-propanol is 2:1 to 1:1, preferably 1.67:1 to 1:1 and particularly preferred 1.43:1 to 1:1.
85. The process according to one of the claims 77 to 80, 83 and 84 or the use according to one of the claims 81 to 84, characterized in that the membrane is a hydrophobic membrane.
86. The process or use according to claim 85, characterized in that the hydrophobic membrane is made from a polymer with polar groups.
87. The process or use according to claim 85 or 86, characterized in that the membrane is a hydrophilic membrane with a hydrophobized surface.
88. The process or use according to one of the claims 85 to 87, characterized in that the membrane consists of nylon, one polysulfone, polyethersulfone, polypropylene, polycarbonate, polyacrylate, acrylate-copolymers, polyurethane, polyamide, polyvinylchloride, polyfluorocarbonate, polytetrafluorethylene, polyvinylidendifluoride, polyethylenetetrafluorethylene-copolymerisate, one polyethylenechlorotrifluorethylene-copolymerisate or polyphenylenesulfide.
89. The process or use according to claim 87 or 88, characterized in that the membrane consists of a hydrophobized nylon.
90. The process or use according to one of the claims 87 to 89, characterized in that the membrane is coated with a hydrophobizing agent selected from the group of paraffins, waxes, metallic soaps, with additions of aluminium- or zirconium salts if applicable, quaternary organic compounds, urea derivatives, lipid modified melamine resins, silicones, organic zink compounds and/or glutaric dialdehyde.
91. The process according to one of the claims 77 to 80, 83 and 84 or use according to one of the claims 81 to 86, characterized in that the membrane is a hydrophilic or hydrophilized membrane.
92. The process or use according to claim 91, characterized in that the membrane consists of hydrophilized nylon, polyethersulfone, polycarbonate, polyacrylate, acrylate-copolymers, polyurethane, polyamide, polyvinylchloride, polyflourocarbonate, polytetrafluoroethylene, polyvinylidendifluoride, polyethylenetetrafluoroethylene-copolymerisate, one polyethylenechlorotrifluoroethylene- copolymerisate, cellulose acetate, cellulose nitrate or polyphenylene sulfide.
93. The process or use according to claim 92, characterized in that the membrane consists of cellulose acetate or cellulose nitrate.
94. The process or use according to one of the claims 77 to 93, characterized in that the membrane shows a pore size of larger than 0.45 µm.
95. The process or use according to claim 94, characterized in that the membrane shows a pore size of larger than 0.6µm.
96. A machine, characterized in that it is capable of carrying out the process of one of the claims 1 to 80 and 83 to 95.
97. The machine according to claim 96,characterized in that it is equipped with at least one suction device, which carries out, or is capable of carrying out, the addition of buffers and solutions onto the surface and away from the surface.
98. An isolation container for the isolation of nucleic acids comprising at least one cylindrical upper part with a top opening, a bottom opening and a membrane which is located at the bottom opening and covers the entire cross section of the upper part;
a lower part with an absorbent material; and a mechanism to connect the upper and lower parts, whereby, with effected connection, the membrane is in contact with the absorbent material and when not connected, the membrane is not in contact with the absorbent material.
99. The isolation container according to claim 98, characterized in that the lower part is a cylinder of the same diameter as the upper part.
100. The isolation container according to claim 98 or 99, characterized in that the mechanism is a coupling which permits a physical separation of the upper and lower parts.
101. The isolation container according to claim 100, characterized in that the coupling is of the bayonet type.
102. The isolation container according to claim 100, characterized in that the coupling is of the screw type.
103. The isolation container according to claim 98 or 99, characterized in that the mechanism is a slide, which can be inserted between the absorbent material and the membrane.
104. The isolation container according to claim 98 or 99, characterized in that the mechanism is the predetermined breaking point between upper part and lower part.
105. The isolation container according to one of the claims 98 to 104, characterized in that the upper part is a tube, which is suitable for placement inside reaction container holders.
106. The isolation container according to one of the claims 98 to 105, characterized in that the upper part and the lower part form a tube, which is suitable for placement inside reaction container holders.
107. The isolation container according to one of the claims 98 to 105, characterized in that several upper parts are located on top of one lower part.
108. The isolation container according to one of the claims 98 to 107, characterized in that the absorbent material comprises a sponge.
109. The isolation container according to one of the claims 98 to 108, characterized in that the absorbant material comprises granules.
110. Use of the isolation container according to one of the claims 98 to 109 for the analysis of the properties of nucleic acids.
111. Use of the isolation container according to one of the claims 98 to 109 for the isolation of nucleic acids.
112. Use of an isolation container according to one of the claims 98 to 111 as reaction container for the durable immobilization of nucleic acids at a membrane, which covers the entire cross section of the isolation container.
113. An isolation container for the isolation of nucleic acids comprising at least an upper part with a top opening, a bottom opening and a membrane, which is located at the bottom opening and covers the entire cross section of the upper part;
a lower part with an absorbant material;
and a jacket to contain a cooling medium, surrounding the upper part, at least in the membrane section.
114. The isolation container according to claim 113, characterized in that the jacket contains two compartments, which are separated by a mechanically destructible partition and each compartment contains a solution, whereby, after the destruction of the partition, the cooling medium is formed through the mixing of both solutions.
115. Use of cellulose acetate, not carboxylated, hydrophobic polyvinylidendifluoride or solid, hydrophobic polytetrafluoroethylene as the material for the attachment and isolation of nucleic acids.
116. Use according to claim 115, characterized in that the material is used in the form of a membrane.
117. Use according to claim 115, characterized in that the; material is used in the form of granules.
118. Use according to claim 115, characterized in that the material is used in the form of fibres.
119. Use according to claim 118, characterized in that the fibres are arranged in a fleece.
120. A kit for the isolation of nucleic acids with - an immobilization buffer;

- an elution buffer; and - at least one isolation container according to one of the claims 98 to 108 or 113.
121. The kit according to claim 120, characterized in that, in addition, it further comprises a washing buffer.
122. The kit according to claims 120 or 121, characterized in that, in addition, it further comprises a lyse buffer.
123. The kit according to one of the claims 120 to 122, characterized in that it is suitable for the carrying out of the processes according to one of the claims 1 to 81 or 85 to 95.
CA2348054A 1998-10-23 1999-04-20 Processes and means for the isolation and purification of nucleic acids at surfaces Expired - Lifetime CA2348054C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA2771000A CA2771000A1 (en) 1998-10-23 1999-04-20 Processes and means for the isolation and purification of nucleic acids at surfaces

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
PCT/EP1998/006756 WO1999022021A1 (en) 1997-10-23 1998-10-23 Method for isolating and purifying nucleic acids on surfaces
EPPCT/EP98/06756 1998-10-23
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CA2348054C (en) 2012-06-19
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ATE474063T1 (en) 2010-07-15

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