Methods for array-based comparitive binding assays

• US 6,916,621 B2
• Filed: 03/27/2002
• Issued: 07/12/2005
• Est. Priority Date: 03/27/2002
• Status: Expired due to Fees

• Current Assignee: PerkinElmer LAS, Inc. (Perkinelmer Incorporated)
• Original Assignee: Spectral Genomics, Inc. (Revvity, Inc.)
• Inventors: Shah, Shishir
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US10/112,657
• Publication Number:
  US 20030186250A1
• Time in Patent Office: 1,203 Days
• Field of Search: 435/6, 435/7.1, 435/174, 435/283.1, 435/287.2, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• 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 array-based method for determining a relative amount of a nucleic acid sequence in two or more samples, the method comprising:

(a) contacting a first sample and a second sample with a first array and a second array respectively, the first and second array comprising a plurality of nucleic acid segments, wherein each nucleic acid segment is immobilized to a discrete and known spot on each of a first substrate surface and a second substrate surface to form the first and the second arrays of nucleic acid segments wherein each said spot comprises a nucleic acid sequence that is a marker for each said spot on the array providing a plurality of markers on the array and the nucleic acid segments immobilized on the second array comprise substantially the same plurality of nucleic acid segments immobilized on the first array, each array further comprising at least one calibration spot comprising a mixture of the same plurality of nucleic acid sequence markers present in all of the other spots of the array, the first sample and second sample each comprising a plurality of nucleic acid segments comprising a detectable label wherein contacting is performed under conditions wherein the labeled nucleic acid segments specifically bind to the nucleic acid segment immobilized on the arrays;

(b) identifying which spots on the first and the second arrays are specifically bound to a labeled nucleic acid segment and measuring the an amount of label on each spot;

(c) comparing the amount of labeled nucleic acid segment specifically bound to immobilized nucleic acid segments on the first array to the amount of labeled nucleic acid segments specifically bound to the same nucleic acid segment immobilized on the second array, and comparing the amount of labeled nucleic acid segments specifically bound to the calibration spot on the first and second arrays; and

(d) normalizing the ratio of the amount of label associated with the nucleic acid segments in the first and second arrays by adjusting the ratio by a figure representing the difference between an expected ratio of label associated with the calibration spot on the first and second arrays and a detected ratio of label associated with the calibration spot on the first and second arrays, thereby determining the relative amount of a nucleic acid sequence complementary to a same nucleic acid segment present in the first sample compared to that present in the second sample.

2. 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.

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

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

5. The method of claim 1, wherein the nucleic acid comprises a nucleic acid analog or a nucleic acid mimetic thereof.

6. The method of claim 1, wherein the nucleic acid comprises a genomic nucleic acid.

7. The method of claim 6, wherein the genomic nucleic acid is a nucleic acid segment that comprises a gene.

8. The method of claim 6, wherein the genomic nucleic acid comprises a substantially complete chromosome or a known subset of a chromosome.

9. The method of claim 8, wherein the genomic nucleic acid comprises a substantially complete genome or a known subset of a genome.

10. The method of claim 6, wherein the genomic nucleic acid is derived from a genome of a normal cell.

11. The method of claim 6, wherein the genomic nucleic acid is derived from a genome of a mammalian cell.

12. The method of claim 11, wherein the mammalian cell is a human cell.

13. The method of claim 1, wherein the labeled nucleic acid segments comprise sequences complementary to a subset of transcripts expressed by a cell.

14. The method of claim 13, wherein the plurality of labeled nucleic acid segments comprise sequences complementary to a substantially complete sample of transcripts expressed by a cell.

15. The method of claim 1, wherein the plurality of labeled nucleic acid sequences comprise genomic sequences.

16. The method of claim 15, wherein the plurality of labeled nucleic acid sequences comprise a substantially complete chromosome or a known subset of a chromosome.

17. The method of claim 16, wherein the plurality of labeled nucleic acid sequences comprise a substantially complete genome or a known subset of a genome.

18. The method of claim 1, wherein the nucleic acid segments immobilized on the first and second arrays comprise nucleic acids comprising a substantially complete genome or a known subset of a genome and the labeled nucleic acid segments 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.

19. The method of claim 1, 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.

20. The method of claim 1, 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.

21. The method of claim 20, wherein the abnormal phenotype comprises a disease phenotype.

22. The method of claim 20, wherein the abnormal phenotype comprises a neoplastic or hyperplastic phenotype.

23. The method of claim 22, wherein neoplastic phenotype is selected from the group consisting of breast cancer, skin cancer and bone cancer.

24. The method of claim 1, 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.

25. The method of claim 1, 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.

26. The method of claim 1, 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.

27. The method of claim 1, 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.

28. The method of claim 27, wherein the environmental stress comprises a high or a low or a change in temperature.

29. The method of claim 27, wherein the environmental stress comprises an exposure to a chemical.

30. The method of claim 29, wherein the chemical is a carcinogen.

31. The method of claim 29, wherein the chemical is a drug or a medicine.

32. The method of claim 1, wherein the sample nucleic acid comprises a DNA.

33. The method of claim 1, wherein the sample nucleic acid comprises a cDNA or an RNA.

34. The method of claim 33, wherein the RNA comprises mRNA.

35. The method of claim 1, wherein the sample nucleic acid comprises oligonucleotides.

36. The method of claim 1, wherein the sample nucleic acid comprises expressed sequence tags (EST).

37. The method of claim 7, wherein the nucleic acid segment immobilized on the first and second arrays comprises genomic nucleic acid cloned in a construct comprising an artificial chromosome.

38. The method of claim 37, wherein the artificial chromosome comprises a bacterial artificial chromosome (BAC).

39. The method of claim 37, 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).

40. The method of claim 7, wherein the nucleic acid segment immobilized on the first and second arrays is cloned in a construct comprising a vector selected from the group consisting of a cosmid vector, a plasmid vector and a viral vector.

41. The method of claim 7, wherein the nucleic acid segment immobilized on the first and second arrays is between about 50 kilobases to about 500 kilobases in length, between about 100 kilobases to about 400 kilobases in length, or, is about 300 kilobases in length.

42. The method of claim 1, wherein a labeled nucleic acid is derived from a body fluid sample, a cell sample or a tissue sample.

43. The method of claim 1, wherein a labeled nucleic acid is derived from a cancer cell or a tumor cell sample.

44. The method of claim 1, wherein a labeled nucleic acid is derived from a biopsy sample.

45. The method of claim 1, wherein a labeled nucleic acid is derived from a blood sample.

46. The method of claim 7, further comprising a washing step, wherein sample nucleic acids not specifically hybridized to the nucleic acid segment immobilized on the first and second arrays are removed before the identifying step.

47. The method of claim 46, 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.

48. The method of claim 46, 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.

49. The method of claim 46, 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.

50. The method of claim 1, wherein the first sample nucleic acid and the second sample nucleic acid comprise the same label.

51. The method of claim 1, wherein the first sample nucleic acid and the second sample nucleic acid comprise different labels.

52. The method of claim 1, wherein the sample nucleic acids are labeled with a fluorochrome.

53. The method of claim 52, wherein the label comprises a Cy3™

or a Cy5™

or an equivalent.

54. The method of claim 1, wherein the sample nucleic acids are labeled with a bioluminescent or a chemiluminescent label.

55. The method of claim 1, further comprising a third array comprising a plurality of nucleic acids and at least one calibration spot, wherein each nucleic acids is immobilized to a discrete and known spot on a third substrate surface to form the third array of nucleic acids, and the nucleic acids immobilized on the third array comprise substantially the same plurality of nucleic acids and nucleic acid sequence markers arrayed as in the first and second arrays, and a third sample comprising a plurality of nucleic acids comprising a detectable label, the method further comprising contacting the third sample with the third array under the same conditions, thereby allowing the labeled nucleic acids to specifically bind to a nucleic acid immobilized on the third array;

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

, comparing the amount of labeled nucleic acids bound by specific binding to the same nucleic acid immobilized on the first array, the second array and the third array and normalizing the ratio of the amount of label associated with the nucleic acid in the first, second and third arrays by adjusting the ratio by a figure representing the difference between an expected ratio of label associated with a calibration spot on the first, second and third ax-rays and a detected ratio of label associated with a calibration spot on the first, second and third arrays, thereby determining the relative amount of a nucleic acid in the first, second and third samples.

56. The method of claim 55, 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.

57. The method of claim 55, wherein the first, second and third array are in separated wells of a multi-well plate.

58. The method of claim 57, wherein the multi-well plate is a multi-titer well plate and each well comprises one array.

59. The method of claim 1, further comprising the step of blocking the hybridization capacity of repetitive nucleic acid sequences in the immobilized nucleic acid.

60. The method of claim 1, further comprising 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.

61. The method of claim 60, wherein the sample nucleic acid sequences are first mixed with unlabeled repetitive nucleic acid sequences before the step comprising contacting with the nucleic acid segments immobilized on the first and second arrays.

62. The method of claim 60 or claim 61, wherein the repetitive nucleic acid sequences comprise Cot-1 DNA or equivalent.

63. The method of claim 60 or claim 61, wherein the repetitive nucleic acid sequences comprise SST sequences or salmon sperm DNA or equivalents.

64. An array-based method for performing comparative genomic hybridization, the method comprising:

(a) contacting a first sample and a second sample with a first array and a second array respectively, each 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 wherein each said spot comprises a nucleic acid sequence that is a marker for each said spot on the array providing a plurality of markers on the array and the plurality of genomic nucleic acid segments comprise a substantially complete genome or a known subset of a genome and each array further comprising at least one calibration spot comprising a mixture of the same plurality of nucleic acid sequence markers present in all of the other spots of the, each sample comprising a plurality of genomic nucleic acid segments comprising a detectable label;

wherein the contacting is performed under conditions wherein the labeled nucleic acid segments specifically bind to a nucleic acid segment immobilized on the first array;

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

(c) 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, and comparing an amount of a labeled nucleic acid sequence bound by specific hybridization to the calibration spots on the first array and the calibration spots on the second array;

(d) normalizing the ratio of the amount of label associated with a nucleic acid segment to each spot on the first and second arrays by a figure representing the difference between the amount of label bound to the calibration spot on the first array and the calibration spot on 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.

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

(a) contacting a first sample and at least a second sample with respectively a first array and at least a second array, each array comprising a plurality of immobilized nucleic acid molecules, wherein the nucleic acid molecules are immobilized to discrete and known spots on a substrate surface to form at least two arrays of nucleic acid molecules, wherein each said spot comprises a nucleic acid sequence that is a marker for each said spot on the array providing a plurality of markers on the array and the second array comprises substantially the same plurality of nucleic acid molecules immobilized in the first array, each array further comprising at least one calibration spot comprising a mixture of the same plurality of nucleic acid sequence markers present in all of the other spots of the array wherein at least two samples are labeled, and the two samples comprise the same label or nucleic acid molecules in the first sample comprise a different label than nucleic acid molecules in the second label, wherein the contacting is performed under conditions wherein the labeled sample nucleic acid molecules specifically bind to the immobilized nucleic acid molecules;

(b) detecting an amount of label associated with each spot and comparing the amount of label associated with an immobilized nucleic acid molecule in the first array to the amount of label associated with the same immobilized nucleic acid molecule in the second array and detecting an amount of label associated with the calibration spots on the first array and on the second array; and

(c) normalizing the amount of label bound to each spot on the first and second arrays by adjusting the ratio by a figure representing the difference between an expected ratio of calibration molecules and a detected ratio of calibration molecules on the first and second arrays, thereby determining the one or more variations in copy numbers of immobilized nucleic acid molecule in the first sample relative to the second sample.

66. The method of claim 65, further comprising determining the ratio of the amount of label associated with the immobilized nucleic acid molecule in the first and the second array, thereby determining a ratio of signal intensity.

67. The method of claim 65, further comprising determining the amount of a calibration molecule, wherein a known amount of a calibration molecule is spotted on the calibration spots on each array.

68. The method of claim 67, wherein a known amount of a calibration molecule-binding composition is mixed with the first and the second samples.

69. The method of claim 65, comprising determining the average copy number of a calibration sequence, wherein a known amount of a calibration sequence is spotted on each array, and a known amount of the calibration sequence is mixed with the first and the second samples, and the calibration sequence is derived from a different source compared to that from which the sample nucleic acids were derived.

70. The method of claim 69, wherein the calibration sequence is derived from an organism different from which the sample nucleic acids were derived.

71. The method of claim 67, wherein the calibration molecule is spotted in titrated concentrations on each of the arrays.

72. The method of claim 67, further comprising determining whether the expected ratio of the known amount of calibration molecule is detected on the two arrays.

73. The method of claim 67, further comprising normalizing the ratio of the amount of label associated with a nucleic acid molecule in the first and second arrays by adjusting the ratio by a figure representing the difference between an expected ratio of calibration molecules and a detected ratio of calibration molecules on the two arrays.