MXPA00005474A - Isolation of nucleic acids - Google Patents

Abstract

A method of extracting nucleic acids from blood comprises contacting blood cells, preferably after lysing with an activated solid phase at one pH to immobilise the nucleic acids and then removing the nucleic acids at a higher pH when the charge has been reversed or neutralised. The solid phase can be glass beads activated by a histidine as a binding agent. The beads can be fluidised by sucking the blood with air up through a column containing the beads to improve contact and prevent clogging.

Description

METHOD FOR ISOLATING NUCLEIC ACIDS Description of the invention: The present invention relates to a method for the extraction of nucleic acids and other biomolecules from biological material, especially blood. There is a great demand for DNA analysis for several objectives and this has led to require fast, safe and high performance methods for the isolation and purification of DNA and other nucleic acids. Samples for use for DNA identification or analysis can be taken from a wide range of sources, such as biological material, such as animal or plant cells, feces, tissue, etc., soil samples can also be obtained, food products, water, etc. Existing methods for DNA extraction include the use of phenol / chloroform, desalting, the use of chaotropic salts and silica resins, the use of affinity resins, ion exchange chromatography and the use of magnetic beads. The methods are described in U.S. Patents 5057426, 4923978, EP 0512767 Al and EP0515484B and WO 95/13368, WO 97/10331 and WO 96/18731. These patents and patent applications describe methods of adsorbing nucleic acids on a solid support and the REF; 120533 isolation after nucleic acids. The previously used methods use some kind of solvent to isolate the nucleic acids and these solvents are flammable, combustible or toxic. Blood is one of the most abundant sources for obtaining samples for DNA analysis, since blood samples are routinely extracted for a wide variety of reasons. However, due to the viscous and protein nature of the blood, the use of known DNA extraction methods has proved difficult to carry out with automation due to the difficulties of handling large volumes containing relatively small amounts of DNA. So far, nucleic acid extraction has been partially automated only using dangerous reagents and slow processing times. I have now conceived an improved method for the extraction of nucleic acids and other biomolecules from blood and other biological materials. According to the invention there is provided a method for the extraction of biomolecules from biological material, which method comprises contacting the biological material with a solid phase which is capable of binding the biomolecules thereto at a first pH, and then extracting the biomolecules bound to the solid phase by elution using an elution solvent at a second pH.

The method is particularly useful if the biological material is blood, but the method can be used for a wide range of substances with applications, such as for the isolation of plasmids and vectors, and the extraction of DNA from plants. Preferably, the cells in the blood are used to release the nucleic acids, and known lysis agents and methods can be used, such as contacting with ionic and nonionic detergents, hypotonic solutions of salts, proteases, chaotropic agents, solvents, using changes in pH or heat. In WO 96/00228 a method of cell lysation for isolating nucleic acids is described. When the biological material consists of blood, the samples can be optionally diluted with water or with another diluent in order to facilitate their handling and processing. Dilutions up to ten times may be used and in general, a higher dilution may be better, and it is a feature of the present invention that low blood dilution is possible. The solid phase with which the blood is contacted may be formed of a material that has a natural affinity for the nucleic acids or may be formed of a material having a surface treated with an agent that will cause the nucleic acids to be join it or increase its affinity for nucleic acids. Suitable materials include controlled porous glass, polysaccharides (agarose or cellulose), other types of silica / glass, ceramic materials, porous plastic materials, such as porous plastic caps included in a single molded part or in the form of an insert in a standard tube, polystyrene beads, paramagnetic beads, etc. The size and porosity are not critical and can vary and be selected for particular applications. Suitable means for treating the surface of the solid phase or for derivatizing it include treating it with a substance that can introduce a charge, for example a positive charge on the surface or a hydrophilic or hydrophobic surface on the surface. solid phase, for example, hydroxyl groups, nitrate groups, autoreactive groups, dyes and other aromatics. In a preferred embodiment of the invention, the solid phase will cause the DNA to bind thereto at a pH, preferably contaminants in the blood sample, and will allow the bound nucleic acid to be released when contacted with a eluent at a different pH. This system can be used with a solid phase incorporating histidine or a polyhistidine, which will tend to bind the nucleic acids at a low pH, for example, less than 6, and then release the bound nucleic acids when the pH increases, for example, when is greater than 8. Alternatively, the nucleic acids bind at a substantially neutral pH to an aminated surface and are released at a very high pH. In another embodiment of the invention, a plastic mold can incorporate a binding agent, for example, in a well in a plate, etc., so that the binding agent is incorporated into the surface, the sample of blood with the surface, in such a way that the union of the nucleic acids to the surface takes place. The blood sample is then removed and the surface is treated with an eluent to release bound nucleic acids. When the surface is a part of a well in a multi-well plate, the total system can be easily adapted to rapid and large-scale sample collection and extraction techniques. The binding agents that can be used include displaceable charge ion exchange resins using a positively charged solid phase which can be inverted or made neutral by changing the pH above its pKa, for example, nucleotides, polyamines, imidazole groups and other reagents similar with an appropriate pKa value. Also, nucleic acids can be joined by intercalation using a variety of intercalating compounds incorporated in the solid phase, for example, actinomycin D, ethidium bromide, etc. In another embodiment of the invention, a plastic surface can be modified to include functional groups. The plastic can be any plastic used to contain samples, for example, polypropylene. The functional groups can be positively or negatively charged, so that they bind the nucleic acids in the correct buffer solution. Alternatively, the functional groups may be chemical groups capable of covalently binding to other ligands or polymers. When the plastic is used in a plastic mold, for example in a well on a plate, or as a tube for the polymerase chain reaction (PCR), the surface characteristics of the plastic can be appropriately modified for use in the present invention including or adding the appropriate chemicals in the molding compound, as, for example, in a compound for injection molding. When used in a PCR tube or in a deep well plate, tubes or wells can be used to isolate and immobilize small amounts of DNA or RNA that generate a pure template for subsequent PCR or other genetic analysis and manipulation. When the plastic is polypropylene, for example, it is in the form of a thin-walled PCR tube, the polypropylene surface can be modified by oxidizing the surface with an oxidizing agent, such as potassium permanganate and sulfuric acid to create a carboxylated surface (COOH groups). This tube can then be used to improve the isolation of DNA from the solutions or from the raw samples, for example, blood. By adjusting the pH, the dielectric constant, the solubility or the ionic power, the DNA or the RNA can be immobilized on the walls of the tube, washed to leave it free of contaminants, ready for PCR or other analytical techniques. The carboxy groups can be further modified by covalent coupling of an anionic group, such as imidazole or polyhistidine, or any strong or weak ion exchanger, to allow binding of the nucleic acids by a charge interaction. This tube could then be used to improve the isolation of the DNA from the solutions or from the crude samples, for example, from blood. Again, by adjusting the pH, the dielectric constant or the ionic power, the DNA or RNA can be immobilized on the walls of the tube, washed to leave it free of contaminants, ready for PCR or other analytical techniques. The nucleic acids can be eluted with a buffer of low salinity, so that it is ready for PCR or other analyzes. The solid phase can be contacted with a blood sample by mixing with the solid phase in a mixing / stirring device, by passing the blood sample over the solid phase or the solid phase can be paramagnetic and manipulated with a magnetic field. Although the invention is particularly suitable for the separation or isolation of nucleic acids from blood, it can be used with a wide range of biomolecules, especially those which require the removal of debris from cell walls or insoluble particles. In a preferred embodiment of the invention the solid phase is in granular form in a column and the blood sample is drawn through the column by applying differential pressure through the column, the blood sample is extracted with air and the solid granular material can be fluidized, thus increasing the mixture and contact percentages and minimizing coagulation. The method of the invention is suitable for use in a multi-well format where a series of extractions of different samples can take place substantially simultaneously and this will facilitate the automation of the extraction process, allowing a rapid high-throughput extraction to take place and that the combinatorial chemistry is allowed. This will allow the performance to be very high in a standard well arrangement, for example, in an arrangement of eight to twelve wells, so that a large number of sample types can be treated automatically at the same time. The invention is described in the Example.

Example 1 Extraction of nucleic acids from whole blood. A displaceable charge ion exchanger was prepared by covalent coupling of polyhistidine to 100 μm glass beads using glutaraldehyde by mixing 1 gram of amber glass beads with 0.01% (v / v) glutaraldehyde in sodium bicarbonate 0.1. M at a pH of 8 containing 20 mg of polyhistidine. After overnight incubation, the beads were thoroughly washed to remove non-covalently bound material and stored in 10 mM MES, pH 5 containing 0.1% Tween 20 (v / v). Approximately 300 mg of derivatized 100 μm glass beads were added to a 1 ml plastic column closed at both ends. A blood sample was incubated with an equal volume of 10 mM MES pH 5, containing 1% Tween 20, proteases (200 μg / ml) and 1 mM EDTA. After the digestion was completed, the blood was sucked into the upper part of the column containing the glass beads and the DNA remained immobilized, allowing the contaminating proteins to pass through and being discarded. The glass beads containing the immobilized DNA were washed with a buffer comprising 10 mM MES pH 5, containing 1% Tween 20 and 1 mM EDTA and this was repeated until the washing solution was colorless. After washing, the beads were air dried and the DNA was eluted with a small amount of 10 M Tris HCl, pH 8.5 and collected in a sterile tube ready for analysis. Thus, the DNA separated from the blood. For different biomolecules, buffer, etc. they can be modified appropriately.

Example 2 One gram of carboxylated paramagnetic beads was washed in 50 mM imidazole buffer pH 6 and then mixed with 100 mg of polyhistidine in 50 ml of 50 mM imidazole buffer pH 6. A chemical coupling agent (EDC) was added at a concentration end of 5 mg per ml and mixed overnight. The beads were washed in water, 0.5 M sodium chloride, 1% Tween 20, 100 mM Tris HCl pH 8 and stored in 10 mM MES, 0.1% Tween 20 pH 5. To extract the DNA from the blood, 1 mg of beads was mixed with blood diluted in 10% Tween 20 with 25 mM MES, 1 mM EDTA, pH 5. The beads were separated with a magnet and washed by resuspending them in 1 mM MES, 0 Tween 20, 1%. To elute the DNA, the beads were resuspended in 10 mM Tris HCl pH 8.5 and separated with a magnet leaving the DNA in solution.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (35)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for extracting biomolecules from biological material, characterized in that it comprises contacting the biological material with a solid phase which is capable of binding the biomolecules thereto at a first pH and then extracting the biomolecules attached to the biomolecule. solid phase by elution, using an elution solvent at a second pH.

2. A method as claimed in claim 1, characterized in that the biomolecule comprises nucleic acids.

3. A method as claimed in claim 1 or 2, characterized in that the biological material is blood.

4. A method as claimed in claim 1, 2 or 3, characterized in that the biomolecule is contacted with the solid phase at a pH of less than 6 to bind the DNA to the solid phase and the DNA is released from the solid phase at a pH higher than 8.

5. A method as claimed in claim 1, 2 or 3, characterized in that the biomolecule is contacted with the solid phase at a substantially neutral pH to bind the DNA to the solid phase and the DNA is released from the solid phase at a pH greater than 10.

6. A method as claimed in any one of claims 3 to 5, characterized in that the cells in the blood are used to release nucleic acids.

7. A method as claimed in claim 6, characterized in that the cells are used by contacting them with an ionic or non-ionic detergent, hypotonic solutions of a salt, a protease, a chaotropic agent or by the use of changes in pH or heat.

8. A method as claimed in any of claims 3 to 7, characterized in that the blood is diluted with water or another diluent in order to make it easier to handle and pour it.

9. A method as claimed in claim 8, characterized in that a dilution of up to ten times is used.

10. A method as claimed in any one of the preceding claims, characterized in that the solid phase with which the biological material is brought into contact is formed of a material having a natural affinity for the nucleic acids.

11. A method as claimed in any one of the preceding claims, characterized in that the solid phase with which the biological material is brought into contact is formed of a material having its surface treated with an agent that will cause the nucleic acids to bind to or increase their affinity for nucleic acids.

12. A method as claimed in claim 11, characterized in that the surface of the solid material is treated with a substance that can introduce a positive charge or a hydrophilic or hydrophobic surface onto the solid phase.

13. A method as claimed in claim 11, characterized in that the binding agent used is a displaceable charge ion exchange resin that uses a positively charged solid phase that can be inverted or neutralized by changing the pH above or below its pKa .

14. A method as claimed in claim 13, characterized in that the binding agent is a nucleotide, a polyamine or a compound containing an imidazole moiety.

15. A method as claimed in claim 11, characterized in that the nucleic acids are joined by intercalation using an intercalating compound incorporated into the solid phase.

16. A method as claimed in claim 15, characterized in that the dye is actinomycin D or ethidium bromide.

17. A method as claimed in claim 11, characterized in that the surface of the solid material is treated with histidine or polyhistidine.

18. A method as claimed in any one of the preceding claims, characterized in that the solid phase is a controlled porous glass, a polysaccharide, a ceramic material or a plastic porous material.

19. A method as claimed in any one of the preceding claims, characterized in that the solid phase comprises polystyrene beads or a paramagnetic material.

20. A method as claimed in any one of the preceding claims, characterized in that the solid phase comprises a plastic surface modified to include functional groups.

21. A method as claimed in claim 20, characterized in that the plastic is polypropylene.

22. A method as claimed in claim 21, characterized in that the polypropylene surface is modified by oxidizing the surface with an oxidizing agent to create a carboxylated surface.

23. A method as claimed in claim 22, characterized in that the carboxyl groups are further modified by covalently coupling an anionic group, such as imidazole or polyhistidine or any strong or weak ion exchanger, to allow the binding of the nucleic acids by an interaction of cargo.

24. A method as claimed in any one of claims 11 to 23, characterized in that the functional groups are positively or negatively charged so that the nucleic acids bind.

25. A method as claimed in any one of claims 11 to 23, characterized in that the functional groups are chemical groups capable of covalently binding to other ligands or polymers.

26. A method as claimed in any one of claims 20 to 25, characterized in that the solid material is a porous plastic stopper, which is a single molded part or that is inserted into a container.

27. A method as claimed in claim 20 to 25, characterized in that the plastic is in a plastic mold.

28. A method as claimed in claim 27, characterized in that the solid material is a tube for the polymerase chain reaction (PCR).

29. A method as claimed in claim 27, characterized in that the solid material is a plate of deep wells.

30. A method as claimed in any one of claims 26 to 29, characterized in that the tubes or wells are used to isolate and immobilize small amounts of DNA or RNA, generating a pure template for subsequent PCR or other genetic analysis and manipulation.

31. A method as claimed in any one of the preceding claims, characterized in that the solid phase comprises solid granular material which is brought into contact with a blood sample by mixing with the solid material in a mixing / stirring device, by passing the sample of Blood on the solid phase or the solid phase is handled on a magnetizable support.

32. A method as claimed in any one of the preceding claims, characterized in that the packages are wells in a multi-well plate, and a series of DNA extractions of different samples takes place substantially simultaneously.

33. A method as claimed in any one of the preceding claims, characterized in that the solid phase is in granular form in a column and the blood sample is drawn through the column by application to the entire differential pressure column.

34. A method as claimed in claim 33, characterized in that the blood sample is extracted with air and the granular solid material is fluidized.

35. A method as claimed in any one of the preceding claims, characterized in that the biomolecules are removed by elution with a buffer solution of low salinity or water.