Comparative fluorescence hybridization to nucleic acid arrays

• US 20050084898A1
• Filed: 12/03/2004
• Published: 04/21/2005
• Est. Priority Date: 12/09/1994
• Status: Abandoned Application

• Current Assignee: Medical Research Council (Government of United Kingdom), Regents of the University of California (University of California)
• Original Assignee: Medical Research Council (Government of United Kingdom), Regents of the University of California (University of California)
• Inventors: Pinkel, Daniel, Gray, Joe W., Albertson, Donna
• Application Number:
  US11/002,789
• Publication Number:
  US 20050084898A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• 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: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

The present invention provides methods of determining relative copy number of target nucleic acids and precise mapping of chromosomal abnormalities associated with disease. The methods of the invention use target nucleic acids immobilized on a solid surface, to which a sample comprising two sets of differentially labeled nucleic acids are hybridized. The hybridization of the labeled nucleic acids to the solid surface is then detected using standard techniques.

Claims

1. A method for comparing copy number of nucleic acid sequences in two or more collections of nucleic acid molecules, the method comprising:

(a) providing an array comprising a plurality of target elements bound to a solid surface, each target element comprising a target nucleic acid, which target nucleic acid is not in a cell nucleus and lacks superstructure of a condensed chromosome;

(b) contacting the target elements with;

(i) a first collection of labeled nucleic acid molecules comprising a plurality of first nucleic acid sequences each of which specifically hybridizes to a different target nucleic acid under pre-selected hybridization conditions, and

(ii) a second collection of labeled nucleic acid molecules comprising a plurality of second nucleic acid sequences each of which specifically hybridizes to a different target nucleic acid under pre-selected hybridization conditions;

wherein the first and second labels are distinguishable from each other and at least one of the first and at least one of the second nucleic acid sequences hybridize to the same target nucleic acid;

(c) detecting the amount of binding of the first and second labeled complementary nucleic acids to the target nucleic acids; and

(d) comparing the amount of binding of the first and second labeled complementary nucleic acids to the target nucleic acids to compare copy number of nucleic acid sequences in the first and second collections that are bound to the target nucleic acids.

2. The method of claim 1, wherein the target nucleic acids are between about 1,000 and about 1,000,000 nucleotides in complexity.

3. The method of claim 2, wherein the target nucleic acids are of a size suitable for cloning in a cosmid.

4. The method of claim 2, wherein the target nucleic acids are of a size suitable for cloning in a P1 phage.

5. The method of claim 1, wherein the first and second collections of labeled nucleic acids are contacted with the target elements simultaneously.

6. The method of claim 1, wherein the first and second collections of nucleic acids are treated to inhibit the binding of repetitive sequences.

7. The method of claim 6, wherein the first and second collections of nucleic acids are mixed with unlabeled blocking nucleic acids comprising repetitive sequences.

8. The method of claim 7, wherein the unlabeled blocking nucleic acids are Cot-1 DNA.

9. The method of claim 1, wherein the first and second collections of labeled nucleic acids are fluorescently labeled.

10. The method of claim 1, wherein the target nucleic acids are human DNA.

11. The method of claim 10, wherein the DNA is cDNA.

12. The method of claim 10, wherein the DNA is genomic DNA.

13. The method of claim 1, wherein the target nucleic acids are RNA.

14. The method of claim 1, wherein the first or second collection of labeled nucleic acids is DNA.

15. The method of claim 14, wherein the DNA is genomic DNA.

16. The method of claim 14, wherein the DNA is human DNA.

17. The method of claim 14, where in the first collection of labeled nucleic acids are from a human test genome and the second collection of labeled nucleic acids are from a normal human reference genome.

18. The method of claim 14, wherein the DNA is cDNA.

19. The method of claim 1, wherein the first or second collection of labeled nucleic acids is RNA.

20. The method of claim 1, wherein the target nucleic acids include a chromosomal region which is present in increased copy number in human cancer cells.

21. The method of claim 1, wherein the target nucleic acids include a known human oncogene.

22. The method of claim 1, wherein the target nucleic acids include a chromosomal region which is present in decreased copy number in human cancer cells.

23. The method of claim 1, wherein the array comprises between about 300 and about 30,000 elements.

25. The method of claim 1, where in the first collection of fluorescently labeled nucleic acids are from a human test genome and the second collection of fluorescently labeled nucleic acids are from a normal human reference genome.

24. A method of comparing copy number of nucleic acid sequences in two or more collections of nucleic acid molecules, the method comprising:

(a) providing an array comprising a plurality of target elements bound to a solid surface, each target element comprising a target nucleic acid, which target nucleic acid is not in a cell nucleus and lacks superstructure of a condensed chromosome;

(b) contacting the target elements with;

(i) a first collection of fluorescently labeled nucleic acid molecules comprising a plurality of first nucleic acid sequences each of which specifically hybridizes to a different target nucleic acid under pre-selected hybridization conditions, and

(ii) at least a second collection of fluorescently labeled nucleic acid molecules comprising a plurality of second nucleic acid sequences each of which specifically hybridizes to a different target nucleic acid under pre-selected hybridization conditions;

wherein the first and second labels are distinguishable from each other and at least one of the first and at least one of the second nucleic acid sequences hybridize to the same target nucleic acid;

(c) detecting the amount of binding of the first and second labeled complementary nucleic acids to the target nucleic acids; and

(d) comparing the amount of binding of the first and second labeled complementary nucleic acids to the target nucleic acids to compare copy number of nucleic acid sequences in the first and second collections that are bound to the target nucleic acids.

26. The method of claim 24, wherein the target nucleic acids include a chromosomal region which is present in increased copy number in cancer cells.

27. The method of claim 24, wherein the target nucleic acids include a known oncogene.

28. The method of claim 24, wherein the target nucleic acids include a chromosomal region which is present in decreased copy number in cancer cells.

29. A method for comparing the relative copy numbers of nucleic acid sequences in a plurality of collections of nucleic acid molecules, the method comprising:

(a) providing an array comprising a plurality of target elements bound to a solid surface, each target element comprising a target nucleic acid, which target nucleic acid is not in a cell nucleus and lacks superstructure of a condensed chromosome;

(b) contacting the target elements with;

(i) a first collection of labeled nucleic acid molecules comprising a plurality of first nucleic acid sequences each of which specifically hybridizes to a different target nucleic acid under pre-selected hybridization conditions, and

(ii) a second collection of labeled nucleic acid molecules comprising a plurality of second nucleic acid sequences each of which specifically hybridizes to a different target nucleic acid under pre-selected hybridization conditions;

wherein the first and second labels are distinguishable from each other, and wherein a plurality of the first and a plurality of the second nucleic acid sequences hybridize to the same target nucleic acids;

(c) detecting the first and second labels bound to each of a plurality of said target elements to determine, for each of such target elements, the binding ratio of the first- and second nucleic acid sequences bound thereto; and

(d) comparing a plurality of said binding ratios with one another to determine the relative copy numbers of nucleic acid sequences in said first and second collections that are bound to such target elements.

30. A process comprising the steps of:

(a) providing an array comprising a plurality of target elements bound to a solid surface, each target element comprising a target nucleic acid, which target nucleic acid is not in a cell nucleus and lacks superstructure of a condensed chromosome;

(b) contacting the target elements with a first collection of nucleic acid molecules comprising a plurality of first nucleic acid sequences each of which specifically hybridizes to a different target nucleic acid under pre-selected hybridization conditions, wherein said first collection of nucleic acid molecules is labeled with a detectable label;

(c) detecting the amount of binding of the first nucleic acid molecules to the target nucleic acids;

(d) storing the data obtained in step (c) in a computer; and

(e) comparing the amount of binding detected in step (c) to the amount of binding of at least a second collection of nucleic acid molecules to the target nucleic acids.

31. The process of claim 30, wherein a computer program is used to analyze the signals produced by the array.

32. The process of claim 31, wherein the computer program is a digital image analysis system.

33. The process of claim 32, wherein the digital image analysis system is a quantitative image processing system.

34. The process of claim 30, wherein the target nucleic acids are between about 1,000 and about 1,000,000 nucleotides in complexity.

35. The process of claim 34, wherein the target nucleic acids are of a size suitable for cloning in a cosmid.

36. The process of claim 34, wherein the target nucleic acids are of a size suitable for cloning in a P1 phage.

37. The process of claim 30, wherein the first collection of nucleic acids is treated to inhibit the binding of repetitive sequences.

38. The process of claim 37, wherein the first collection of nucleic acids is mixed with unlabeled blocking nucleic acids comprising repetitive sequences.

39. The process of claim 38, wherein the unlabeled blocking nucleic acids are Cot-1 DNA.

40. The process of claim 30, wherein the detectable label is a fluorescent label.

41. The process of claim 30, wherein the target nucleic acids are human DNA.

42. The process of claim 41, wherein the DNA is cDNA.

43. The process of claim 41, wherein the DNA is genomic DNA.

44. The process of claim 30, wherein the target nucleic acids are RNA.

45. The process of claim 30, wherein the first or second collection of nucleic acids is DNA.

46. The process of claim 45, wherein the DNA is genomic DNA.

47. The process of claim 45, wherein the DNA is human DNA.

48. The process of claim 45, where in the first collection of nucleic acids are from a human test genome and the second collection of nucleic acids is from a normal human reference genome.

49. The process of claim 45, wherein the DNA is cDNA.

50. The process of claim 30, wherein the first or second collection of nucleic acids is RNA.

51. The process of claim 30, wherein the target nucleic acids include a chromosomal region which is present in increased copy number in human cancer cells.

52. The process of claim 30, wherein the target nucleic acids include a known human oncogene.

53. The process of claim 30, wherein the target nucleic acids include a chromosomal region which is present in decreased copy number in human cancer cells.

54. The process of claim 30, wherein the array comprises between about 300 and about 30,000 elements.