Arrays, computer program products and methods for in silico array-based comparative binding assays

• US 20030186250A1
• Filed: 03/27/2002
• Published: 10/02/2003
• Est. Priority Date: 03/27/2002
• Status: Active Grant

• Current Assignee: PerkinElmer LAS, Inc. (Perkinelmer Incorporated)
• Original Assignee: Spectral Genomics, Inc. (Revvity, Inc.)
• Inventors: Shah, Shishir
• Granted Patent: US 6,916,621 B2
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

The invention provides computer systems, computer program products and methods for in silico array-based methods for determining the relative amount of biological molecules (e.g., nucleic acid sequences) in two or more samples. The invention also provides novel arrays comprising immobilized calibration molecules (e.g., nucleic acids) for normalizing the results of array-based binding assays (e.g., hybridization reactions).

Claims

1. An in silico, array-based method for determining the relative amount of a biological molecule in two or more samples, the method comprising:

(a) providing a first array comprising a plurality of biological molecules, wherein each biological molecule is immobilized to a discrete and known spot on a first substrate surface to form a first array of biological molecules;

(b) providing a second array comprising a plurality of biological molecules, wherein each biological molecule is immobilized to a discrete and known spot on a second substrate surface to form a second array of biological molecules,

and the biological molecules immobilized on the second array comprise substantially the same plurality of biological molecules arrayed in step (a);

(c) providing a first sample comprising a plurality of biological molecules comprising a detectable label;

(d) providing a second sample comprising a plurality of biological molecules comprising a detectable label;

(e) contacting the first sample of step (c) with the first array of step (a) under conditions wherein the labeled biological molecules can specifically bind to a biological molecule immobilized on the first array;

(f) contacting the second sample of step (d) with the second array of step (b) under the same conditions as in step (e), thereby allowing the labeled biological molecules to specifically bind to a biological molecule immobilized on the second array;

(g) identifying which spots on the first and the second substrate surfaces are specifically bound to a labeled biological molecule and measuring the amount of label on each spot; and

,

(h) comparing the amount of labeled biological molecule specifically bound to immobilized biological molecules on the first array to the amount of labeled biological molecules specifically bound to the same biological molecule immobilized on the second array, thereby determining the relative amount of sample biological molecule able to bind to the same immobilized biological molecule in the first sample compared to the second sample.

3. The method of claim 1, wherein the first substrate surface and the second substrate surface comprise one surface and the first array and the second array are separated by a hydrophobic barrier such that a first sample can be applied to the first array at the same time a second sample is applied to the second array without the two samples mixing together.

4. The method of claim 1, wherein the first array and the second array are in separated wells of a multi-well plate.

5. The method of claim 3, wherein the multi-well plate is a multi-titer well plate and each well comprises one array.

6. The method of claim 1, wherein the biological molecule comprises a nucleic acid or an analog or a mimetic thereof.

7. The method of claim 1, wherein the biological molecule comprises a lipid or a polysaccharide or an analog or a mimetic thereof.

8. The method of claim 1, wherein the biological molecule comprises a polypeptide or an analog or a mimetic thereof.

9. The method of claim 6, wherein the nucleic acid comprises a genomic nucleic acid.

10. The method of claim 9, wherein the cloned genomic nucleic acid segment comprises a gene.

11. The method of claim 1, wherein the array-immobilized biological molecules comprise cloned genomic nucleic acid.

12. The method of claim 9, wherein the genomic nucleic acid comprises a substantially complete chromosome or a known subset of a chromosome.

13. The method of claim 12, wherein the genomic nucleic acid comprises a substantially complete genome or a known subset of a genome.

14. The method of claim 9, wherein the genomic nucleic acid is derived from a genome of a normal cell.

15. The method of claim 9, wherein the genomic nucleic acid is derived from a genome of a mammalian cell.

16. The method of claim 15, wherein the mammalian cell is a human cell.

17. The method of claim 1, wherein the labeled biological molecules comprise a plurality of labeled nucleic acid sequences comprising sequences complementary to a subset of transcripts expressed by a cell.

18. The method of claim 17, wherein the plurality of labeled nucleic acid sequences comprise sequences complementary to a substantially complete sample of transcripts expressed by a cell.

19. The method of claim 1, wherein the labeled biological molecules comprise a plurality of labeled nucleic acid sequences comprising genomic sequences.

20. The method of claim 19, wherein the plurality of labeled nucleic acid sequences comprise a substantially complete chromosome or a known subset of a chromosome.

21. The method of claim 20, wherein the plurality of labeled nucleic acid sequences comprise a substantially complete genome or a known subset of a genome.

22. The method of claim 1, wherein the array-immobilized biological molecules comprise nucleic acids comprising a substantially complete genome or a known subset of a genome and the labeled biological molecules from each sample comprise a plurality of labeled nucleic acid sequences comprising a substantially complete genome or a known subset of a genome, thereby performing a comparative genomic hybridization.

23. The method of claim 6, wherein the nucleic acid from the first sample is derived from a cell with a normal genotype and the nucleic acid from the second sample is derived from a cell with an abnormal genotype.

24. The method of claim 6, wherein the nucleic acid from the first sample is derived from a cell with a normal phenotype and the nucleic acid from the second sample is derived from a cell with an abnormal phenotype.

25. The method of claim 24, wherein the abnormal phenotype comprises a disease phenotype.

26. The method of claim 24, wherein the abnormal phenotype comprises a neoplastic or hyperplastic phenotype.

27. The method of claim 26, wherein neoplastic phenotype is selected from the group consisting of breast cancer, skin cancer and bone cancer.

28. The method of claim 6, wherein the nucleic acid from the first sample is derived from an unstimulated cell and the nucleic acid from the second sample is derived from the unstimulated cell after stimulation.

29. The method of claim 6, wherein the nucleic acid from the first sample is derived from an undifferentiated cell and the nucleic acid from the second sample is derived from the undifferentiated cell after stimulation.

30. The method of claim 6, wherein the nucleic acid from the first sample is derived from a normal cell and the nucleic acid from the second sample is derived from the normal cell after an injury.

31. The method of claim 6, wherein the nucleic acid from the first sample is derived from a normal cell and the nucleic acid from the second sample is derived from the normal cell after an environmental stress.

32. The method of claim 31, wherein the environmental stress comprises a high or a low or a change in temperature.

33. The method of claim 31, wherein the environmental stress comprises an exposure to a chemical.

34. The method of claim 33, wherein the chemical is a carcinogen.

35. The method of claim 33, wherein the chemical is a drug or a medicine.

36. The method of claim 6, wherein the sample nucleic acid comprises a DNA.

37. The method of claim 6, wherein the sample nucleic acid sequence comprises a cDNA or an RNA.

38. The method of claim 37, wherein the RNA comprises mRNA.

39. The method of claim 6, wherein the sample nucleic acid sequence comprises oligonucleotides.

40. The method of claim 6, wherein the sample nucleic acid sequence comprises expressed sequence tags (EST).

41. The method of claim 6, wherein the array-immobilized nucleic acid segment comprises genomic nucleic acid cloned in a construct comprising an artificial chromosome.

42. The method of claim 41, wherein the artificial chromosome comprises a bacterial artificial chromosome (BAC).

43. The method of claim 41, wherein the artificial chromosome is selected from the group consisting of a human artificial chromosome (HAC) a yeast artificial chromosome (YAC), a transformation-competent artificial chromosome (TAC) and a bacteriophage P1-derived artificial chromosome (PAC).

44. The method of claim 6, wherein the array-immobilized nucleic acid segment is cloned in a construct comprising a vector selected from the group consisting of a cosmid vector, a plasmid vector and a viral vector.

45. The method of claim 6, wherein the array-immobilized nucleic acid segment is between about 50 kilobases (0.5 megabase) to about 500 kilobases (5 megabases) in length, between about 100 kilobases (1 megabase) to about 400 kilobases (4 megabases) in length, or, is about 300 kilobases (3 megabases) in length.

46. The method of claim 1, wherein a labeled biological molecule is derived from a body fluid sample, a cell sample or a tissue sample.

47. The method of claim 1, wherein a labeled biological molecule is derived from a cancer cell or a tumor cell sample.

48. The method of claim 1, wherein a labeled biological molecule is derived from a biopsy sample.

49. The method of claim 1, wherein a labeled biological molecule is derived from a blood sample.

50. The method of claim 1, further comprising a washing step, wherein sample biological molecule not specifically bound to array-immobilized biological molecule are removed before the identifying step (g).

51. The method of claim 6, further comprising a washing step, wherein sample nucleic acids not specifically hybridized to array-immobilized nucleic acid segments are removed before the identifying step (g).

52. The method of claim 51, wherein the washing step comprises use of a solution comprising a salt concentration of about 0.02 molar at pH 7 at a temperature of at least about 50°

C.

53. The method of claim 51, wherein the washing step comprises use of a solution comprising a salt concentration of about 0.15 M at a temperature of at least about 72°

C. for about 15 minutes.

54. The method of claim 51, wherein the washing step comprises use of a solution comprising a salt concentration of about 0.2×

SSC at a temperature of at least about 50°

C. for at least about 15 minutes.

55. The method of claim 1, wherein the first sample biological molecule and the second sample biological molecule comprise the same label.

56. The method of claim 1, wherein the first sample biological molecule and the second sample biological molecule comprise different labels.

57. The method of claim 1, wherein the sample biological molecules are labeled with a fluorochrome.

58. The method of claim 57, wherein the label comprises a Cy3™

or a Cy5™

or an equivalent.

59. The method of claim 1, wherein the sample biological molecules are labeled with a bioluminescent or a chemiluminescent label.

60. The method of claim 1, further comprising

providing a third array comprising a plurality of biological molecules, wherein each biological molecule is immobilized to a discrete and known spot on a third substrate surface to form a third array of biological molecules, and the biological molecules immobilized on the third array comprise substantially the same plurality of biological molecules arrayed in step (a), and a third sample comprising a plurality of biological molecules comprising a detectable label,

contacting the third sample with the third array under the same conditions as in step (e), thereby allowing the labeled biological molecule to specifically bind to a biological molecule immobilized on the third array;

identifying which spots on the first, second and third substrate surfaces are specifically bound to a labeled sample biological molecule and measuring the amount of label on each spot; and

,

comparing the amount of labeled biological molecules bound by specific binding to the same biological molecule immobilized on the first array, the second array and the third array, thereby determining the relative amount of a biological molecule in the first, second and third samples.

61. The method of claim 60, wherein the first, second and third substrate surfaces comprise one surface and the first, second and third arrays are separated by a hydrophobic barrier such that the samples can be applied to the arrays at the same time without the three samples mixing together.

62. The method of claim 60, wherein the first, second and third array are in separated wells of a multi-well plate.

63. The method of claim 62, wherein the multi-well plate is a multi-titer well plate and each well comprises one array.

64. The method of claim 1, wherein the biological molecules comprise nucleic acid sequences and the method further comprises the step of blocking the hybridization capacity of repetitive nucleic acid sequences in the immobilized nucleic acid.

65. The method of claim 1, wherein the biological molecules comprise nucleic acid sequences and the method further comprises the step of blocking the hybridization capacity of repetitive nucleic acid sequences in the sample nucleic acid sequences by mixing the sample nucleic acid sequences with unlabeled repetitive nucleic acid sequences.

67. The method of claim 65 and claim 66, wherein the repetitive nucleic acid sequences comprise Cot-1 DNA or equivalent.

68. The method of claim 65 and claim 66, wherein the repetitive nucleic acid sequences comprise SST sequences or salmon sperm DNA or equivalents.

80. A kit comprising the following components:

(a) (i) at least two arrays, each comprising a plurality of biological molecules, wherein each biological molecules is immobilized to a discrete and known spot on a substrate surface to form an array, or (ii) a biochip comprising a first substrate surface comprising a first array and a second substrate surface comprising a second array, wherein the first and second arrays are separated by a hydrophobic barrier such that a first sample can be applied to the first array at the same time a second sample is applied to the second array without the two samples mixing together; and

,

(b) instructions for using the arrays comprising a method as set forth in claim 1.

81. A kit comprising the following components:

(a) (i) at least two arrays, each comprising a plurality of cloned genomic nucleic acid segments, wherein each genomic nucleic acid segment is immobilized to a discrete and known spot on a substrate surface to form an array and the cloned genomic nucleic acid segments comprise a substantially complete genome or a known subset of a genome, or, (ii) a biochip comprising a first substrate surface comprising a first array and a second substrate surface comprising a second array, wherein the first and second arrays are separated by a hydrophobic barrier such that a first sample can be applied to the first array at the same time a second sample is applied to the second array without the two samples mixing together; and

,

(b) instructions for using the arrays comprising a method as set forth in claim 6.

82. The kit of claim 80, further comprising materials to prepare a sample for application to the array.

83. The kit of claim 81, further comprising a sample of wild type, or normal, nucleic acid.

84. The kit of claim 83, wherein the wild type, or normal, nucleic acid comprises a label.

85. The kit of claim 83, wherein the wild type, or normal, nucleic acid comprises a human wild type genomic nucleic acid.

86. The kit of claim 81, wherein the array comprises a mouse BAC array or a human BAC array.

87. A computer program product in a computer readable medium for determining the relative amount of a nucleic acid sequence in two or more samples comprising:

a computer useable. medium comprising a computer readable program code embodied therein, wherein the computer program product is capable of determining the relative amount of a nucleic acid sequence in two or more samples by a process comprising the following steps;

(a) collecting data comprising which spots on a first array substrate surface and at least a second array substrate surface are specifically hybridized to a labeled nucleic acid sequence and the amount of labeled nucleic acid on each spot, wherein the data is generated by a method as set forth in claim 1, claim 69 or claim 70;

and

(b) comparing the amount of labeled nucleic acid sequence bound by specific hybridization to a nucleic acid segment immobilized on the first array to the amount of labeled nucleic acid sequence bound by specific hybridization to the same nucleic acid segment immobilized on the second array by comparing the data collected in step (a), thereby determining the relative amount of a nucleic acid sequence complementary to the same nucleic acid segment in the first sample compared to the second sample.

88. A computer-implemented method for determining the relative amount of a nucleic acid sequence in two or more samples comprising the following steps:

(a) identifying which spots on a first array substrate surface and at least a second array substrate surface are specifically hybridized to a labeled nucleic acid segment and the amount of labeled nucleic acid on each spot, wherein the data is generated by a method as set forth in claim 1, claim 69 or claim 70, and communicating this data to a computer program product;

(b) comparing the amount of labeled nucleic acid sequence bound by specific hybridization to a nucleic acid segment immobilized on the first array to the amount of labeled nucleic acid sequence bound by specific hybridization to the same nucleic acid segment immobilized on the second array by comparing the data communicated in step (a) and using a computer program product implementing a method as-set forth in claim 1, claim 69 or claim 70, thereby determining the relative amount of a nucleic acid sequence complementary to the same nucleic acid segment in the first sample compared to the second sample.

89. A computer system, comprising:

(a) a processor; and

(b) a computer program product as set forth in claim 87.

2. An in silico, array-based method for determining the relative amount of a nucleic acid sequence in two or more samples, the method comprising:

(a) providing a first array comprising a plurality of nucleic acid segments, wherein each nucleic acid segment is immobilized to a discrete and known spot on a first substrate surface to form a first array of nucleic acid segments;

(b) providing a second array comprising a plurality of nucleic acid segments, wherein each nucleic acid segment is immobilized to a discrete and known spot on a second substrate surface to form a second array of nucleic acid segments,

and the nucleic acid segments immobilized on the second array comprise substantially the same plurality of nucleic acid segments arrayed in step (a);

(c) providing a first sample comprising a plurality of nucleic acid sequences comprising a detectable label;

(d) providing a second sample comprising a plurality of nucleic acid sequences comprising a detectable label;

(e) contacting the first sample of step (c) with the first array of step (a) under conditions wherein the labeled nucleic acid can specifically hybridize to a nucleic acid segment immobilized on the first array;

(f) contacting the second sample of step (d) with the second array of step (b) under the same conditions as in step (e), thereby allowing the labeled nucleic acid to specifically hybridize to a nucleic acid segment immobilized on the second array;

(g) identifying which spots on the first and the second substrate surfaces are specifically hybridized to a labeled nucleic acid segment and measuring the amount of label on each spot; and

,

(h) comparing the amount of labeled nucleic acid sequence bound by specific hybridization to a nucleic acid segment immobilized on the first array to the amount of labeled nucleic acid sequence bound by specific hybridization to the same nucleic acid segment immobilized on the second array, thereby determining the relative amount of a nucleic acid sequence complementary to the same nucleic acid segment in the first sample compared to the second sample.

66. The method of claim 66, wherein the sample nucleic acid sequences are first mixed with unlabeled repetitive nucleic acid sequences before the step comprising contacting with the array-immobilized nucleic acid segments.

69. An in silico, array-based method for performing comparative genomic hybridization, the method comprising:

(a) providing a first array comprising a plurality of genomic nucleic acid segments, wherein each nucleic acid segment is immobilized to a discrete and known spot on a first substrate surface to form a first array of genomic nucleic acid segments and the plurality of genomic nucleic acid segments comprise a substantially complete genome or a known subset of a genome;

(b) providing a second array comprising substantially the same plurality of genomic nucleic acid segments arrayed in step (a), wherein each nucleic acid segment is immobilized to a discrete and known spot on a second substrate surface to form a second array of genomic nucleic acid segments;

(c) providing a first sample comprising a plurality of genomic nucleic acid sequences comprising a detectable label;

(d) providing a second sample comprising a plurality of genomic nucleic acid sequences comprising a detectable label;

(e) contacting the first sample of step (c) with the first array of step (a) under conditions wherein the labeled nucleic acid can specifically hybridize to the nucleic acid segments immobilized on the first array;

(f) contacting the second sample of step (d) with the second array of step (b) under the same conditions as in step (e), thereby allowing the labeled nucleic acid to specifically hybridize to the nucleic acid segments immobilized on the second array;

(g) identifying which spots on the first and the second substrate surfaces are specifically hybridized to a labeled nucleic acid segment and measuring the amount of label on each spot; and

,

(h) comparing the amount of labeled nucleic acid sequence bound by specific hybridization to a nucleic acid segment in the first array to the amount of labeled nucleic acid sequence bound by specific hybridization to a nucleic acid segment in the second array, thereby determining the relative amount of a nucleic acid sequence complementary to the nucleic acid segment in the first sample compared to the second sample and performing a comparative genomic hybridization.

70. An in silico, array-based method of determining one or more variations in copy numbers of biological molecules in a first sample relative to copy numbers of substantially identical biological molecules in at least a second sample, the method comprising the steps of:

(a) providing a first array and at least a second array, each comprising a plurality of immobilized biological molecules, wherein the biological molecules are immobilized to discrete and known spots on a substrate surface to form at least two arrays of biological molecules, and the second array comprises substantially the same plurality of biological molecules immobilized in the first array;

(a) providing at least two samples comprising biological molecules and labeling biological molecules from each sample, wherein biological molecules in all samples comprise the same label or biological molecules in the first sample comprise a different label than biological molecules in the second label;

(b) contacting the first sample of labeled biological molecules to the first array and the second sample of labeled biological molecules to the second array under conditions wherein the labeled sample biological molecules can specifically bind to the immobilized biological molecules; and

(c) detecting the amount of label associated with each spot and comparing the amount of label associated with an immobilized biological molecule in the first array to the amount of label associated with the same immobilized biological molecule in the second array, thereby determining the amount of immobilized biological molecule in the first sample relative to the second sample.

71. The method of claim 70, further comprising determining the ratio of the amount of label associated with the immobilized biological molecule in the first and the second array, thereby determining a ratio of signal intensity.

72. The method of claim 70, further comprising determining the amount of a calibration molecule, wherein a known amount of a calibration molecule is spotted on each array.

73. The method of claim 72, wherein a known amount of a calibration molecule-binding composition is mixed with the first and the second samples.

74. The method of claim 72, wherein each array comprises a calibration spot, wherein the calibration spot comprises a biological molecule from each spot on an array.

75. The method of claim 70, wherein the biological molecule comprises a nucleic acid and the method further comprises determining the average copy number of a calibration sequence, wherein a known amount of a calibration sequence is spotted on each array, and (i) a known amount of a calibration sequence is mixed with the first and the second samples, and the calibration sequence is derived from an different source from which the sample nucleic acids were derived, or, (ii) the calibration sequences spotted on the array comprise at least one sequence of a nucleic acid from each of the array spots.

76. The method of claim 75, wherein the calibration sequence is derived from an organism different from which the sample nucleic acids were derived.

77. The method of claim 72, wherein the calibration molecule is spotted in titrated concentrations on each of the arrays.

78. The method of claim 72, further comprising determining whether the expected ratio of the known amount of calibration molecule is detected on the two arrays.

79. The method of claim 72, further comprising normalizing the ratio of the amount of label associated with a biological molecule in the first and second arrays by adjusting the ratio by a figure representing the difference between the expected ratio of calibration molecules and the detected ratio of calibration molecules on the two arrays.

90. An array for determining the relative amount of a biological molecule a sample comprising a plurality of biological molecules immobilized to a plurality of discrete and known spots on a substrate surface to form an array of biological molecules, wherein the array of spots comprises a plurality of test spots and at least one calibration spot, and the calibration spot comprises at least one copy of a sequence from each test spot on the array.

91. The array of claim 90, wherein the calibration spot comprises an equimolar mixture of all the biological molecules spotted on the array.

92. The array of claim 90, comprising at least a second calibration spot comprising an equimolar dilution of the mixture of biological molecules spotted on a first calibration spot.

93. The array of claim 92, comprising a plurality of calibration spots, wherein each calibration spot represents a different equimolar dilution of the mixture of biological molecules spotted on the array.

94. The array of claim 92, wherein the array comprises a first substrate surface comprising a first array and a second substrate surface comprising a second array, wherein the first and second arrays are separated by a hydrophobic barrier such that a first sample can be applied to the first array at the same time a second sample is applied to the second array without the two samples mixing together, and the first and the second arrays comprise the same calibration spots.

95. The array of claim 92, wherein the biological molecule comprises a nucleic acid.

96. The array of claim 95, wherein the nucleic acid comprises a DNA or an RNA.

97. The array of claim 92, wherein the biological molecule comprises a polypeptide or a peptide.

98. The array of claim 92, wherein the biological molecule comprises a lipid or a polysaccharide.

99. A multiplexed system for performing comparative binding assays using an array comprising:

(a) an array comprising a plurality of biological molecules immobilized to a plurality of discrete and known spots on a substrate surface to form an array of biological molecules, wherein the array of spots comprises a plurality of test spots and at least one calibration spot, and the calibration spot comprises at least one copy of a biological molecule from each test spot on the array, or (ii) a first substrate surface comprising a first array and a second substrate surface comprising a second array, wherein the first and second arrays are separated by a hydrophobic barrier such that a first sample can be applied to the first array at the same time a second sample is applied to the second array without the two samples mixing together, and the first and the second arrays comprise the same calibration spots;

(b) a device for detecting a detectable label, wherein the device can measure which detectable labels are on which spots on the substrate surface.

100. A multiplexed system for performing comparative genomic hybridization (CGH) using an array comprising:

(a) an array comprising (i) a plurality of nucleic acids immobilized to a plurality of discrete and known spots on a substrate surface to form an array of nucleic acids, wherein the array of spot comprises a plurality of test spots and at least one calibration spot, and the calibration spot comprises at least one copy of a sequence from each test spot on the array, or (ii) a first substrate surface comprising a first array and a second substrate surface comprising a second array, wherein the first and second arrays are separated by a hydrophobic barrier such that a first sample can be applied to the first array at the same time a second sample is applied to the second array without the two samples mixing together, and the first and the second arrays comprise the same calibration spots;

(b) a device for detecting a detectable label, wherein the device can measure which detectable labels are on which spots on the substrate surface.