Multiplex nucleic acid analysis using archived or fixed samples

• US 20040259105A1
• Filed: 10/03/2003
• Published: 12/23/2004
• Est. Priority Date: 10/03/2002
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Fan, Jian-Bing, Bibikova, Marina
• Application Number:
  US10/678,608
• Publication Number:
  US 20040259105A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present invention is directed to compositions and methods for multiplex analyses of nucleic acids from archival tissues.

Claims

1. A method for determining the expression level of target sequences in an archived tissue sample, said method comprising:

(a) contacting a nucleic acid molecule derived from an archived tissue sample with a plurality of probe sets under conditions where complementary probes form hybridization complexes with said target sequences, each of said probe sets comprising at least two universal priming sites and a target-specific sequence;

(b) amplifying said probes forming said hybridization complexes to produce amplicons, each amplicon corresponding to one of said probe sets;

(c) detecting said amplicons, wherein the detection of each of said amplicons indicates the presence of one of said plurality of target sequence in said archived tissue sample; and

(d) determining the expression level of said target sequences.

2. The method of claim 1, wherein each of said probes further comprises an adapter sequence distinctive of said target-specific sequence

3. The method of claim 2, further comprising contacting said adapter sequence, or amplicon thereof, with a capture probe to form a hybridization complex comprising said adapter sequence and said capture probe.

4. The method of claim 1, wherein said target sequence comprises DNA.

5. The method of claim 4, further comprising denaturing said target sequence prior to said contacting with said set of probes.

6. The method of claim 1, wherein said target sequence comprises RNA.

7. The method of claim 6, wherein said universal priming site comprises an RNA polymerase priming site.

8. The method of claim 7, wherein said RNA polymerase priming site comprises an RNA polymerase promoter sequence corresponding to T7, T4, T3, SP6, RNA polymerase from Thermus species or Q beta replicase from bacteriophage.

9. The method of claim 1, wherein said amplifying comprises linear amplification.

10. The method of claim 9, wherein said probe is converted to a double-stranded molecule prior to linear amplification.

11. The method of claim 10, wherein said linear amplification comprises in vitro transcription (IVT).

12. The method of claim 1, wherein said amplicons comprise a label.

13. The method of claim 1, further comprising removing non-hybridized probes.

14. The method of claim 1, wherein said probes further comprise a solid support.

15. The method of claim 1, further comprising modifying said probes.

16. The method of claim 15, wherein said modifying comprises polymerase extension, ligation or both.

17. The method of claim 1, further comprising contacting a plurality of archived tissue samples with a plurality of probe sets to detect the presence of said target sequence in a plurality of archived tissue samples.

18. The method of claim 17, wherein said plurality of archived samples is contacted individually.

19. The method of claim 17, wherein said plurality comprises 8, 16, 48, 96, 192, 384, 1152 or 1536.

20. The method of claim 1, further comprising producing a report of said detected amplicons.

21. The method of claim 1, wherein said detection comprises a microarray, liquid chromatography, mass spectrometry or electrophoresis.

22. The method of claim 21, wherein said detection comprises a microarray.

23. The method of claim 22, wherein said microarray comprises a plurality of microspheres, a probe hybridization buffer, an amplification buffer, and said set of probes.

24. A method for determining the expression level of target sequences in an archived tissue sample, said method comprising:

(a) contacting a nucleic acid molecule derived from an archived tissue sample with a plurality of probe sets under conditions where complementary probes form hybridization complexes with said target sequences, each of said probe sets comprising at least two universal priming sites and a target-specific sequence;

(b) amplifying said probes forming said hybridization complexes to produce amplicons, each amplicon corresponding to one of said probe sets; and

(c) detecting said amplicons, wherein the detection of each of said amplicons indicates the presence of one of said plurality of target sequence in said archived tissue sample.

25. The method of claim 24, further comprising the step of determining the expression level of each of said target sequences, wherein said expression level comprises at least five target sequences.

26. The method of claim 24, wherein each of said probes further comprises an adapter sequence distinctive of said target-specific sequence

27. The method of claim 26, further comprising contacting said adapter sequence, or amplicon thereof, with a capture probe to form a hybridization complex comprising said adapter sequence and said capture probe

28. The method of claim 24, wherein said target sequence comprises DNA.

29. The method of claim 28, further comprising denaturing said target sequence prior to said contacting with said set of probes.

30. The method of claim 24, wherein said target sequence comprises RNA.

31. The method of claim 30, wherein said universal priming site comprises an RNA polymerase priming site.

32. The method of claim 31, wherein said RNA polymerase priming site comprises an RNA polymerase promoter sequence corresponding to T7, T4, T3, SP6, RNA polymerase from Thermus species or Q beta replicase from bacteriophage.

33. The method of claim 24, wherein said amplifying comprises linear amplification.

34. The method of claim 33, wherein said probe is converted to a double-stranded molecule prior to linear amplification.

35. The method of claim 34, wherein said linear amplification comprises in vitro transcription (IVT).

36. The method of claim 24, wherein said amplicons comprise a label.

37. The method of claim 24, further comprising removing non-hybridized probes.

38. The method of claim 24, wherein said probes further comprise a solid support.

39. The method of claim 24, further comprising modifying said probes.

40. The method of claim 39, wherein said modifying comprises polymerase extension, ligation or both.

41. The method of claim 24, further comprising contacting a plurality of archived tissue samples with a plurality of probe sets to detect the presence of said target sequence in a plurality of archived tissue samples.

42. The method of claim 41, wherein said plurality of archived samples is contacted individually.

43. The method of claim 41, wherein said plurality comprises 8, 16, 48, 96, 192, 384, 1152 or 1536.

44. The method of claim 24, further comprising producing a report of said detected amplicons.

45. The method of claim 24, wherein said detection comprises a microarray, liquid chromatography, mass spectrometry or electrophoresis.

46. The method of claim 45, wherein said detection comprises a microarray.

47. The method of claim 46, wherein said microarray comprises a plurality of microspheres, a probe hybridization buffer, an amplification buffer, and said set of probes.

48. A method for determining the expression level of target sequences in an archived tissue sample, said method comprising:

(a) contacting a nucleic acid molecule derived from an archived tissue sample with a plurality of probe sets under conditions where complementary probes form hybridization complexes with said target sequences, each of said probe sets comprising at least two universal priming sites and a target-specific sequence having less than fifty nucleotides;

(b) amplifying said probes forming said hybridization complexes to produce amplicons, each amplicon corresponding to one of said probe sets;

(c) detecting said amplicons, wherein the detection of each of said amplicons indicates the presence of one of said plurality of target sequence in said archived tissue sample; and

(d) determining the expression level of said target sequences.

49. The method of claim 48, wherein each of said probes further comprises an adapter sequence distinctive of said target-specific sequence

50. The method of claim 49, further comprising contacting said adapter sequence, or amplicon thereof, with a capture probe to form a hybridization complex comprising said adapter sequence and said capture probe

51. The method of claim 48, wherein said target sequence comprises DNA.

52. The method of claim 51, further comprising denaturing said target sequence prior to said contacting with said set of probes.

53. The method of claim 48, wherein said target sequence comprises RNA.

54. The method of claim 53, wherein said universal priming site comprises an RNA polymerase priming site.

55. The method of claim 54, wherein said RNA polymerase priming site comprises an RNA polymerase promoter sequence corresponding to T7, T4, T3, SP6, RNA polymerase from Thermus species or Q beta replicase from bacteriophage.

56. The method of claim 48, wherein said amplifying comprises linear amplification.

57. The method of claim 56, wherein said probe is converted to a double-stranded molecule prior to linear amplification.

58. The method of claim 57, wherein said linear amplification comprises in vitro transcription (IVT).

59. The method of claim 48, wherein said amplicons comprise a label.

60. The method of claim 48, further comprising removing non-hybridized probes.

61. The method of claim 48, wherein said probes further comprise a solid support.

62. The method of claim 48, further comprising modifying said probes.

63. The method of claim 62, wherein said modifying comprises polymerase extension, ligation or both.

64. The method of claim 48, further comprising contacting a plurality of archived tissue samples with a plurality of probe sets to detect the presence of said target sequence in a plurality of archived tissue samples.

65. The method of claim 64, wherein said plurality of archived samples is contacted individually.

66. The method of claim 64, wherein said plurality comprises 8, 16, 48, 96, 192, 384, 1152 or 1536.

67. The method of claim 48, further comprising producing a report of said detected amplicons.

68. The method of claim 48, wherein said detection comprises a microarray, liquid chromatography, mass spectrometry or electrophoresis.

69. The method of claim 68, wherein said detection comprises a micro array.

70. The method of claim 69, wherein said microarray comprises a plurality of microspheres, a probe hybridization buffer, an amplification buffer, and said set of probes.

71. A method for detecting a target sequence in an archived tissue sample, said method comprising:

(a) contacting a nucleic acid molecule derived from an archived tissue sample with a set of ligation probe pairs, each of said pairs capable of forming a ligation complex with said target sequence, each of said ligation probes of said pair comprising a universal priming site and a target-specific sequence;

(b) ligating said ligation complexes to form ligated probes;

(c) amplifying said ligated probes to produce amplicons, each amplicon corresponding to one of said sets of probe pairs;

(d) detecting said amplicons, wherein the detection of said amplicons indicates the presence of said target sequence in said archived tissue sample; and

(e) determining the expression level of said target sequences.

72. The method of claim 71, wherein said ligation probe pair further comprises an adapter sequence distinctive of said target-specific sequence.

73. The method of claim 71, wherein said probes comprise DNA.

74. The method of claim 71, wherein said ligation probes hybridize contiguously to said target sequence to form said complex.

75. The method of claim 71, wherein wherein said ligation probes hybridize non-contiguously to said target sequence to form said complex.

76. The method of claim 71, further comprising adding a polymerase and nucleotides.

77. The method of claim 72, further comprising contacting said adapter sequence, or amplicon thereof, with a capture probe to form a hybridization complex comprising said adapter sequence and said capture probe

78. The method of claim 71, wherein said target sequence comprises DNA.

79. The method of claim 78, further comprising denaturing said target sequence prior to said contacting with said set of probes.

80. The method of claim 71, wherein said target sequence comprises RNA.

81. The method of claim 80, wherein said universal priming site comprises an RNA polymerase priming site.

82. The method of claim 81, wherein said RNA polymerase priming site comprises an RNA polymerase promoter sequence corresponding to T7, T4, T3, SP6, RNA polymerase from Thermus species or Q beta replicase from bacteriophage.

83. The method of claim 71, wherein said amplifying comprises linear amplification.

84. The method of claim 83, wherein said probe is converted to a double-stranded molecule prior to linear amplification.

85. The method of claim 84, wherein said linear amplification comprises in vitro transcription (IVT).

86. The method of claim 71, wherein said amplicons comprise a label.

87. The method of claim 71, further comprising removing non-hybridized probes.

88. The method of claim 71, wherein said probes further comprise a solid support.

89. The method of claim 71, further comprising modifying said probes.

90. The method of claim 89, wherein said modifying comprises polymerase extension, ligation or both.

91. The method of claim 71, further comprising contacting a plurality of archived tissue samples with a plurality of probe sets to detect the presence of said target sequence in a plurality of archived tissue samples.

92. The method of claim 91, wherein said plurality of archived samples is contacted individually.

93. The method of claim 97, wherein said plurality comprises 8, 16, 48, 96, 192, 384, 1152 or 1536.

94. The method of claim 71, further comprising producing a report of said detected amplicons.

95. The method of claim 71, wherein said detection comprises a microarray, liquid chromatography, mass spectrometry or electrophoresis.

96. The method of claim 95, wherein said detection comprises a microarray.

97. The method of claim 96, wherein said microarray comprises a plurality of microspheres, a probe hybridization buffer, an amplification buffer, and said set of probes.

98. A method for detecting a plurality of target sequences in an archived tissue sample, said method comprising:

(a) contacting a nucleic acid molecule derived from an archived tissue sample with a plurality of sets of ligation probe pairs, each set comprising ligation probe pairs capable of forming a ligation complex with one of said plurality of target sequences, each of said ligation probes of said pair comprising a universal priming site and a target-specific sequence;

(b) ligating said ligation probe pairs of said ligation complexes to form ligated probes;

(c) amplifying said ligated probes to produce amplicons, each amplicon corresponding to one of said sets of ligation probe pairs; and

(d) detecting said amplicons, wherein the detection of each of said amplicons indicates the presence of one of said plurality of target sequences in said archived tissue sample.

99. The method of claim 98, wherein said ligation probe pair further comprises an adapter sequence distinctive of said target-specific sequence.

100. The method of claim 98, wherein said probes comprise DNA.

101. The method of claim 98, wherein said ligation probes hybridize contiguously to said target sequence to form said complex.

102. The method of claim 98, wherein wherein said ligation probes hybridize non-contiguously to said target sequence to form said complex.

103. The method of claim 98, further comprising adding a polymerase and nucleotides.

104. The method of claim 99, further comprising contacting said adapter sequence, or amplicon thereof, with a capture probe to form a hybridization complex comprising said adapter sequence and said capture probe

105. The method of claim 98, wherein said target sequence comprises DNA.

106. The method of claim 105, further comprising denaturing said target sequence prior to said contacting with said set of probes.

107. The method of claim 98, wherein said target sequence comprises RNA.

108. The method of claim 107, wherein said universal priming site comprises an RNA polymerase priming site.

109. The method of claim 108, wherein said RNA polymerase priming site comprises an RNA polymerase promoter sequence corresponding to T7, T4, T3, SP6, RNA polymerase from Thermus species or Q beta replicase from bacteriophage.

110. The method of claim 98, wherein said amplifying comprises linear amplification.

111. The method of claim 110, wherein said probe is converted to a double-stranded molecule prior to linear amplification.

112. The method of claim 111, wherein said linear amplification comprises in vitro transcription (IVT).

113. The method of claim 98, wherein said amplicons comprise a label.

114. The method of claim 98, further comprising removing non-hybridized probes.

115. The method of claim 98, wherein said probes further comprise a solid support.

116. The method of claim 98, further comprising modifying said probes.

117. The method of claim 116, wherein said modifying comprises polymerase extension, ligation or both.

118. The method of claim 98, further comprising contacting a plurality of archived tissue samples with a plurality of probe sets to detect the presence of said target sequence in a plurality of archived tissue samples.

119. The method of claim 118, wherein said plurality of archived samples is contacted individually.

120. The method of claim 118, wherein said plurality comprises 8, 16, 48, 96, 192, 384, 1152 or 1536.

121. The method of claim 98, further comprising producing a report of said detected amplicons.

122. The method of claim 98, wherein said detection comprises a microarray, liquid chromatography, mass spectrometry or electrophoresis.

123. The method of claim 122, wherein said detection comprises a micro array.

124. The method of claim 123, wherein said microarray comprises a plurality of microspheres, a probe hybridization buffer, an amplification buffer, and said set of probes.