Methods for identifying low-abundance polynucleotides and related compositions

• US 20040006033A1
• Filed: 06/30/2003
• Published: 01/08/2004
• Est. Priority Date: 08/06/2001
• Status: Abandoned Application

• Current Assignee: Genemed Biotechnologies, Inc. (Sakura Finetechnical Co., Ltd.)
• Original Assignee: Genemed Biotechnologies, Inc. (Sakura Finetechnical Co., Ltd.)
• Inventors: Zhu, York Yuan-Yuan
• Application Number:
  US10/343,345
• Publication Number:
  US 20040006033A1
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2539/101 Subtraction analysis

Abstract

This invention provides novel methods for producing a plurality of polynucleotides prepared from a polynucleotide sample and the plurality of polynucleotides so produced. In one embodiment, the plurality of polynucleotides is prepared by subtractive hybridization between test and reference polynucleotide samples and is substantially enriched in sequences that are either not present in the reference polynucleotide sample or are present in the reference polynucleotide sample in substantially lower concentration than in the test polynucleotide sample. The plurality of polynucleotides is also substantially enriched in low-abundance sequences, relative to the test polynucleotide sample. The invention also provides kits useful in the methods of the invention and for using the polynucleotides produced thereby. The polynucleotides are useful in a wide variety of applications, such as cloning, expression, and hybridization studies.

Claims

1. A subtractive hybridization method for identifying one or more polynucleotides in a test sample that are absent from, or less abundant in, a reference sample, said method comprising:

a) providing high-abundance enriched polynucleotide strands of, or prepared from, a pool of test or reference polynucleotides that is enriched in high-abundance polynucleotide sequences relative to a test or reference polynucleotide sample, respectively;

b) contacting the high-abundance enriched polynucleotide strands with test polynucleotide strands of, or prepared from, the test polynucleotide sample under hybridization conditions to form a first hybridization mixture, thereby producing unhybridized test polynucleotide strands that are enriched in low-abundance polynucleotide sequences relative to the test polynucleotide sample;

c) synthesizing polynucleotide strands from the unhybridized test polynucleotide strands, thereby producing low-abundance enriched test polynucleotide strands;

d) providing low-abundance-enriched reference polynucleotide stands of, or prepared from, a reference pool of polynucleotides that is enriched in low-abundance polynucleotide sequences relative to the reference polynucleotide sample;

e) contacting the low-abundance enriched reference polynucleotide strands with the low-abundance enriched test polynucleotide strands under hybridization conditions to form a second hybridization mixture, thereby producing hybrid duplexes, unhybridized low-abundance enriched reference polynucleotide strands, and unhybridized low-abundance enriched test polynucleotide strands;

f) removing or digesting the hybrid duplexes; and

g) producing test-specific duplexes from the unhybridized low-abundance enriched test polynucleotide strands.

2. The method of claim 1 wherein the high-abundance enriched polynucleotide strands are of, or prepared from, a pool of test polynucleotides that is enriched in high-abundance polynucleotide sequences relative to a test polynucleotide sample.

3. The method of claim 1 wherein:

the high-abundance enriched polynucleotide strands of (a) comprise high-abundance enriched antisense polynucleotide strands;

the test polynucleotide strands of (b) comprise sense test polynucleotide strands;

the low-abundance enriched test polynucleotide strands of (c) comprise antisense test polynucleotide strands; and

the low-abundance enriched reference polynucleotide strands of (d) comprise sense reference polynucleotide strands.

4. The method of claim 3 wherein the antisense test polynucleotide strands are synthesized from the unhybridized sense test polynucleotide strands using a first antisense primer or a first antisense primer complex, said first antisense primer complex comprising a first antisense primer operably linked to a first RNA promoter sequence, wherein the first RNA promoter sequence is 5′

of the first antisense primer.

5. The method of claim 4 wherein the first antisense primer or first antisense primer complex comprises:

a) a sequence that binds to a primer site in the unhybridized sense test polynucleotide strands;

b) a first restriction site 5′

of the sequence of (a), wherein the first restriction site is cleaved by a restiction endonuclease that cleaves double-stranded polynucleotides, but leaves single-stranded polynucleotides substantially intact; and

c) a first universal primer site 5′

of the restriction site of (b).

6. The method of claim 5 wherein the antisense test polynucleotide strands are synthesized from the unhybridized sense test polynucleotide strands using a first antisense primer, and the first universal primer site of (c) comprises a second restriction site, wherein said second restriction site is different from said first restriction site.

7. The method of claim 5 wherein the sense reference polynucleotide strands comprise a third restriction site previously added to the 5′

ends of the sense reference polynucleotide strands, wherein the third restriction site is cleaved by a restriction endonuclease that cleaves double-stranded polynucleotides, but leaves single-stranded polynucleotides substantially intact.

8. The method of claim 1 wherein the hybrid duplexes are digested with at least one enzyme.

9. The method of claim 8 wherein the enzyme is a restriction endonuclease.

10. The method of claim 7 wherein the removal or digestion of the hybrid duplexes of (f) comprises:

i) treating the second hybridization mixture with an enzyme that renders single-stranded portions of the hybrid duplexes double-stranded, thereby producing double-stranded first, second, and third restriction sites in the hybrid duplexes;

ii) adding a second universal primer site to the 3′

ends of the polynucleotide strands in the second hybridization mixture; and

iii) treating the second hybridization mixture with one or more restriction endonuclease(s), wherein the restriction endonuclease(s) cleave(s) the hybrid duplexes at the double-stranded first and third restriction sites, but leaves the antisense test polynucleotide strands and the sense reference polynucleotide strands substantially intact.

11. The method of claim 10 wherein the test-specific duplexes are produced from the unhybridized antisense test polynucleotide strands by amplification.

12. The method of claim 11 wherein the test-specific duplexes are synthesized from the unhybridized antisense test polynucleotide stands using a first universal primer that hybridizes to the first universal primer site as the 3′

primer and a second universal primer that hybridizes to the second universal primer site as the 5′

primer.

13. The method of claim 11 wherein the amplification is performed by an enhanced polymerase chain reaction.

14. The method of claim 1 wherein the molar ratio of the high-abundance enriched polynucleotide strands to the test polynucleotide strands in the first hybridization mixture is between about 1 and about 100 to 1.

15. The method of claim 1 wherein the molar ratio of the low-abundance enriched reference polynucleotide strands to the low-abundance enriched test polynucleotide strands in the second hybridization mixture is between about 1 and about 100 to 1.

16. The method of claim 1 wherein the test and reference polynucleotide samples are mRNA samples.

17. The method of claim 3 wherein the high-abundance enriched antisense polynucleotide strands are antisense RNA molecules.

18. The method of claim 3 wherein the sense test polynucleotide strands are mRNA molecules.

19. The method of claim 3 wherein the antisense test polynucleotide strands are antisense cDNA strands.

20. The method of claim 3 wherein the sense reference polynucleotide strands are sense cDNA strands.

21. The method of claim 3 wherein the synthesis of the antisense test polynucleotide strands is primed using oligonucleotide-dT priming.

22. The method of claim 5 wherein the test and reference polynucleotide samples are test and reference mRNA samples and the high-abundance enriched antisense polynucleotide strands are test or reference antisense RNA molecules prepared by a method comprising:

a) synthesizing first antisense cDNA strands from the test or reference mRNA sample using a second antisense primer complex comprising a second antisense primer operably linked to a second RNA promoter sequence, wherein the second RNA promoter sequence is 5′

of the second antisense primer;

b) adding a third universal primer site to the 3′

ends of the first antisense cDNA strands;

c) diluting the first antisense cDNA strands to substantially eliminate at least some low-abundance antisense cDNA strands;

d) producing first double-stranded cDNA molecules from the remaining first antisense cDNA strands, wherein the first double-stranded cDNA molecules are enriched in high-abundance polynucleotide sequences relative to the starting mRNA sample; and

e) synthesizing high-abundance enriched antisense RNA molecules from the double-stranded cDNA molecules.

23. The method of claim 22 wherein the sense reference polynucleotides are sense cDNA strands prepared by a method comprising:

a) providing high-abundance enriched antisense RNA molecules of, or prepared from, a pool of reference polynucleotides that is enriched in high-abundance polynucleotide sequences relative to the reference polynucleotide sample;

b) contacting the high-abundance enriched antisense RNA molecules with the reference mRNA sample under hybridization conditions to form a hybridization mixture, thereby producing unhybridized mRNA molecules that are enriched in low-abundance polynucleotide sequences relative to the reference mRNA sample;

c) synthesizing second antisense cDNA strands from the unhybridized mRNA molecules using a third antisense primer or a third antisense primer complex, said third antisense primer complex comprising a third antisense primer operably linked to a third RNA promoter sequence, wherein the third RNA promoter sequence is 5′

of the third antisense primer;

d) adding a fourth universal primer site to the 3′

ends of the second antisense cDNA strands;

e) producing second double-stranded cDNA molecules from the second antisense cDNA strands;

f) synthesizing second antisense RNA molecules from the second double-stranded cDNA molecules; and

g) synthesizing first sense cDNA strands from the second antisense RNA molecules.

24. The method of claim 1 further comprising cloning at least one of the test-specific duplexes into a vector.

25. The method of claim 24 additionally comprising producing a polynucleotide library from said test-specific duplexes.

26. The method of claim 24 wherein the cloned test-specific duplex encodes a polypeptide, and the vector is an expression vector.

27. The method of claim 26 further comprising introducing the expression vector into a host cell and expressing the protein encoded by the cloned test-specific duplex.

28. The method of claim 4 wherein the test-specific duplexes are synthesized from the unhybridized antisense test polynucleotide strands using a first antisense primer complex, said method further comprising synthesizing one or more antisense RNA molecules from the test-specific duplexes.

29. The method of claim 28 additionally comprising introducing the one or more antisense RNA molecules synthesized from the test-specific duplexes into a cell.

30. The method of claim 29 wherein the cell is in vitro.

31. The method of claim 1 comprising employing one or more test-specific duplexes or one or more polynucleotides produced directly or indirectly therefrom in a hybridization reaction.

32. The method of claim 31 wherein at least one of the test-specific duplexes or a polynucleotide produced therefrom is labeled with a detectable label.

33. The method of claim 1 further comprising attaching a plurality of the test-specific duplexes or polynucleotides produced therefrom to a substrate to produce a polynucleotide array.

34. The method of claim 1 further comprising amplifying one or more of the test-specific duplexes.

35. The method of claim 34 wherein said one or more test-specific duplexes are amplified using one or more gene-specific primers.

36. The method of claim 1 wherein the test and reference polynucleotide samples are different samples.

37. The method of claim 36 wherein the test and reference polynucleotide samples are selected from one of the following:

mRNA from a first cell or tissue and mRNA from a second, different cell or tissue;

mRNA from a cell or tissue at a first stage of differentiation or development and mRNA from the cell or tissue at a second, different stage of differentiation or development;

mRNA from a cell or tissue treated with an active agent and mRNA from a cell or tissue that is untreated or treated with a second, different active agent; and

mRNA from a normal cell or tissue and mRNA from a diseased cell or tissue.

38. The method of claim 37 wherein the test and reference polynucleotide samples are mRNA from a normal cell or tissue and mRNA from a diseased cell or tissue, wherein the disease is an infectious disease or a cancer.

42. A plurality of polynucleotides prepared by subtractive hybridization between test and reference polynucleotide samples according to the method of claim 1, wherein the plurality of polynucleotides includes at least 10³ different polynucleotides and is substantially enriched in sequences that are:

either not present in the reference polynucleotide sample or are present in the reference polynucleotide sample in substantially lower concentration than in the test polynucleotide sample; and

low-abundance sequences, relative to the test polynucleotide sample.

43. The plurality of polynucleotides of claim 42 wherein the polynucleotides each comprise an RNA promoter sequence and a universal primer site.

44. The plurality of polynucleotides of claim 42 wherein the polynucleotides are double-stranded cDNA molecules.

45. The plurality of polynucleotides of claim 42 wherein the polynucleotides are antisense RNA molecules.

46. A kit comprising:

a) an antisense primer or antisense primer complex comprising;

i) a sequence that binds to a primer site in the unhybridized sense test polynucleotide strands;

ii) a first restriction site 5′

of the sequence of (i), wherein the first restriction site is cleaved by a restiction endonuclease that cleaves double-stranded polynucleotides, but leaves single-stranded polynucleotides substantially intact; and

iii) a first universal primer site 5′

of the restriction site of (iii); and

b) instructions for performing the method of claim 3.

47. A kit comprising:

a) the plurality of polynucleotides of claim 42;

b) an antisense primer complex comprising a antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5′

of the antisense primer; and

c) a sense primer.

48. A kit comprising:

a) the plurality of polynucleotides of claim 42;

and

b) an RNA polymerase capable of transcribing antisense RNA from the plurality of polynucleotides.

39. A method for functionally isolating single-stranded polynucleotides in a mixture of single- and double-stranded polynucleotides, said method comprising contacting a mixture of single- and double-stranded polynucleotides with one or more restriction endonucleases under conditions sufficient to allow digestion of double-stranded polynucleotides to a form that cannot serve as a template for a nucleotide synthesis reaction that uses the single-stranded polynucleotides as a template.

40. The method of claim 39 wherein said restriction endonuclease(s) cleave(s) a primer site from double-stranded polynucleotides in said mixture.

41. The method of claim 40 wherein uncleaved single-stranded polynucleotides are converted to double-stranded polynucleotides by amplification.

49. A method for preparing a selected polynucleotide pool from a polynucleotide sample comprising:

a) synthesizing first antisense polynucleotide strands from sense polynucleotides of, or prepared from, the polynucleotide sample using an antisense primer complex comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5′

of the antisense primer;

b) adding a universal primer site to the 3′

ends of the first antisense polynucleotide strands;

c) diluting the first antisense polynucleotide strands to substantially eliminate at least some low-abundance first antisense polynucleotide strands; and

d) producing first double-stranded polynucleotides from the remaining first antisense polynucleotide strands, wherein the first double-stranded polynucleotides are enriched in high-abundance polynucleotide sequences relative to the polynucleotide sample.

50. The method of claim 49 wherein the polynucleotide sample is an mRNA sample, the first antisense polynucleotide strands are first antisense cDNA strands, and the first double-stranded polynucleotides are first double-stranded cDNA molecules.

51. The method of claim 50 wherein the synthesis of first antisense cDNA strands is primed using a random primer or an oligonucleotide-dT primer.

52. The method of claim 50 wherein the universal primer site is added to the 3′

end of the first antisense cDNA strands by template switching, oligonucleotide-tailing, or ligation.

53. The method of claim 49 wherein the RNA promoter sequence is an RNA promoter sequence recognized by a bacteriophage RNA polymerase selected from the group consisting of T7, T3, and SP6.

54. The method of claim 49 wherein the first double-stranded polynucleotides are produced by amplifying the remaining first antisense polynucleotide strands, and wherein the amplification is carried out using a universal primer that hybridizes to the universal primer site as the 5′

primer and using the antisense primer complex as the 3′

primer.

55. The method of claim 54 wherein the amplification is performed by enhanced polymerase chain reaction.

56. The method of claim 49 wherein the first double-stranded polynucleotides are produced using an enzyme mixture comprising a DNA polymerase, a DNA ligase, and an RNase.

57. The method of claim 49 further comprising synthesizing first antisense RNA molecules from the first double-stranded polynucleotides.

58. The method of claim 57 further comprising:

a) contacting the first antisense RNA molecules with sense polynucleotide strands of, or prepared from, the polynucleotide sample under hybridization conditions to form a hybridization mixture, thereby producing unhybridized sense polynucleotide strands that are enriched in low-abundance polynucleotide sequences relative to the polynucleotide sample;

b) synthesizing second antisense polynucleotide strands from the unhybridized sense polynucleotide strands using an antisense primer or an antisense primer complex, said antisense primer complex comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5′

of the antisense primer;

c) adding a universal primer site to the 3′

ends of the second antisense polynucleotide strands; and

d) producing second double-stranded polynucleotides from the second antisense polynucleotide strands.

59. The method of claim 58 wherein the molar ratio of the first antisense RNA molecules to the other polynucleotides in the hybridization mixture is between about 1 and about 100 to 1.

60. The method of claim 58 wherein the polynucleotide sample is an mRNA sample, the sense polynucleotide strands are the mRNA molecules in the mRNA sample, the second antisense polynucleotide strands are second antisense cDNA strands, and the second double-stranded polynucleotides are second double-stranded cDNA molecules.

61. The method of claim 60 wherein the synthesis of second antisense cDNA strands is primed using oligonucleotide-dT priming.

62. The method of claim 60 wherein the universal primer site is added to the 3′

end of the second antisense cDNA strands by template switching, oligonucleotide-tailing, or ligation.

63. The method of claim 58 wherein the second antisense polynucleotide strands are synthesized from the unhybridized sense polynucleotide strands using an antisense primer complex, and the RNA promoter sequence of (b) comprises an RNA promoter sequence for an RNA polymerase selected from the group consisting of T7, T3, and SP6.

64. The method of claim 58 wherein the second double-stranded polynucleotides are produced by amplifying the second antisense polynucleotide strands, and wherein the amplification is carried out using a universal primer that hybridizes to the universal primer site as the 5′

primer and using the antisense primer or antisense primer complex as the 3′

primer.

65. The method of claim 64 wherein the amplification is performed by enhanced polymerase chain reaction.

66. The method of claim 58 wherein the second double-stranded polynucleotides are produced using an enzyme mixture comprising a DNA polymerase, a DNA ligase, and an RNase.

67. The method of claim 58 wherein the universal primer and/or the antisense primer or antisense primer complex each comprise a restriction site.

68. The method of claim 58 wherein the second antisense polynucleotide strands are synthesized from the unhybridized sense polynucleotide strands using an antisense primer complex, said method further comprising synthesizing antisense RNA molecules from the second double-stranded polynucleotides.

69. The method of claim 58 comprising employing second double-stranded polynucleotides or a polynucleotide produced directly or indirectly therefrom in a hybridization reaction.

90. A kit comprising:

a) an antisense primer complex comprising a antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5′

of the antisense primer;

b) a sense primer; and

c) instructions for performing the method of claim 49.

70. A method for preparing a selected polynucleotide pool from a polynucleotide sample comprising:

a) hybridizing first antisense polynucleotide strands prepared from a first polynucleotide sample to sense polynucleotide strands of, or prepared from, a second polynucleotide sample, wherein the first antisense polynucleotide strands are enriched in high-abundance polynucleotide sequences relative to the first polynucleotide sample, thereby producing unhybridized sense polynucleotide strands that are enriched in low-abundance polynucleotide sequences relative to the second polynucleotide sample; and

b) synthesizing second antisense polynucleotide strands from the unhybridized sense polynucleotide strands using an antisense primer or an antisense primer complex, said antisense primer complex comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5′

of the antisense primer;

c) adding a universal primer site to the 3′

ends of the second antisense polynucleotide strands;

d) producing double-stranded polynucleotides from the second antisense polynucleotide strands.

71. The method of claim 70 wherein the molar ratio of the first antisense polynucleotide strands to the other polynucleotides in the hybridization mixture is between about 1 and about 100 to 1.

72. The method of claim 70 wherein the first and second polynucleotide samples are mRNA samples, the sense polynucleotide strands are the mRNA molecules, the first antisense polynucleotide strands are antisense RNA, the second antisense polynucleotide strands are antisense cDNA strands, and the double-stranded polynucleotides are double-stranded cDNA molecules.

73. The method of claim 70 wherein the double-stranded polynucleotides are produced by amplifying the second antisense polynucleotide strands, and wherein the amplification is carried out using a universal primer that hybridizes to the universal primer site as the 5′

primer and using the antisense primer or antisense primer complex as the 3′

primer.

74. The method of claim 70 wherein the universal primer and/or the antisense primer or antisense primer complex each comprise a restriction site.

75. The method of claim 70 further comprising cloning at least one of the double-stranded polynucleotides into a vector.

76. The method of claim 75 wherein the cloned polynucleotide encodes a polypeptide, and the vector is an expression vector.

77. The method of claim 76 further comprising introducing the expression vector into a host cell and expressing the protein encoded by the cloned double-stranded polynucleotide.

78. The method of claim 70 wherein the second antisense polynucleotide strands are synthesized from the unhybridized sense polynucleotide strands using an antisense primer complex, said method further comprising synthesizing antisense RNA molecules from the double-stranded polynucleotides.

79. The method of claim 70 comprising employing one or more of the double-stranded polynucleotides or a polynucleotide produced directly or indirectly therefrom in a hybridization reaction.

80. The method of claim 79 wherein at least one of the double-stranded polynucleotides or a polynucleotide produced therefrom is labeled with a detectable label.

81. The method of claim 70 further comprising attaching a plurality of the double-stranded polynucleotides or polynucleotides produced therefrom to a substrate to produce a polynucleotide array.

82. The method of claim 70 further comprising amplifying at least one of the double-stranded polynucleotides.

83. The method of claim 82 wherein said one or more double-stranded polynucleotides are amplified using one or more gene-specific primers.

84. The method of claim 70 wherein the first and second polynucleotide samples are different samples.

91. A kit comprising:

a) an antisense primer complex comprising a antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5′

of the antisense primer;

b) a sense primer; and

c) instructions for performing the method of claim 70.

85. A plurality of polynucleotides prepared from a polynucleotide sample, wherein the plurality of polynucleotides includes at least 103 different polynucleotides and is substantially enriched in high-abundance polynucleotide sequences relative to the polynucleotide sample, wherein the polynucleotides each comprise a RNA promoter sequence and a universal primer site.

86. A plurality of polynucleotides prepared from a polynucleotide sample, wherein the plurality of polynucleotides includes at least 10³ different polynucleotides and is substantially enriched in low-abundance polynucleotide sequences relative to the polynucleotide sample.

87. The plurality of polynucleotides of claim 86 wherein the polynucleotides each comprise a RNA promoter sequence and a universal primer site.

88. The plurality of polynucleotides of claim 86 wherein the polynucleotides are double-stranded cDNA.

89. The plurality of polynucleotides of claim 86 wherein the polynucleotides are antisense RNA.

92. A kit comprising:

a) the plurality of polynucleotide of claim 86;

b) an antisense primer complex comprising a antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5′

of the antisense primer; and

c) a sense primer.

93. A kit comprising:

a) the plurality of polynucleotides of claim 86;

and

b) an RNA polymerase capable of transcribing antisense RNA from the plurality of polynucleotides.

94. A method for preparing a selected polynucleotide pool from a polynucleotide sample comprising:

a) synthesizing first antisense polynucleotide strands from sense polynucleotides of, or prepared from, the polynucleotide sample;

b) diluting the first antisense polynucleotide strands to substantially eliminate at least some low-abundance first antisense polynucleotide strands; and

c) producing first double-stranded polynucleotides from the remaining first antisense polynucleotide strands, wherein the first double-stranded polynucleotides are enriched in high-abundance polynucleotide sequences relative to the polynucleotide sample;

d) producing second antisense polynucleotide strands from the first double-stranded polynucleotides;

e) contacting the second antisense polynucleotide strands with sense polynucleotide strands of, or prepared from, the polynucleotide sample under hybridization conditions to form a hybridization mixture, thereby producing unhybridized sense polynucleotide strands that are enriched in low-abundance polynucleotide sequences relative to the polynucleotide sample;

f) synthesizing third antisense polynucleotide strands from the unhybridized sense polynucleotide strands;

g) producing second double-stranded polynucleotides from the third antisense polynucleotide strands.