Isolation of nucleic acids
View Patent ↗A method for extracting nucleic acids from a biological material such as blood comprises contacting the mixture with a material at a pH such that the material is positively charged and will bind negatively charged nucleic acids and then eluting the nucleic acids at a pH when the said materials possess a neutral or negative charge to release the nucleic acids. The nucleic acids can be removed under mildly alkaline conditions to the maintain integrity of the nucleic acids and to allow retrieval of the nucleic acids in reagents that are immediately compatible with either storage or analytical testing.
1. A product comprising a solid phase for use in a method in which the solid phase reversibly binds nucleic acid present in a sample, the product comprising a plurality of positively ionizable groups, wherein the ionizable groups are immobilized on the solid phase and are effective at a first pH at which the ionizable groups are positively charged to bind nucleic acid present in a sample and are effective to release the nucleic acid at a second, higher, pH at which the charge on the ionizable groups is negative, neutral or less positive, the ionizable groups being provided by a chemical species selected from the group consisting of biological buffers, polyhydroxylated amines, histidine and polyhistidine, wherein said plurality of positively ionizable groups have a pKa between about 4.5 and about 8.5.
2. A product according to claim 1 , wherein the plurality of positively charged groups are provided by a biological buffer which is selected from the group consisting of:
N-2-acetamido-2-aminoethanesulfonic acid (ACES);
N-2-acetamido-2-iminodiacetic acid (ADA);
N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES);
N,N-bis-2-hydroxyethylglycine (BICINE);
bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane);
3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO);
-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS);
-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
2-N-morpholinoethanesulfonic acid (MES);
4-N--morpholinobutanesulfonic acid (MOBS);
3-N-morpholinopropanesulfonic acid (MOPS);
3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO);
N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO);
N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES);
N-trishydroxymethylmethylglycine (TRICINE);
trishydroxymethylaminomethane (Tris);
polyhydroxylated imidazoles; and
triethanolamine dimers and polymers.
3. A product according to claim 1 , wherein the plurality of ionizable groups are separately immobilised on a solid support by covalent or ionic bonding or by adsorption.
4. A product according to claim 1 , wherein the plurality of ionizable groups are separately attached to a polymer, said polymer being immobilised on a solid support by covalent or ionic bonding or by adsorption.
5. A product according to claim 1 , wherein the ionizable groups are polymerised, optionally by means of cross-linking reagents.
6. A product according to claim 5 , wherein the polymer is immobilised on a solid support by covalent or ionic bonding or by adsorption.
7. A product according to claim 5 , wherein the polymer is a solid.
8. A product according to claim 5 which is a container.
9. A container according to claim 8 which is a polymerase chain reaction or storage tube or well, or a pipette tip.
10. A water soluble product for use in a method of extracting nucleic acid from a sample, the product comprising a plurality of positively ionizable groups, the ionizable groups being provided by a biological buffers;
wherein the plurality of ionizable groups are (i) separately attached to a polymer or (ii) polymerized, optionally by means of cross-linking reagents or (iii) separately attached to a polymer and polymerized, optimally by means of cross-linking reagents; and wherein said plurality of positively ionizable groups have a pKa between about 4.5 and 8.5 and the biological buffer is selected from the group consisting of:
N-2-acetamido-2-aminoethanesulfonic acid (ACES);
N-2-acetamido-2-iminodiacetic acid (ADA);
N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES);
N,N-bis-2-hydroxyethylglycine (BICINE);
bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane);
3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO);
-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS);
-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
2-N-morpholinoethanesulfonic acid (MES);
4-N-morpholinobutanesulfonic acid (MOBS);
3-N-morpholinopropanesulfonic acid (MOPS);
3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO);
N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO);
N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES);
N-trishydroxymethylmethylglycine (TRICINE);
trishydroxymethylaminomethane (Tris);
polyhydroxylated imidazoles; and
triethanolamine dimers and polymers.
11. A product comprising a solid phase for use in which the solid phase reversibly binds nucleic acid present in a sample, the product comprising a plurality of positively ionizable groups, wherein the ionizable groups are immobilised on the solid phase and are capable at a first pH at which the ionizable groups are positively charged of binding nucleic acid present in a sample and are capable of releasing the nucleic acid at a second, higher, pH at which the charge on the ionizable groups is negative, neutral or less positive, wherein said plurality of positively ionizable groups have a pKa between about 4.5 and about 8.5, the ionizable groups being provided by a biological buffer selected from the group consisting of:
N-2-acetamido-2-aminoethanesulfonic acid (ACES);
N-2-acetamido-2-iminodiacetic acid (ADA);
N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES);
N,N-bis-2-hydroxyethylglycine (BICINE);
bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane);
3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO);
-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS);
-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
2-N-morpholinoethanesulfonic acid (MES);
4-N-morpholinobutanesulfonic acid (MOBS);
3-N-morpholinopropanesulfonic acid (MOPS);
3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO);
N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO);
N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES);
N-trishydroxymethylmethylglycine (TRICINE);
trishydroxymethylaminomethane (Tris);
polyhydroxylated imidazoles; and
triethanolamine dimers and polymers.
12. A product according to claim 11 , wherein the biological buffer is bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris).
13. A product according to claim 11 , wherein the plurality of ionizable groups are separately immobilised on a solid support by covalent or ionic bonding or by adsorption.
14. A product according to claim 11 , wherein the plurality of ionizable groups are separately attached to a polymer, said polymer being immobilised on a solid support by covalent or ionic bonding or by adsorption.
15. A product according to claim 11 , wherein the ionizable groups are polymerised, optionally by means of cross-linking reagents.
16. A product according to claim 15 , wherein the polymer is immobilised on a solid support by covalent or ionic bonding or by adsorption.
17. A product according to claim 11 , wherein the solid phase is selected from the group consisting of beads, particles, tubes, wells, probes, dipsticks, pipette tips, slides, fibers, membranes, papers, glass and plastics.
18. A product according to claim 17 , wherein the solid phase is magnetic beads.
19. A product according to claim 17 , wherein the solid phase is paramagnetic beads.
20. A product comprising a solid phase for use in which the solid phase reversibly binds nucleic acid present in a sample, the product comprising a plurality of positively ionizable groups, wherein the ionizable groups are immobilised on the solid phase and are capable at a first pH at which the ionizable groups are positively charged of binding nucleic acid present in a sample and are capable of releasing the nucleic acid at a second, higher, pH at which the charge on the ionizable groups is negative, neutral or less positive, the ionizable groups being provided by bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris) and having a pKa between about 4.5 and 8.5.
21. A product according to claim 20 , wherein the bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris) groups are separately immobilised on a solid support by covalent or ionic bonding or by adsorption.
22. A product according to claim 20 , wherein the bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris) groups are separately attached to a polymer, said polymer being immobilised on a solid support by covalent or ionic bonding or by adsorption.
23. A product according to claim 20 , wherein the bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris) is polymerised, optionally by means of cross-linking reagents.
24. A product according to claim 23 , wherein the polymer is immobilised on a solid support by covalent or ionic bonding or by adsorption.
25. A product according to claim 20 , wherein the solid phase is selected from the group consisting of beads, particles, tubes, wells, probes, dipsticks, pipette tips, slides, fibers, membranes, papers, glass and plastics.
26. A product according to claim 25 , wherein the solid phase is magnetic beads.
27. A product according to claim 25 , wherein the solid phase is paramagnetic beads.
28. A product comprising a solid phase for use in which the solid phase reversibly binds nucleic acid present in a sample, the product comprising a plurality of positively ionizable groups, wherein the ionizable groups are immobilised on the solid phase and are capable at a first pH at which the ionizable groups are positively charged of binding nucleic acid present in a sample and are capable of releasing the nucleic acid at a second, higher, pH at which the charge on the ionizable groups is negative, neutral or less positive, the ionizable groups being provided by bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris) and having a pKa between about 4.5 and 8.5. and the solid phase comprises beads.
29. A product according to claim 28 , wherein the bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris) groups are separately immobilised on the beads by covalent or ionic bonding or by adsorption.
30. A product according to claim 28 , wherein the bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris) groups are separately attached to a polymer, said polymer being immobilised on the bead by covalent or ionic bonding or by adsorption.
31. A product according to claim 28 , wherein the bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris) is polymerised, optionally by means of cross-linking reagents.
32. A product according to claim 31 , wherein the polymerized Bis-Tris is immobilised on the beads by covalent or ionic bonding or by adsorption.
33. A product according to claim 28 , wherein the solid phase is magnetic beads.
34. A product according to claim 28 , wherein the solid phase is paramagnetic beads.
35. A product according to claim 1 , wherein the solid phase is selected from the group consisting of beads, particles, tubes, wells, probes, dipsticks, pipette tips, slides, fibers, membranes, papers, glass and plastics.
36. A product according to claim 35 , wherein the solid phase is magnetic beads.
37. A product according to claim 35 , wherein the solid phase is paramagnetic beads.
38. A product according to claim 1 , wherein the pKa of the plurality of ionizable groups is between 5.0 and 6.5.
39. A product according to claim 10 , wherein the pKa of the plurality of ionizable groups is between 5.0 and 6.5.
40. A product according to claim 11 , wherein the pKa of the plurality of ionizable groups is between 5.0 and 6.5.
41. A product according to claim 20 , wherein the pKa of the plurality of ionizable groups is between 5.0 and 6.5.
42. A product according to claim 28 , wherein the pKa of the plurality of ionizable groups is between 5.0 and 6.5.