Combinatorial array for nucleic acid analysis

• US 20020012926A1
• Filed: 03/05/2001
• Published: 01/31/2002
• Est. Priority Date: 03/03/2000
• Status: Abandoned Application

• Current Assignee: California Institute of Technology
• Original Assignee: Mycometrix Incorporated
• Inventors: Quake, Stephen R., Van Dam, Robert Michael
• Application Number:
  US09/800,435
• Publication Number:
  US 20020012926A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/007 Simulation or vitual synthesis
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00729 Peptide nucleic acids [PNA]
• CPC Class Codes: B01J 2219/00731 Saccharides
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

This invention relates to an array, including a universal micro-array, for the analysis of nucleic acids, such as DNA. The devices and methods of the invention can be used for identifying gene expression patterns in any organism. More specifically, all possible oligonucleotides (n-mers) necessary for the identification of gene expression patterns are synthesized. According to the invention, n is large enough to give the specificity to uniquely identify the expression pattern of each gene in an organism of interest, and is small enough that the method and device can be easily and efficiently practiced and made. The invention provides a method of analyzing molecules, such as polynucleotides (e.g., DNA), by measuring the signal of an optically-detectable (e.g., fluorescent, ultraviolet, radioactive or color change) reporter associated with the molecules. In a polynucleotide analysis device according to the invention, levels of gene expression are correlated to a signal from an optically-detectable (e.g. fluorescent) reporter associated with a hybridized polynucleotide. The invention includes an algorithm and method to interpret data derived from a micro-array or other device, including techniques to decode or deconvolve potentially ambiguous signals into unambiguous or reliable gene expression data.

Claims

1. A method for analyzing data from hybridization of a sample to an array of oligonucleotide probes,

wherein the sample comprises a plurality of nucleotide sequences, each nucleotide sequence corresponding to a particular gene,

wherein some or all of the oligonucleotide probes are assigned to invertible subblocks such that each gene which hybridizes to an oligonucleotide probe assigned to a particular subblock does not hybridize to the oligonucleotide probes in the other subblocks, and

which method comprises a step of separately analyzing the data for the oligonucleotide probes in each subblock.

2. A method according to claim 1, wherein the array comprises a plurality (N₀) of oligonucleotide probes having a particular sequence length n so that all nucleic acid sequences having the particular sequence length are present on the array.

3. A method according to claim 2 wherein the particular sequence length n is from about 6 to about 20.

4. A method according to claim 3 wherein the particular sequence length n is from about 9 to about 16.

5. A method according to claim 4 wherein the particular sequence length n is from about 10 to about 12.

6. A method according to claim 4 wherein the particular sequence length n is from about 12 to about 15.

7. A method according to claim 1 wherein oligonucleotide probes are assigned to subblocks according to a method which comprises, for each subblock, steps of:

(a) associating a gene g_a with a gene list for a subblock, wherein the gene g_a is not already associated with a gene list for a subblock; and

(b) assigning an oligonucleotide probe o_x to the subblock, wherein the oligonucleotide probe o_x hybridizes to the gene g_a,

wherein the steps are repeated for each subblock until each gene is associated with a gene list for a subblock.

8. A method according to claim 7, further comprising steps of:

(c) for each probe o_x assigned to the subblock, associating genes g_b with the gene list for the subblock, wherein each gene g_b hybridizes to the probe o_x; and

(d) for each gene g_b associated with the gene list, assigning an oligonucleotide probe o_y to the subblock, wherein the oligonucleotide probe o_y hybridizes to the gene g_b.

9. A method according to claim 8 wherein the steps of:

(c) associating genes g_b with the gene list for the subblock; and

(d) assigning an oligonucleotide probe o_y for each gene g_b associated with the gene list

are iteratively repeated for each oligonucleotide probe o_y assigned in step (d).

10. A method according to claim 9 wherein the steps (c)-(d) are repeated for not more than 100 iterations.

11. A method according to claim 10 wherein the steps (c)-(d) are repeated for not more than 50 iterations.

12. A method according to claim 11 wherein the steps (c)-(d) are repeated for not more than 20 iterations.

13. A method according to claim 12 wherein the steps (c)-(d) are repeated for not more than 15 iterations.

14. A method according to claim 13 wherein the steps (c)-(d) are repeated for not more than ten iterations.

15. A method according to claim 14 wherein the steps (c)-(d) are repeated for not more than five iterations.

16. A method according to claim 15 wherein the steps (c)-(d) are repeated for not more than four iterations.

17. A method according to claim 16 wherein the steps (c)-(d) are repeated for not more than three iterations.

18. A method according to claim 17 wherein the steps (c)-(d) are repeated for not more than two iterations.

19. A method according to claim 9 wherein the steps (c)-(d) are iteratively repeated until, for each oligonucleotide probe o_x assigned to the particular subblock, all genes g_a that hybridize to the oligonucleotide probe o_x are associated with the gene list for the particular subblock.

20. A method according to claim 8 wherein:

(i) each oligonucleotide probe assigned to a subblock has a degeneracy value indicating the number of different genes that hybridize to that oligonucleotide probe; and

(ii) the steps (c)-(d) are iteratively repeated until for each oligonucleotide probe o_x assigned to the particular subblock, all genes g_a that hybridize to the oligonucleotide probe o_x are associated with the gene list for the particular subblock.

21. A method according to claim 7 in which:

(i) each oligonucleotide probe assigned to the subblock has a degeneracy value indicating the number of different genes that hybridize to that oligonucleotide probe, the degeneracy value being equal to or below a particular threshold T for each oligonucleotide probe assigned to the subblock; and

(ii) each gene g_a associated with the gene list for the subblock hybridizes to at least one oligonucleotide probe o_x having a degeneracy value less than the particular threshold T.

22. A method according to claim 1 wherein:

(i) each oligonucleotide probe assigned to a subblock has a degeneracy value indicating the number of different genes that hybridize to that oligonucleotide probe, and (ii) the degeneracy value is equal to or below a particular threshold T for each oligonucleotide probe assigned to the subblock.

23. A method according to claim 22 wherein the particular threshold T is no more than 100.

24. A method according to claim 23 wherein the particular threshold T is no more than 50.

25. A method according to claim 24 wherein the particular threshold T is no more than 20.

26. A method according to claim 25 wherein the particular threshold T is no more than ten.

27. A method according to claim 26 wherein the particular threshold T is no more than five.

28. A method according to claim 27 wherein the particular threshold T is no more than four.

29. A method according to claim 28 wherein the particular threshold T is no more than three.

30. A method according to claim 29 wherein the particular threshold T is no more than two.

31. A method according to claim 30 wherein the particular threshold T is one.

32. A method according to claim 1 in which expression levels are determined for each gene g_i that hybridizes to oligonucleotide probes assigned to a particular subblock by a method which comprises solving a system of linear equations for the hybridization of each gene g_i to each oligonucleotide probe o_j assigned to the particular subblock.

33. A method according to claim 32 wherein the system of linear equations is of the form

{right arrow over (E)}=(Ĥ

′

)<sup>−

1</sup>·

{right arrow over (S)}′

wherein;

(a) each element E_i of the vector {right arrow over (E)} indicates abundance of a nucleotide sequence in the sample corresponding to a particular gene g_i;

(b) each element S_j of the vector {right arrow over (S)}′

indicates a level of hybridization to a particular oligonucleotide probe o_j; and

(c) each element H_ij of the matrix Ĥ

′

indicates hybridization affinity of the nucleotide sequence corresponding to said particular particular gene g_i for the particular oligonucleotide probe o_j.

34. A method according to claim 1 wherein each of the nucleotide sequences has a length l_i equal to the length of the corresponding gene.

35. A method according to claim 1 wherein the length of each different nucleic acid is decreased before hybridization so that each different nucleic acid has a decreased length L_i=l_i−

Δ

L_i that is less than the length of the corresponding gene.

36. A method according to claim 35 wherein the length is decreased by enzymatic digestion.

37. A method according to claim 35 wherein the length of each different nucleic acid is decreased, on average, by a controled amount <

Δ

L>

.

38. A method according to claim 37 wherein the amount <

Δ

L>

is between about 50 and about 500 bases.

39. A method according to claim 38 wherein the amount <

Δ

L>

is between about 50-100 bases.

40. A method according to claim 38 wherein the amount <

Δ

L>

is between about 100-500 bases.

41. A method according to claim 35 wherein the length of each different nucleic acid is decreased by a method which comprises:

(i) protecting each nucleic acid along a particular length; and

(ii) removing the unprotected portion.

42. A method according to claim 35 wherein the average decreased length <

L>

is controled.

43. A method according to claim 42 wherein the average decreased length <

L>

is less than or equal to about 500 bases.

44. A method according to claim 43 wherein the average decreased length <

L>

is less than or equal to about 100 bases.

45. A method according to claim 44 wherein the average decreased length <

L>

is about 50 bases.

46. A method according to claim 42 wherein the average decreased length <

L>

is between about 50 and 100 bases.

47. A method according to claim 42 wherein the average decreased length <

L>

is between about 100 and 500 bases.

48. A method for assigning all or some of a plurality of oligonucleotide probes to subblocks suitable for analyzing data from hybridization of a sample to an array of the oligonucleotide probes,

wherein the sample comprises a plurality of nucleotide sequences, each nucleotide sequence corresponding to a particular gene,

which method comprises steps of:

(a) associating a gene g_a with a gene list for a subblock, wherein the gene g_a is not already associated with a gene list for a subblock; and

(b) assigning an oligonucleotide probe o_x to the subblock, wherein the oligonucleotide probe o_x hybridizes to the gene g_a,

wherein the steps are repeated for each subblock until each gene is associated with a gene list for a subblock.

49. A method according to claim 48 further comprising steps of:

(c) for each probe o_x assigned to the subblock, associating genes g_b with the gene list for the subblock, wherein each gene g_b hybridizes to the probe o_x; and

(d) for each gene g_b associated with the gene list, assigning an oligonucleotide probe o_y to the subblock, wherein the oligonucleotide probe o_y hybridizes to the gene g_b.

50. A method according to claim 49 wherein the steps of:

(c) associating genes g_b with the gene list for the subblock; and

(d) assigning an oligonucleotide probe o_y for each gene g_b associated with the gene list are iteratively repeated.

51. A method according to claim 50 wherein the step (c)-(d) are repeated for not more than 100 iterations.

52. A method according to claim 51 wherein the steps (c)-(d) are repeated for not more than 50 iterations.

53. A method according to claim 52 wherein the steps (c)-(d) are repeated for not more than 20 iterations.

54. A method according to claim 53 wherein the steps (c)-(d) are repeated for not more than ten iterations.

55. A method according to claim 54 wherein the steps (c)-(d) are repeated for not more than five iterations.

56. A method according to claim 55 wherein the steps (c)-(d) are repeated for not more than four iterations.

57. A method according to claim 56 wherein the steps (c)-(d) are repeated for not more than three iterations.

58. A method according to claim 57 wherein the steps (c)-(d) are repeated for not more than two iterations.

59. A method according to claim 50 wherein the steps (c)-(d) are iteratively repeated until, for each oligonucleotide probe o_x assigned to the particular subblock, all genes g_a that hybridize to the oligonucleotide probe o_x are associated with the gene list for the particular subblock.

60. A method according to claim 51 wherein:

(i) each oligonucleotide probe assigned to a subblock has a degeneracy value indicating the number of different genes that hybridize to that oligonucleotide probe; and

(ii) the steps (c)-(d) are iteratively repeated until, for each oligonucleotide probe o_x assigned to the particular subblock, all genes g_a that hybridize to the oligonucleotide probe o_x are associated with the gene list for the particular subblock.

61. A method according to claim 48 in which:

(i) each oligonucleotide rpboe assigned to the subblock has a degeneracy value indicating the number of different genes that hybridize to that oligonucleotide probe, the degeneracy value being equal to or below a particular threshold T for each oligonucleotide probe assigned to the subblock; and

(ii) each gene g_a associated with the gene list for the subblock hybridizes to at least one oligonucleotide probe o_x having a degeneracy value less than the particular threshold T.

62. A method according to claim 48 wherein:

(i) each oligonucleotide probe assigned to a subblock has a degeneracy value indicating the number of different genes that hybridize to that oligonucleotide probe, and

(ii) the degeneracy value is equal to or below a particular threshold T for each oligonucleotide probe assigned to the subblock.

63. A method according to claim 48, wherein the array comprises a plurality (N₀) of oligonucleotide probes having a particular sequence length n so that all nucleic acid sequences having the particular sequence length are present on the array.

64. A method for selecting a particular sequence length n for an array comprising a plurality (N₀) of oligonucleotide probes having the particular sequence length n, which method comprises:

(a) identifying a sequence length n providing an average probe degeneracy <

d(n)>

suitable for analyzing nucleic acid expression using the array; and

(b) selecting the identified sequence length n,

wherein the average probe degeneracy <

d(n)>

indicates the number of different nucleic acids that hybridize, on average, to a particular oligonucleotide probe.

65. A method according to claim 64 wherein each of the different nucleic acids corresponds to a gene in a plurality(N_g) of different genes.

66. A method according to claim 65 wherein each of the nucleotide sequences has a length l_i equal to the length of the corresponding gene.

67. A method according to claim 66 wherein the length of each different nucleic acid is decreased before hybridization so that each different nucleic acid has a decreased length L_i=l_i−

Δ

L_i that is less than the length of the corresponding gene.

68. A method according to claim 67 wherein the length is decreased by enzymatic digestion.

69. A method according to claim 67 wherein the length of each different nucleic acid is decreased, on average, by a controled amount <

Δ

L>

.

70. A method according to claim 69 wherein the amount <

Δ

L>

is between about 50 and about 500 bases.

71. A method according to claim 70 wherein the amount <

Δ

L>

is between about 50-100 bases.

72. A method according to claim 70 wherein the amount <

Δ

L>

is between about 100-500 bases.

73. A method according to claim 67 wherein the length of each different nucleic acid is decreased by a method which comprises:

(i) protecting each nucleic acid along a particular length; and

(ii) removing the unprotected portion.

74. A method according to claim 67 wherein the average decreased length <

L>

is controled.

75. A method according to claim 74 wherein the average decreased length <

L>

is less than or equal to about 500 bases.

76. A method according to claim 75 wherein the average decreased length <

L>

is less than or equal to about 100 bases.

77. A method according to claim 76 wherein the average decreased length <

L>

is about 50 bases.

78. A method according to claim 74 wherein the average decreased length <

L>

is between about 50 and 100 bases.

79. A method according to claim 74 wherein the average decreased length <

L>

is between about 100 and 500 bases.

80. A method according to claim 64 wherein the nucleic acids hybridize to the oligonucleotide probes with no more than a particular number (m) of base-pair mismatches.

81. A method according to claim 80 wherein the average probe degeneracy

<

d(n)>

is provided by the relation

$〈<br><br>d<br><br>\left(n\right)〉<br><br>=\frac{N_g}{N_0}<br><br>\left(1-n+〈<br><br>L〉<br><br>\right)\times <br><br>c$

c is provided by the relation

$c=\underset{k=0}{\overset{m}{\sum <br><br>}}<br><br>\left(\begin{array}{c}n\\ k\end{array}\right)<br><br>3^k,$

 

and

<

L>

indicates the average length of the different nucleic acids.

82. A method according to claim 64 wherein the average probe degeneracy <

d(n)>

is provided by the relation

83. A method according to claim 64 wherein the step (a) of identifying a sequence length n comprises:

(i) comparing oligonucleotide sequences having a particular sequence length n with sequences of the different nucleic acids, so that nucleic acids which hybridize to each oligonucleotide sequence are identified; and

(ii) determining the average probe degeneracy <

d(n)>

from the number of different nucleic acids that hybridize to each oligonucleotide sequence.

84. A method according to claim 64 wherein the identified sequence length n provides an average probe degeneracy <

d(n)>

that is less than or equal to about five.

85. A method according to claim 64 wherein the identified sequence length n provides an average probe degeneracy <

d(n)>

that is less than or equal to about four.

86. A method according to claim 64 wherein the identified sequence length n provides an average probe degeneracy <

d(n)>

that is less than or equal to about three.

87. A method according to claim 64 wherein the identified sequence length n provides an average probe degeneracy <

d(n)>

that is less than or equal to about two.

88. A method according to claim 64 wherein the identified sequence length n provides an average probe degeneracy <

d(n)>

of about one.

89. A method according to claim 64 wherein the step (a) of identifying a sequence length n comprises:

(i) assigning all or some of a plurality of oligonucleotide probes having a particular sequence length n to subblocks suitable for analyzing data from hybridization of a sample to an array of the oligonucleotide probes; and

(ii) determining the average probe degeneracy <

d(n)>

from the oligonucleotide probes assigned to the subblocks.

90. A method according to claim 89 wherein the plurality of oligonucleotide probes is a plurality all nucleic acid sequences having the particular length n.

91. A method according to claim 89 wherein the oligonucleotide probes are assigned to subblocks according to a method which comprises steps of:

(a) associating a gene g_a with a gene list for a subblock, wherein the gene g_b is not already associated with a gene list for a subblock; and

(b) assigning an oligonucleotide probe o_x to the subblock, wherein the oligonucleotide probe o_x hybridizes to the gene g_a,

wherein the steps are repeated for each subblock until each gene is associated with a gene list for a subblock.

92. A method according to claim 91 further comprising steps of:

(c) for each probe o_x assigned to the subblock, associating genes g_b with the gene list for the subblock, wherein each gene g_b hybridizes to the probe o_x; and

(d) for each gene g_b associated with the gene list, assigning an oligonucleotide probe o_y to the subblock, wherein the oligonucleotide probe o_y hybridizes to the gene g_b.

93. A method according to claim 92 wherein the steps of:

(c) associating genes g_b with the gene list for the subblock; and

(d) assigning an oligonucleotide probe o_y for each gene g_b associated with the gene list

are iteratively repeated.

94. A method according to claim 93 wherein the step (c)-(d) are repeated for not more than 100 iterations.

95. A method according to claim 94 wherein the steps (c)-(d) are repeated for not more than 50 iterations.

96. A method according to claim 95 wherein the steps (c)-(d) are repeated for not more than 20 iterations.

97. A method according to claim 96 wherein the steps (c)-(d) are repeated for not more than ten iterations.

98. A method according to claim 97 wherein the steps (c)-(d) are repeated for not more than five iterations.

99. A method according to claim 98 wherein the steps (c)-(d) are repeated for not more than four iterations.

100. A method according to claim 99 wherein the steps (c)-(d) are repeated for not more than three iterations.

102. A method according to claim 93 wherein the steps (c)-(d) are iteratively repeated until, for each oligonucleotide probe o_x assigned to the particular subblock, all genes g_a that hybridize to the oligonucleotide probe o_x are associated with the gene list for the particular subblock.

103. A method according to claim 93 wherein

(i) each oligonucleotide probe assigned to a subblock has a degeneracy value indicating the number of different genes that hybridize to that oligonucleotide probe;

and

(ii) the steps (c)-(d) are iteratively repeated until, for each oligonucleotide probe o_x assigned to the particular subblock, all genes g_a that hybridize to the oligonucleotide probe o_x are associated with the gene list for the particular subblock.

104. A method according to claim 91 in which:

(i) each oligonucleotide probe assigned to the subblock has a degeneracy value indicating the number of different genes that hybridize to that oligonucleotide probe, the degeneracy value being equal to or below a particular threshold T for each oligonucleotide probe assigned to the subblock; and

(ii) each gene g_a associated with the gene list for the subblock hybridizes to at least one oligonucleotide probe o_x having a degeneracy less than the particular threshold T.

105. A method according to claim 91 wherein:

(i) each oligonucleotide probe assigned to a subblock has a degeneracy value indicating the number of different genes that hybridize to that oligonucleotide probe, and

(ii) the degeneracy value is equal to or below a particular threshold T for each oligonucleotide probe assigned to the subblock.

101. A method according to claim 101 wherein the steps (c)-(d) are repeated for not more than two iterations.