Modified surfaces as solid supports for nucleic acid purification

• US 20050208510A1
• Filed: 09/30/2004
• Published: 09/22/2005
• Est. Priority Date: 03/18/2004
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Ambion Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Kemppainen, Jon A., Latham, Gary J., Pasloske, Brittan L., Conrad, Richard C., Fang, Xingwang, Setterquist, Robert A.
• Granted Patent: US 8,426,126 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

The invention relates to methods of separating polynucleotides, such as DNA, RNA and PNA, from solutions containing polynucleotides by reversibly binding the polynucleotides to a solid surface, such as a magnetic microparticle.

Claims

1. A method of reversibly binding at least one polynucleotide to one or more surface comprising a polymer or resin, comprising:

contacting the surface with a solution containing at least one polynucleotide; and

adjusting a salt concentration and/or an organic solvent concentration of the solution to allow for binding of the polynucleotide onto the surface;

wherein, if the surface comprises a polymer, the polymer is further defined as comprising a polyvinylpyrrolidone, a polyethylene glycol, or a polymeric polyol, with the proviso that any polymeric polyol cannot be cellulose.

2. The method of claim 1, wherein the surface comprises a polymer.

3. The method of claim 2, wherein the polymer is a polymeric polyol.

4. The method of claim 3, wherein the polymeric polyol is Dextran.

5. The method of claim 3, wherein the polymeric polyol is a polysaccharide.

6. The method of claim 2, wherein the polysaccharide is xanthum gum, glycogen, ficoll, gum arabic, carageenan, amylose, agar, amylopectin, xylans, or a beta-glucan.

7. The method of claim 2, wherein the polymer is polyethylene glycol.

8. The method of claim 2, wherein the polymer is polyvinylpyrrolidone.

9. The method of claim 2, wherein the polymer is hydrophilic.

10. The method of claim 2, wherein the polymer binds at least 10% of the polynucleotides in the solution.

11. The method of claim 2, wherein the polymer is further defined as a polymer that will co-precipitate with nucleic acid.

12. The method of claim 1, wherein the surface comprises a resin.

13. The method of claim 12, wherein the resin is isocyanate, glycerol(Diol), piperidino-methyl, polyDMAP, DIPAM, aminomethyl, polystyrene aldehyde, Tris(2-aminomethyl)amine, morpholino-methyl, BOBA, triphenyl-phosphine, or benzylthio-methyl.

14. The method of claim 12, wherein the resin is phenylsepharose, glycerol(diol), morpholino-methyl, piperidino-methyl, or polystyrene aldehyde.

15. The method of claim 1, wherein the surface is on one or more magnetic microparticles.

16. The method of claim 1, wherein the salt concentration is a concentration of one or more salts comprising at least one of sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride, cesium chloride, sodium perchlorate, guanidinium isothiocyanate, guanidinium hydrochloride, potassium iodide, and sodium iodide.

17. The method of claim 16, wherein the salt concentration is from 0.1M to 5M.

18. The method of claim 16, wherein the organic solvent concentration is a concentration of one or more organic solvents comprising at least one of a C1 to C5 alcohol, a polyethylene glycol, a dimethyl sulfoxide, or an acetone.

19. The method of claim 18, wherein the concentration of the organic solvent is adjusted to between about 20% and 50%.

20. The method of claim 18, wherein the salt is guanidinium isothiocyanate.

21. The method of claim 1, wherein the sample is a biosample or a lysate of a biosample.

22. The method of claim 21, wherein the polynucleotide solution comprises a lysate of blood, a blood fraction, fresh tissue, fixed tissue, or a cultured cell.

23. The method of claim 1, wherein the polynucleotide is a bacterial polynucleotide or a polynucleotide encoding a bacterial component.

24. The method of claim 1, wherein the polynucleotide is a eukaryotic polynucleotide or a polynucleotide encoding a eukaryotic component.

25. The method of claim 1, wherein the polynucleotide is a viral polynucleotide or a polynucleotide encoding a viral component.

26. The method of claim 1, wherein the polynucleotide is a DNA.

27. The method of claim 1, wherein the polynucleotide is a RNA.

28. The method of claim 1, further comprising preparing the solution.

29. The method of claim 28, wherein preparing the solution comprises performing an RNA amplification reaction, an in vitro transcription reaction, a reverse transcription reaction, a second strand DNA synthesis reaction, a DNase reaction, and/or or a PCR reaction.

30. The method of claim 28, wherein preparing the solution comprises lysing a cell.

31. The method of claim 28, wherein preparing the solution comprises conjugating RNA, DNA, or PNA in the solution to a reporter dye.

32. The method of claim 1, further defined as a method of isolating or concentrating RNA, DNA, or PNA.

33. The method of claim 32, further comprising conjugating the RNA, DNA, or PNA to a reporter dye.

34. The method of claim 32, further comprising fragmenting the RNA, DNA, or PNA and then hybridizing at least a portion of the fragmented RNA, DNA, or PNA to a microarray.

35. The method of claim 32, further comprising hybridizing at least a portion of the RNA, DNA, or PNA to a microarray.

36. The method of claim 1, further defined as a method of separating at least one polynucleotide from a solution containing the polynucleotide, comprising:

admixing the at least one surface comprising a polymer or resin and a solution containing at least one polynucleotide to produce an admixture;

adjusting a salt concentration and an organic solvent concentration of the admixture to concentrations wherein the at least one polynucleotide binds to the at least one surface;

separating the at least one modified surface from the remainder of the admixture while the at least one polynucleotide is bound to the surface; and

contacting the surface with an elution buffer, thereby separating the at least one polynucleotide from the at least one surface.

37. The method of claim 36, wherein the surface is on a magnetic microparticle.

39. The method of claim 36, wherein the at least one polynucleotide bound to the at least one surface is washed with a buffer solution that dissolves one or more impurities bound to the surface while leaving the at least one polynucleotide bound to the at least one surface.

40. The method of claim 36, wherein the at least one polynucleotide is further defined as at least one RNA.

41. The method of claim 40, further defined as comprising binding the at least one RNA bound to the at least one surface in a method comprising using a selective binding solution that does not allow substantial DNA binding to the surfaces but does allow RNA binding to the surface.

42. The method of claim 40, wherein the RNA bound to the surfaces is washed with a selective wash solution, wherein the selective binding solution substantially dissolves DNA fragments bound to the surfaces while leaving RNA fragments substantially bound to the surfaces.

43. The method of claim 40, wherein the at least one RNA bound to the at least one surface is eluted with a selective elution solution that elutes the at least one RNA fragment from the surface while leaving DNA fragments substantially bound to the surface.

44. The method of claim 36, wherein the at least one polynucleotide is digested by one or more enzymes while bound to the at least one support.

45. The method of claim 36, wherein the polynucleotide is a viral polynucleotide or a polynucleotide encoding a viral component.

46. The method of claim 36, wherein the solution is a biosample or a lysate of a biosample.

47. The method of claim 36, wherein the solution is an in vitro transcription reaction, a reverse transcription reaction, a second strand DNA synthesis reaction, a DNase reaction, a PCR reaction, or a RNA amplification reaction.

48. The method of claim 36, further defined as a method of isolating or concentrating DNA, RNA, and/or PNA.

49. The method of claim 36, further defined as preparing the solution.

50. The method of claim 49, wherein preparing the solution comprises performing an RNA amplification reaction, an in vitro transcription reaction, a reverse transcription reaction, a second strand DNA synthesis reaction, a DNase reaction, and/or or a PCR reaction.

51. The method of claim 49, wherein preparing the solution comprises lysing a cell.

38. The method of claim 38, wherein the separation of the surface is performed using a magnet.

52. A kit comprising a binding buffer or one or more components thereof, and at least one polymer-modified surface, wherein the binding buffer comprises a suitable salt and a suitable organic solvent at concentrations suitable for binding polynucleotides to the surface of the surface during use, wherein the polymer-modified surface is further defined as comprising a polyvinylpyrrolidone, a polyethyleneglycol, or a polymeric polyol, with the proviso that any polymeric polyol cannot be cellulose.

53. The kit of claim 52, wherein the kit further comprises an elution buffer or one or more components thereof, wherein the elution buffer is capable of dissolving polynucleotides bound to the surface during use.

54. The kit of claim 52, wherein the kit further comprises a wash buffer or one or more components thereof, wherein the wash buffer is capable of dissolving impurities bound to the surface but is incapable of dissolving selective polynucleotides bound to the surface during use.

55. The kit of claim 53, wherein the kit additionally comprises a reagent or one or more components thereof that preserve the intactness of RNA or DNA during use.

56. A method of reversibly binding at least one polynucleotide to one or more surface comprising a polymer, comprising:

contacting the surface with a solution containing at least one polynucleotide; and

adjusting a salt concentration and/or a concentration of a non-polyethelyene glycol nucleic acid coprecipitant in the solution to allow for binding of the polynucleotide onto the surface;

wherein the polymer is further defined as comprising a polyvinylpyrrolidone, a polyethylene glycol, or a polymeric polyol, with the proviso that any polymeric polyol cannot be cellulose.

57. The method of claim 56, wherein the non-polyethelyene glycol nucleic acid coprecipitant is a PVP, ficoll, or gum arabic.