Normalized nucleic acid libraries and methods of production thereof

• US 20020177155A1
• Filed: 05/13/2002
• Published: 11/28/2002
• Est. Priority Date: 09/24/1997
• Status: Abandoned Application

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Jessee, Joel, Li, Wu-Bo, Nisson, Paul E.
• Application Number:
  US10/143,007
• Publication Number:
  US 20020177155A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C40B 30/06 by measuring effects on living organisms, tissues or cells
• CPC Class Codes: C40B 30/08 by measuring catalytic activity

Abstract

The present invention relates generally to methods for producing normalized nucleic acid libraries in which each member of the library can be isolated with approximately equivalent probability. In particular, the present methods comprise subtractive hybridization of a nucleic acid library with haptenylated (e.g., biotinylated, avidinated or streptavidinated) nucleic acid molecules that are complementary to one or more of the nucleic acid molecules of the library, such that the variation in the abundances of the individual nucleic acid molecules in the library is reduced. The invention also relates to production of normalized nucleic acid libraries (particularly cDNA libraries) in which contaminating nucleic acid molecules have been reduced or eliminated, and to normalized nucleic acid libraries produced by such methods.

Claims

1. A method for normalization of a nucleic acid library comprising:

(a) synthesizing one or more nucleic acid molecules complementary to all or a portion of the nucleic acid molecules of said library, wherein said synthesized nucleic acid molecules comprise at least one hapten, thereby producing haptenylated nucleic acid molecules;

(b) incubating a nucleic acid library to be normalized with said haptenylated nucleic acid molecules under conditions favoring the hybridization ofthe more highly abundant molecules of said library with the haptenylated nucleic acid molecules; and

(c) removing said hybridized molecules, thereby producing a normalized library.

2. The method of claim 1, wherein said nucleic acid library is a cDNA library.

3. The method of claim 2, wherein the nucleic acid molecules of said cDNA library are single-stranded.

4. The method of claim 2, wherein the nucleic acid molecules of said cDNA library are double-stranded.

5. The method of claim 2, wherein said cDNA library is produced by a method comprising incubating a population of mRNA molecules under conditions sufficient to produce a cDNA library from said population of mRNA molecules.

6. The method of claim 1, wherein said haptenylated nucleic acid molecules are RNA molecules.

7. The method of claim 1, further comprising reduction or removal of contaminating nucleic acid molecules from said library.

8. The method of claim 7, wherein said reduction or removal is performed before or after normalization of said library.

9. The method of claim 7, wherein the contaminating nucleic acid molecules are one or more vectors.

10. The method of claim 7, wherein said reduction or removal comprises incubating said library with at least one haptenylated probe.

11. The method of claim 10, wherein said probe hybridizes to nucleic acid molecules of said library.

12. The method of claim 11, wherein said probe is not capable of hybridizing to vector sequences of said library.

13. The method of claim 10, wherein the haptens of said haptenylated probe are used to isolate a normalized library having substantially reduced contaminating nucleic acid molecules, thereby producing a selected normalized library.

14. The method of claim 13, wherein said haptens are selected from the group consisting of avidin, streptavidin, protein A, protein G, a cell-surface Fc receptor, an antibody-specific antigen, an enzyme-specific substrate, polymyxin B, endotoxin-neutralizing protein (ENP), Fe⁺⁺⁺, a transferrin receptor, an insulin receptor, a cytokine receptor, CD4, spectrin, fodrin, ICAM-1, ICAM-2, C3bi, fibrinogen, Factor X, ankyrin, an integrin, vitronectin, fibronectin, collagen, laminin, glycophorin, Mac-1, LFA-1, β

-actin, gp120, a cytokine, insulin, ferrotransferrin, apotransferrin, lipopolysaccharide, an enzyme, an antibody, biotin and combinations thereof.

15. The method of claim 14, wherein said hapten is biotin.

16. The method of claim 13, wherein said selected normalized library is isolated by hapten-ligand interactions and/or extraction.

17. The method of claim 16, wherein isolation comprises the use of a solid support comprising at least one ligand that binds said hapten.

18. The method of claim 13, wherein said selected normalized library is single-stranded.

19. The method of claim 18, further comprising incubating said single-stranded selected normalized library under conditions sufficient to render said molecules double stranded.

20. The method of claim 19, wherein said conditions comprise incubating said single-stranded selected normalized library with one or more nucleotides, one or more polypeptides having polymerase activity and one or more primers.

21. The method of claim 20, wherein said one or more nucleotides are nucleotide analogues which confer nuclease resistance on said double stranded molecules.

22. The method of claim 21, further comprising digesting a sample comprising said double stranded molecules with said nuclease.

23. The method of claim 22, further comprising transforming said double-stranded molecules into one or more host cells.

24. The method of claim 19, further comprising transforming said double stranded molecules into one or more host cells.

25. The method of claim 20, wherein said primers are target specific primers.

26. The method of claim 25, further comprising transforming said double stranded molecules into one or more host cells.

27. The method of claim 6, wherein said RNA molecules are produced by one or more RNA polymerases.

28. The method of claim 27, wherein said RNA polymerases are selected from the group consisting of SP6, T7 and T3 RNA polymerases.

29. The method of claim 6, wherein said RNA molecules are produced with one or more promoters.

30. The method of claim 29, wherein said promoters are provided by one or more vectors or by one or more adapters.

31. The method of claim 30, wherein said promoters allows the synthesis of at least one RNA molecule from all or a portion of the nucleic acid molecules of said library.

32. The method of claim 1, wherein said hybridized molecules are removed by hapten-ligand interactions and/or extraction.

33. The method of claim 32, wherein said removal comprises the use of a solid support comprising at least one ligand.

34. The method of claim 4, further comprising treating said double-stranded cDNA library under conditions sufficient to render said molecules single-stranded.

35. The method of claim 34, wherein said conditions comprise degradation of one strand of said double-stranded molecules.

36. The method of claim 34, wherein said conditions comprise denaturing said double-stranded molecules.

37. The method of claim 35, wherein said degradation is accomplished with gene II and Exonuclease III.

38. The method of claim 1, wherein said hybridization conditions are selected from the group consisting of:

(a) a COT equal to or greater than 25;

(b) a COT equal to or greater than 50;

(c) a COT equal to or greater than 100;

(d) a COT f rom about 10 to 10,000;

(e) a COT from about 25 to 10,000;

(f) a COT from about 50 to 10,000;

(g) a COT from about 100 to 10,000; and

(h) a COT of less than 10,000.

39. The method of claim 7, wherein said reduction or removal comprises incubating said library with at least one primer and at least one nucleotide which confers nuclease resistance under condition sufficient to make double stranded nucleic acid molecules.

40. The method of claim 39, wherein said primer hybridizes to nucleic acid molecules of said library.

41. The method of claim 39, wherein said primer is not capable of hybridizing to vector sequences of said library.

42. The method of claim 39, wherein said nucleotide is a nucleotide analog.

43. The method of claim 42, wherein said nucleotide analog is a methylated nucleotide.

44. The method of claim 43, wherein said methylated nucleotide is 5-methyldeoxycytosine.

45. The method of claim 39, further comprising digesting said double-stranded nucleic acid molecules with one or more nucleases.

46. The method of claim 45, further comprising transforming said digested molecules into one or more host cells.

47. A normalized nucleic acid library produced by the method of claim 1.

48. A selected normalized library produced by the method of claim 7.

49. A transformed host cell produced by the method of claim 23.

50. A transformed host cell produced by the method of claim 24.

51. A transformed host cell produced by the method of claim 26.

52. A transformed host cell produced by the method of claim 46.