Methods of isolating nucleic acids using multifunctional group-coated solid phase carriers

• US 7,527,929 B2
• Filed: 07/29/2005
• Issued: 05/05/2009
• Est. Priority Date: 07/30/2004
• Status: Active Grant

• Current Assignee: Agencourt Bioscience Corporation (Danaher Corporation)
• Original Assignee: Agencourt Bioscience Corporation (Danaher Corporation)
• Inventors: Brand, Adrianne D., Gustafson, Erik, McKernan, Kevin J.
• Primary Examiner(s): Martinell; James
• Application Number:
  US11/194,072
• Publication Number:
  US 20060177836A1
• Time in Patent Office: 1,376 Days
• Field of Search: None
• US Class Current: 435/6.16
• 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
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

The present invention is directed to a method of isolating a target species (e.g., target nucleic acid species) from a mixture. In the methods of the present invention, the mixture is combined with solid phase carriers having a surface comprising multiple functional groups one of which reversibly and selectively binds the target species. In a particular embodiment, the mixture is combined with solid phase carriers having a first functional group which reversibly binds nucleic acids and a second functional group which selectively and reversibly binds the target nucleic acid species, thereby producing a first combination. The first combination is maintained under conditions appropriate for binding of the nucleic acids to the first functional group and binding of the target nucleic acid species to the second functional group. The solid phase carriers are separated from the first combination, and combined with an agent (e.g., buffer) that selectively removes (e.g., elutes) either the nucleic acid from the first functional group or the target nucleic acid species from the second functional group of the solid phase carriers, thereby isolating a target nucleic acid species from a mixture comprising a plurality of nucleic acid species.

Claims

1. A method of selectively isolating a target species of nucleic acid molecule present in a mixture, comprising:

a) combining the mixture with solid phase carriers having a surface comprising a first functional group which binds nucleic acids and a second functional group which selectively binds the target species of nucleic acid, thereby producing a first combination;

b) maintaining the first combination under conditions appropriate for binding of the nucleic acids to the first functional group;

c) separating the solid phase carriers from the first combination;

d) combining the solid phase carriers with at least one agent that removes the nucleic acid from the first functional group of the solid phase carriers and binds the target species of nucleic acid to the second functional group of the solid phase carriers, thereby producing a second combination; and

e) separating the solid phase carriers from the second combination, thereby isolating the target species of nucleic acid present in the mixture comprising nucleic acids.

2. The method of claim 1 wherein in step d) the solid phase carriers are combined with a first agent that removes the nucleic acid from the first functional group of the solid phase carriers, and a second agent that allows binding of the target species of nucleic acid to the second functional group of the solid phase carriers, thereby producing a second combination.

3. The method of claim 2 wherein the target species of nucleic acid is a nucleic acid species selected from the group consisting of:

DNA, RNA and PNA.

4. The method of claim 2 wherein the first agent is a buffer comprising Tris, Tris and EDTA, formamide or a combination thereof.

5. The method of claim 4 wherein the buffer further comprises an enzyme selected from the group consisting of:

DNase and Rnase.

6. The method of claim 2 wherein the solid phase carriers are magnetic microparticles.

7. The method of claim 2 wherein the first functional group is COOH and the second functional group is a nucleic acid sequence that is complementary to the target species of nucleic acid.

8. The method of claim 1 further comprising a step of lysing a cell, a host cell, an organism selected from the group consisting of a virus, a bacteria, a fungus, and a parasite, or a combination thereof, so as to release a target species of nucleic acid molecule.

9. A method of isolating mRNA present in a mixture comprising nucleic acids, comprising:

a) combining the mixture with solid phase carriers having a surface comprising a first functional group which binds nucleic acids and a second functional group which selectively binds mRNA, thereby producing a first combination;

b) maintaining the first combination under conditions appropriate for binding of the nucleic acids to the first functional group;

c) separating the solid phase carriers from the first combination;

d) combining the solid phase carriers with at least one agent that removes the nucleic acid from the first functional group of the solid phase carriers and binds the mRNA to the second functional group of the solid phase carriers, thereby producing a second combination; and

e) separating the solid phase carriers from the second combination, thereby isolating mRNA present in a mixture comprising nucleic acids.

10. The method of claim 9 wherein in step d) the solid phase carriers are combined with a first agent that removes the nucleic acid from the first functional group of the solid phase carriers, and a second agent that allows binding of the mRNA to the second functional group of the solid phase carriers, thereby producing a second combination.

11. The method of claim 10 wherein the mixture is a biological sample.

12. The method of claim 11 wherein the biological sample is selected from the group consisting of:

whole cells, tissue, a lysate and blood.

13. The method of claim 10 wherein the solid phase carriers are magnetic microparticles.

14. The method of claim 10 wherein the first functional group is COOH and the second functional group is selected from the group consisting of:

oligo-dT, modified oligo-dT and a combination thereof.

15. The method of claim 10 wherein the buffer that elutes the nucleic acid from the first functional group of the solid phase carriers comprises water.

16. The method of claim 10 wherein the solid phase carriers are removed using a method selected from the group consisting of:

applying a magnetic field, applying a vacuum and applying centrifugation.

17. The method of claim 10 which further comprises contacting the solid phase carriers with a wash buffer to remove contaminants prior to eluting the nucleic acid from the first functional group.

18. The method of claim 10 which further comprises contacting the solid phase carriers with an agent that digests DNA prior to eluting the nucleic acid from the first functional group of the solid phase carriers.

19. The method of claim 18 wherein the agent is DNase.

20. The method of claim 10 further comprising eluting the mRNA from the second functional group of the solid phase carriers.

21. The method of claim 20 wherein the mRNA is eluted in a buffer selected from the group consisting of:

water, Tris, Tris and EDTA, and formamide.

22. A method of separating globin RNA from nucleic acid present in a mixture, comprising:

a) combining the mixture with solid phase carriers having a surface comprising a first functional group which binds nucleic acids and a second functional group which selectively binds globin RNA, thereby producing a first combination;

b) maintaining the first combination under conditions appropriate for binding of the nucleic acids to the first functional group;

c) separating the solid phase carriers from the first combination;

d) combining the solid phase carriers with at least one agent that removes the nucleic acid from the first functional group of the solid phase carriers and binds the globin RNA to the second functional group of the solid phase carriers, thereby producing a second combination; and

e) separating the solid phase carriers from the second combination, thereby separating globin RNA present in a mixture comprising nucleic acids.

23. The method of claim 22 wherein in step d) the solid phase carriers are combined with a first agent that removes the nucleic acid from the first functional group of the solid phase carriers, and a second agent that allows binding of the globin RNA to the second functional group of the solid phase carriers, thereby producing a second combination.

24. The method of claim 22 wherein the solid phase carriers are magnetic microparticles.

25. The method of claim 22 wherein the first functional group is COOH and the second functional group is an oligonucleotide comprising a sequence that is complementary to a beta globin RNA sequence.

26. A method of separating globin RNA from nucleic acid present in a mixture, comprising:

a) combining the mixture with biotin labeled oligonucleotides comprising sequences that are complementary to globin RNA sequences present in the mixture, thereby producing a first combination;

b) maintaining the first combination under conditions in which hybridization occurs between the oligonucleotides and the globin RNA;

c) combining the first combination with solid phase carriers having a first functional group that binds nucleic acid and a second functional group that selectively binds biotin, thereby producing a second combination;

d) maintaining the second combination under conditions in which the nucleic acid binds to the first functional groups and the oligonucleotides which are hybridized to the globin RNA, bind to the second functional group of the solid phase carriers;

e) separating the solid phase carriers from the second combination;

f) combining the solid phase carriers with an agent that elutes the nucleic acid from the first functional group,

thereby separating globin RNA from nucleic acid present in the mixture.

27. The method of claim 26 wherein the globin RNA is beta globin RNA.

28. A method of separating endotoxin from nucleic acid in a mixture, comprising:

a) combining the mixture with solid phase carriers having a surface comprising a first functional group which binds nucleic acids and a second functional group which selectively binds endotoxin, thereby producing a first combination;

b) maintaining the first combination under conditions appropriate for binding of the nucleic acids to the first functional group and endotoxin to the second functional group;

c) separating the solid phase carriers from the first combination;

d) combining the solid phase carriers with at least one agent that removes the nucleic acid from the first functional group of the solid phase carriers, thereby producing a second combination; and

e) separating the solid phase carriers from the second combination,

thereby separating endotoxin from nucleic acid present in the mixture.

29. The method of claim 28 wherein the solid phase carriers are magnetic microparticles.

30. The method of claim 29 wherein the first functional group is COOH and the second functional group is selected from the group consisting of:

polymyxin B, native Limulus anti-LPS factor (LALF) and recombinant LALF.

31. A method of separating endotoxin from nucleic acid in a mixture comprising:

a) combining the mixture with solid phase carriers having a surface comprising a first functional group which binds nucleic acids and a second functional group which selectively binds endotoxin, thereby producing a first combination;

b) maintaining the first combination under conditions appropriate for binding of endotoxin to the second functional group;

c) separating the solid phase carriers from the first combination;

d) combining solid phase carriers having a surface comprising a functional group which binds nucleic acids with the first combination, thereby producing a second combination;

e) maintaining the second combination under conditions appropriate for binding of nucleic acid to the functional group of the solid phase carriers of step d);

f) separating the solid phase carriers from the second combination,

thereby separating endotoxin from nucleic acid present in the mixture.

32. The method of claim 31 further comprising eluting the nucleic acid from the solid phase carriers of step f).

33. The method of claim 31 wherein the first functional group is COOH and the second functional group is selected from the group consisting of:

polymyxin B, native Limulus anti-LPS factor (LALF) and recombinant LALF.

34. A method of isolating nucleic acid of an organism comprising:

a) combining the organism with solid phase carriers having a surface comprising a first functional group which binds the organism and a second functional group that binds nucleic acid, thereby producing a first combination;

b) maintaining the first combination under conditions in which the organism binds to the first functional group;

c) separating the solid phase carriers from the first combination;

d) combining the solid phase carriers with an agent that lyses the organism and binds the nucleic acid of the organism to the second functional group, thereby producing a second combination;

e) maintaining the second combination under conditions in which the organism is lysed and the nucleic acid of the organism binds to the second functional group,

thereby isolating the nucleic acid of the organism.

35. The method of claim 34 further comprising eluting the nucleic acid from the solid phase carriers of step e).

36. The method of claim 34 wherein the first functional group is COOH and the second functional group is polyethyleneimine.

37. A method of separating forward extension products and reverse extension products of a sequencing reaction comprising:

a) combining a sequencing reaction mixture which comprises forward extension products and reverse extension products with solid phase carriers having a surface comprising a first functional group which selectively binds the forward extension products and a second functional group which binds nucleic acid, thereby producing a first combination;

b) maintaining the first combination under conditions appropriate for binding of the forward extension products to the first functional group and binding of the reverse extension products to the second functional group;

c) separating the solid phase carriers from the first combination;

d) combining the solid phase carriers with a buffer that selectively elutes the reverse extension products from the second functional group of the solid phase carriers, thereby producing a second combination; and

e) separating the solid phase carriers from the second combination,

thereby separating forward extension products and reverse extension products of the sequencing reaction.

38. The method of claim 37 wherein the first functional group is streptavidin and the second functional group is COOH.

39. The method of claim 38 wherein the forward extension products are biotinylated.

40. The method of claim 37 wherein the solid phase carriers are magnetic microparticles.

41. The method of claim 37 wherein the solid phase carriers are removed using a method selected from the group consisting of:

applying a magnetic field, applying a vacuum and applying centrifugation.

42. The method of claim 37 which further comprises contacting the solid phase carriers with a wash buffer to remove contaminants prior to eluting the reverse extension products from the second functional group.

43. The method of claim 37 wherein the buffer that selectively elutes the reverse extension products from the second functional group of the solid phase carriers is water.

44. The method of claim 37 further comprising eluting the forward extension products from the solid phase carriers of e).

45. The method of claim 44 further comprising sequencing the reverse extension products and the forward extension products.

46. A method of separating forward extension products and reverse extension products of a sequencing reaction comprising:

a) combining a sequencing reaction mixture which comprises forward extension products and reverse extension products with solid phase carriers having a surface comprising a first functional group which selectively binds the forward extension products and a second functional group which binds nucleic acid, thereby producing a first combination;

b) maintaining the first combination under conditions appropriate for binding of the forward extension products to the first functional group;

c) separating the solid phase carriers from the first combination, thereby producing a second mixture comprising the reverse extension products;

d) combining the solid phase carriers with a buffer that selectively elutes the forward extension products from the first functional group of the solid phase carriers, thereby producing a second combination;

e) separating the solid phase carriers from the second combination;

f) combining the solid phase carriers with the second mixture of c), thereby producing a third combination;

g) maintaining the third combination under conditions appropriate for binding of the reverse extension products to the second functional group;

h) separating the solid phase carriers from the third combination; and

i) combining the solid phase carriers with a buffer that selectively elutes the reverse extension products from the second functional group of the solid phase carriers,

thereby separating forward extension products and reverse extension products of the sequencing reaction.