Comparative Genomic Hybridization (CGH)

• US 5,976,790 A
• Filed: 11/27/1995
• Issued: 11/02/1999
• Est. Priority Date: 03/04/1992
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Waldman, Frederick, Sakamoto, Masaru, Pinkel, Daniel, Gray, Joe W., Kallioniemi, Anne, Kallioniemi, Olli-Pekka
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/565,304
• Time in Patent Office: 1,436 Days
• Field of Search: 435/5, 435/6, 435/71.2, 536/22.1, 536/23.1, 536/24.3, 536/24.31, 536/24.33, 536/24.1
• 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 sample by detecting an amplification of unique sequences of 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 a genome being tested, said method comprising the steps of:

(a) differently labelling DNA sequences from the test genome and a normnal human genome;

(b) hybridizing said labelled DNA sequences from each of said genomes to a reference genome under the following conditions;

(i) either the labelled DNA sequences or the reference genome, or both, have their repetitive sequences blocked and/or removed; and

(ii) unique DNA sequences in the reference genome are retained; and

(c) comparing the intensities of the signals from the labelled DNA sequences as a function of position on the reference genome, thereby allowing detection of the presence or absence of the amplification in the test genome.

2. The method of claim 1, wherein the step of comparing the intensities of the signals from the labelled DNA sequences comprises determining the ratio of the intensities of the signals as a function of position in the reference genome.

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

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

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

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

7. The method of claim 1, wherein the amplification is detected at position q13-qter on human chromosome 9 in the test genome.

8. The method of claim 1, wherein the reference genome comprises at least one metaphase chromosome.

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

9. A method of detecting a chromosomal abnormality in a suspected bladder cancer sample by detecting a deletion of a unique sequence of 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, p12-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, q21-q23 on human chromosome 6, 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 a genome being tested, said method comprising the steps of:

(a) differently labelling DNA sequences from the test, genome and a normal human genome;

(b) hybridizing said labelled DNA sequences from each of said genomes to a reference genome under the following conditions;

(i) either the labelled DNA sequences or the reference genome, or both, have their repetitive sequences blocked and/or removed; and

(ii) unique DNA sequences in the reference genome are retained; and

(c) comparing the intensities of the signals from the labelled DNA sequences as a function of position on the reference genome, thereby allowing detection of the presence or absence of the deletion in the test genome.

10. The method of claim 9, wherein the step of comparing the intensities of the signals from the labelled DNA sequences comprises determining the ratio of the intensities of the signals as a function of position in the reference genome.

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

12. The method of claim 9, wherein the deletion is detected at position p22-pter of human chromosome 8.

13. The method of claim 9, wherein the deletion is detected on the p arm of human chromosome 17.

14. The method of claim 9, wherein the deletion is detected at position p21 on human chromosome 3.

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

16. The method of claim 9, wherein the deletion is detected at position q26 on human chromosome 10.

17. The method of claim 9, wherein the deletion is detected at position q23-qter of human chromosome 12.

18. The method of claim 9, wherein the deletion is detected at position p12-pter of human chromosome 16.

19. The method of claim 9, wherein the deletion is detected on the p arm of human chromosome 3.

20. The method of claim 9, wherein the deletion is detected at position p16 on human chromosome 4.

21. The method of claim 9, wherein the deletion is detected on the p arm of human chromosome 5.

22. The method of claim 9, wherein the deletion is detected at position q21-q22 on human chromosome 6.

23. The method of claim 9, wherein the deletion is detected at position q11-q13 on human chromosome 15.

24. The method of claim 9, wherein the deletion is detected at position p13.1-qter on human chromosome 19.

25. The method of claim 9, wherein the reference genome comprises at least one metaphase chromosome.

37. The method of claim 9, wherein the deletion is detected at position q21-q23 on human chromosome 6.

38. The method of claim 9 wherein the deletion is detected at position p15 on human chromosome 11.

39. The method of claim 9 wherein the deletion is detected at position q23-qter on human chromosome 11.

40. The method of claim 9 wherein the deletion is detected at position q11-qter on human chromosome 15.

26. A method for detecting a chromosomal abnormality in a suspected bladder cancer sample by detecting an amplification of unique sequences of at least one position selected from the group consisting of q21 on human chromosome 8, and q31-qter on human chromosome 13, said method comprising the steps of:

contacting a chromosome sample from a patient with a composition consisting essentially of one or more labeled nucleic acid probes each of which binds selectively to a target polynucleotide sequence of at least one position selected from the group consisting of q21 on human chromosome 8, and q31-qter on human chromosome 13, under conditions in which the probe forms a stable hybridization complex with the target sequence; and

detecting the hybridization complex, thereby detecting the presence or absence of the amplification.

27. The method of claim 26, wherein the amplification is detected at position q21 of human chromosome 8.

28. The method of claim 26, wherein the amplification is detected at position q31-qter of human chromosome 13.

42. The method of claim 28 wherein the deletion is detected at position q11-qter on human chromosome 15.

29. A method of detecting a chromosomal abnormality in a suspected bladder cancer sample by detecting a deletion of a unique 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, q23-qter on human chromosome 11, q11-qter on human chromosome 15 and p13.1-pter on human chromosome 19, said method comprising the steps of:

contacting a chromosome sample from a patient with a composition consisting essentially of one or more labeled nucleic acid probes each of which 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, q23-qter on human chromosome 11, q11-qter on human chromosome 15 and p13.1-pter on human chromosome 19, under conditions in which the probe forms a stable hybridization complex with the target sequence; and

detecting the hybridization complex, thereby detecting the presence or absence of the deletion.

30. The method of claim 29, wherein the deletion is detected at position q23-qter of human chromosome 12.

31. The method of claim 29, wherein the deletion is detected at position p12-pter of human chromosome 16.

32. The method of claim 29, wherein the deletion is detected at position p16 on human chromosome 4.

33. The method of claim 29, wherein the deletion is detected at position q11-q13 on human chromosome 15.

34. The method of claim 29, wherein the deletion is detected at position p13.1-pter on human chromosome 19.

41. The method of claim 29 wherein the deletion is detected at position q23-qter on human chromosome 11.

35. A method for detecting bladder cancer in a patient comprising detecting an amplification of unique sequences of at least one position selected from the group consisting of q21 on human chromosome 8, and q31-qter on human chromosome 13, or a deletion of a unique 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, q23-qter on human chromosome 11, q11-ter on human chromosome 15 and p13.1-pter on human chromosome 19, said method comprising the steps of:

contacting a chromosome sample from a patient with a composition consisting essentially of one or more labeled nucleic acid probes each of which binds selectively to a target polynucleotide sequence of at least one position selected from the group consisting of q21 on human chromosome 8, and q31-qter on human chromosome 13, or a deletion of a unique 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, q23-qter on human chromosome 11, q11-qter on human chromosome 15 and p13.1-pter on human chromosome 19, under conditions in which the probe forms a stable hybridization complex with the target sequence; and

detecting the hybridization complex, thereby detecting the presence or absence of the amplification or deletion.