Comparative genomic hybridization (CGH)

• US 6,335,167 B1
• Filed: 05/14/1999
• Issued: 01/01/2002
• Est. Priority Date: 03/04/1992
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Waldman, Frederic, Kallioniemi, Ollie-Pekka, Kallioniemi, Anne, Sakamoto, Masaru, Pinkel, Daniel, Gray, Joe W.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/311,835
• Time in Patent Office: 963 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G06F 15/025 adapted to a specific application
• CPC Class Codes: Y10S 436/813 Cancer

Abstract

Disclosed are new methods comprising the use of in situ hybridization to detect abnormal nucleic acid sequence copy numbers in one or more genomes wherein repetitive sequences that bind to multiple loci in a reference chromosome spread are either substantially removed and/or their hybridization signals suppressed. The invention termed Comparative Genomic Hybridization (CGH) provides for methods of determining the relative number of copies of nucleic acid sequences in one or more subject genomes or portions thereof (for example, a tumor cell) as a function of the location of those sequences in a reference genome (for example, a normal human genome). The intensity(ies) of the signals from each labeled subject nucleic acid and/or the differences in the ratios between different signals from the labeled subject nucleic acid sequences are compared to determine the relative copy numbers of the nucleic acid sequences in the one or more subject genomes as a function of position along the reference chromosome spread. Amplifications, duplications and/or deletions in the subject genome(s) can be detected. Also provided is a method of determining the absolute copy numbers of substantially all RNA or DNA sequences in subject cell(s) or cell population(s).

Claims

1. A method of detecting a chromosomal abnormality in a suspected bladder cancer test sample by detecting an amplification of a unique sequence at at least one position selected from the group consisting of q21 on human chromosome 8, q31-qter on human chromosome 13, p15-pter on human chromosome 7, q24-qter on human chromosome 8, cen-p13 on human chromosome 11 and q13-qter on human chromosome 9, in the test sample, said method comprising the steps of:

(a) labelling nucleic acids from the test sample and from a control sample with different labels;

(b) contacting said labelled nucleic acids from each sample with a plurality of target nucleic acids, wherein either the labelled nucleic acids or the target nucleic acids, or both, have had repetitive sequences, if initially present, blocked and/or removed; and

(c) comparing the intensities of the signals from labelled nucleic acids hybridized to each target nucleic acid, thereby allowing detection of the presence or absence of the amplification in the test sample.

2. The method of claim 1, wherein the step of comparing the intensities of the signals from the labelled nucleic acids comprises determining the ratio of the intensities of the signals as a function of position in the target nucleic acids.

3. The method of claim 1, wherein the amplification is detected at position q21 of human chromosome 8.

4. The method of claim 1, wherein the amplification is detected at position q31-qter of human chromosome 13.

5. The method of claim 1, wherein the amplification is detected at position p15-pter on human chromosome 7.

6. The method of claim 1, wherein the amplification is detected at position q24-qter on human chromosome 8.

7. The method of claim 1, wherein the amplification is detected at position cen-p13 on human chromosome 11.

8. The method of claim 1, wherein the amplification is detected at about position q13-qter on human chromosome 9.

9. The method of claim 1, wherein the target nucleic acids comprise at least one metaphase chromosome.

30. The method of claim 1 wherein said nucleic acid sample comprises genomic DNA molecules.

31. The method of claim 1 wherein said nucleic acid sample comprises DNA amplified from said suspected bladder cancer sample.

32. The method of claim 1 wherein said nucleic acid sample comprises complementary DNA.

10. A method of detecting a chromosomal abnormality in a suspected bladder cancer test sample by detecting a deletion of a unique sequence at at least one position selected from the group consisting of q34 on human chromosome 9, p22-pter on human chromosome 8, the p arm of human chromosome 17, p21 on human chromosome 3, the p arm of human chromosome 9, q26 on human chromosome 10, q23-qter on human chromosome 12, p 12-pter on human chromosome 16, the p arm of human chromosome 3, p16 on human chromosome 4, the p arm of human chromosome 5, q21-q22 on human chromosome 6, q11-q13 on human chromosome 15, p15 on human chromosome 11, q23-qter on human chromosome 11, q11-qter on human chromosome 15 and p13.1-pter on human chromosome 19, in the test sample, said method comprising the steps of:

(a) labelling nucleic acids from the test sample and from a control sample with different labels;

(b) contacting said labelled nucleic acids from each sample with a plurality of target nucleic acids, wherein either the labelled nucleic acids or the target nucleic acids, or both, have had repetitive sequences, if initially present, blocked and/or removed; and

(c) comparing the intensities of the signals from labelled nucleic acids hybridized to each target nucleic acid, thereby allowing detection of the presence or absence of the deletion in the test sample.

11. The method of claim 10 wherein the step of comparing the intensities of the signals from the labelled nucleic acids comprises determining the ratio of the intensities of the signals as a function of position in the target nucleic acids.

12. The method of claim 10, wherein the deletion is detected at position q34 of human chromosome 9.

13. The method of claim 10, wherein the deletion is detected at position p22-pter of human chromosome 8.

14. The method of claim 10, wherein the deletion is detected on the p arm of human chromosome 17.

15. The method of claim 10, wherein the deletion is detected at position p21 on human chromosome 3.

16. The method of claim 10, wherein the deletion is detected on the p arm of human chromosome 9.

17. The method of claim 10, wherein the deletion is detected at position q26 on human chromosome 10.

18. The method of claim 10, wherein the deletion is detected at position q23-qter of human chromosome 12.

19. The method of claim 10, wherein the deletion is detected at position p12-pter of human chromosome 16.

20. The method of claim 10, wherein the deletion is detected on the p arm of human chromosome 3.

21. The method of claim 10, wherein the deletion is detected at position p16 on human chromosome 4.

22. The method of claim 10, wherein the deletion is detected on the p arm of human chromosome 5.

23. The method of claim 10, wherein the deletion is detected at position q21-q22 on human chromosome 6.

24. The method of claim 10, wherein the deletion is detected at position q11-q13 on human chromosome 15.

25. The method of claim 10, wherein the deletion is detected at position p15 on human chromosome 11.

26. The method of claim 10, wherein the deletion is detected at position q23-qter on human chromosome 11.

27. The method of claim 10, wherein the deletion is detected at position q11-qter on human chromosome 15.

28. The method of claim 10, wherein the deletion is detected at position p13.1-pter on human chromosome 19.

29. The method of claim 10, wherein the target nucleic acids comprise at least one metaphase chromosome.

33. The method of claim 10 wherein said nucleic acid sample comprises genomic DNA molecules.

34. The method of claim 10 wherein said nucleic acid sample comprises DNA amplified from said suspected bladder cancer sample.

35. The method of claim 10 wherein said nucleic acid sample comprises complementary DNA.

36. A method for detecting a copy number variation in a suspected bladder cancer sample by detecting an amplification of a unique sequence at q21 on human chromosome 8, said method comprising:

contacting a probe that binds selectively to a target polynucleotide sequence of q21 on human chromosome 8 with a nucleic acid sample prepared, directly or indirectly, from said suspected bladder cancer sample, wherein said nucleic acid sample comprises said target polynucleotide sequence and said probe is contacted with said sample under conditions in which said probe forms a stable hybridization complex with said target nucleic acid sequence; and

detecting said hybridization complex.

37. The method of claim 36 wherein said probe is labeled.

38. The method of claim 36 wherein said nucleic acid sample is labeled.

39. The method of claim 36 wherein said nucleic acid sample comprises genomic DNA molecules.

40. The method of claim 36 wherein said nucleic acid sample comprises DNA amplified from said suspected bladder cancer sample.

41. The method of claim 36 wherein said nucleic acid sample comprises complementary DNA.

42. A method for detecting a copy number variation in a suspected bladder cancer sample by detecting a deletion of a unique sequence at at least one position selected from the group consisting of q23-qter on human chromosome 12, p12-pter on human chromosome 16, p16 on human chromosome 4, q11-q13 on human chromosome 15, q11-qter on human chromosome 15, and p13.1-pter on human chromosome 19, said method comprising:

contacting a probe that binds selectively to a target polynucleotide sequence of at least one position selected from the group consisting of q23-qter on human chromosome 12, p12-pter on human chromosome 16, p16 on human chromosome 4, q11-q13 on human chromosome 15, q11-qter on human chromosome 15, and p13.1-pter on human chromosome 19 with a nucleic acid sample prepared, directly or indirectly, from said suspected bladder cancer sample, wherein said nucleic acid sample comprises said target polynucleotide sequence and said probe is contacted with said sample under conditions in which said probe forms a stable hybridization complex with said target nucleic acid sequence; and

detecting said hybridization complex.

43. The method of claim 42 wherein said probe is labeled.

44. The method of claim 42 wherein said nucleic acid sample is labeled.

45. The method of claim 42 wherein said nucleic acid sample comprises genomic DNA molecules.

46. The method of claim 42 wherein said nucleic acid sample comprises DNA amplified from said suspected bladder cancer sample.

47. The method of claim 42 wherein said nucleic acid sample comprises complementary DNA.

48. The method of claim 42 wherein the deletion is detected at position q23-qter on human chromosome 12.

49. The method of claim 42 wherein the deletion is detected at position p12-pter on human chromosome 16.

50. The method of claim 42 wherein the deletion is detected at position p16 on human chromosome 4.

51. The method of claim 42 wherein the deletion is detected at position q11-q13 on human chromosome 15.

52. The method of claim 42 wherein the deletion is detected at position q11-qter on human chromosome 15.

53. The method of claim 42 wherein the deletion is detected at position p13.1-pter on human chromosome 19.