Comparative genomic hybridization (CGH)

• US 5,856,097 A
• Filed: 11/27/1995
• Issued: 01/05/1999
• 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: Sakamoto, Masaru, Waldman, Frederic, Pinkel, Daniel, Gray, Joe W., Kallioniemi, Anne, Kallioniemi, Olli-Pekka
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/562,898
• Time in Patent Office: 1,135 Days
• Field of Search: 435/5, 435/6, 435/91.2, 536/24.3, 536/24.32, 536/24.33, 536/23.1, 536/24.31
• 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 an amplification of unique sequences of at least one position selected from the group consisting of about q21 on human chromosome 8, about q31-qter on human chromosome 13, about p15-pter on human chromosome 7, about q24-qter on human chromosome 8, about q13-qter on human chromosome 9 and about q21-23 on human chromosome 6, 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) DNA unique 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 about position q26 of human chromosome 3 in the test genome.

4. The method of claim 1, wherein the amplification is detected at about position q24 of human chromosome 8 in the test genome.

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

21. The method of claim 1, wherein the probe is labeled with a direct label or an affinity label.

22. The method of claim 1, wherein the probe is labeled with digoxigenin or biotin.

6. A method of detecting a deletion of unique sequences of at least one position selected from the group consisting of the p arm of human chromosome 3, the q arm of human chromosome 16, about position q11.2-q21 of human chromosome 17, about position p12-p13 of human chromosome 17, about position p35-p36.1 of human chromosome 1, about position p21 of human chromosome X, about position q14-q21.1 of human chromosome 13, about position p21-p22 of human chromosome 8, and about position q13-q23 of human chromosome 16, in a genome being tested, said method comprising the steps of:

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

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

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

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

(c) comparing the intensities of the signals from the labeled 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.

7. The method of claim 6, 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.

8. The method of claim 6, wherein the deletion is the p arm of human chromosome 3.

9. The method of claim 6, wherein the deletion is the q arm of human chromosome 16.

10. The method of claim 6, wherein the reference genome comprises at least one metaphase chromosome.

11. The method of claim 6, wherein the deletion is at about position q11.2-q21 of human chromosome 17.

12. The method of claim 6, wherein the deletion is at about position p12-p13 of human chromosome 17.

13. The method of claim 6, wherein the amplification is at about position p35-p36.1 of human chromosome 1.

14. The method of claim 6, wherein the amplification is at about position p21 of human chromosome X.

15. The method of claim 6, wherein the amplification is at about position q14-q21.1 of human chromosome 13.

16. The method of claim 6, wherein the amplification is at about position p21-p22 of human chromosome 8.

17. The method of claim 6, wherein the amplification is at about position q13-q23 of human chromosome 16.

18. The method of claim 6, wherein the reference genome comprises at least one metaphase chromosome.

19. The method of claim 6, wherein the probe is labeled with a direct label or an affinity label.

20. The method of claim 6, wherein the probe is labeled with digoxigenin or biotin.

23. A method for detecting ovarian cancer in a patient comprising detecting a deletion of unique sequences of at least one position selected from the group consisting of about position q26 of human chromosome 3, about position q24 of human chromosome 8, about position q11.2-q21 of human chromosome 17, about position p12-p13 of human chromosome 17, about position p35-p36.1 of human chromosome 1, about position p21 of human chromosome X, about position q14-q21.1 of human chromosome 13, about position p21-p22 of human chromosome 8, and about position q13-q23 of human chromosome 16, in a genome being tested, said method comprising the steps of:

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

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

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

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

(c) comparing the intensities of the signals from the labeled 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.

24. A method for detecting ovarian cancer in a patient comprising detecting an amplification of unique sequences of at least one position selected from the group consisting of the p arm of human chromosome 3 and the q arm of human chromosome 16, in a genome being tested, said method comprising the steps of:

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

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

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

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

(c) comparing the intensities of the signals from the labeled 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.