Comparative genomic hybridization

• US 6,159,685 A
• Filed: 07/30/1997
• Issued: 12/12/2000
• Est. Priority Date: 01/16/1986
• 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, Frederic, Pinkel, Daniel, Gray, Joe W., Kallioniemi, Anne, Kallioniemi, Olli-Pekka
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/903,095
• Time in Patent Office: 1,231 Days
• Field of Search: 435/4, 435/5, 435/6, 435/91.1, 435/91.2, 435/91.5, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• 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 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

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 determining one or more variations in copy numbers of unique nucleic acid sequences in a first sample relative to copy numbers of substantially identical sequences in a second sample, said method comprising the steps of:

(a) labeling nucleic acids from each sample with a different label;

(b) contacting said labeled nucleic acids from each sample with a plurality of target nucleic acids, wherein either the labeled 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 labeled nucleic acids hybridized to each target nucleic acid to determine one or more variations in copy numbers of sequences in the first sample relative to copy numbers of substantially identical sequences in the second sample.

2. The method of claim 1, wherein said labeled nucleic acids comprise genomic DNA molecules.

3. The method of claim 1, wherein said labeled nucleic acids comprise RNA molecules.

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

5. The method of claim 1, further comprising the step of amplifying the nucleic acids from at least one of said first sample and said second sample prior to said contacting step.

6. The method of claim 1, wherein repetitive sequences in said labeled nucleic acids are blocked;

and unique sequences in the labeled nucleic acids are retained.

7. The method of claim 1, wherein repetitive sequences in said target nucleic acids are blocked;

and unique sequences in the target nucleic acids are retained.

8. The method of claim 1, wherein repetitive sequences in said labeled genomic DNA molecules are removed;

and unique DNA sequences in the labeled genomic DNA molecules are retained.

9. The method of claim 1, wherein repetitive sequences in said target nucleic acids are removed;

and unique sequences in the target nucleic acids are retained.

10. The method according to claim 1, wherein the hybridization is done in situ, the target nucleic acids comprise reference metaphase chromosomes, and the intensities of signals from labeled nucleic acids are compared as a function of position on the metaphase chromosomes.

11. The method of claim 1, wherein copy numbers of unique sequences from more than two samples are compared, wherein the comparing is done pairwise between signals from each set of labeled nucleic acids.

12. The method of claim 11, wherein the labeled nucleic acids are DNA molecules.

13. The method of claim 1, wherein the target nucleic acids comprise a reference genome or portion thereof.

14. The method of claim 1, wherein the nucleic acids are labeled with fluorochromes.

15. The method of claim 1, wherein said comparing step comprises determining the ratio of signal intensity of the labeled nucleic acids from said first and second samples to each target nucleic acid.

16. The method of claim 15, wherein said comparing step additionally comprises comparing the ratio for one target nucleic acid with the ratio for another target nucleic acid.

17. The method of claim 15, additionally comprising:

(d) determining the average copy number of a calibration sequence in said first and second samples, said calibration sequence being substantially identical to a unique sequence in the target nucleic acids; and

(e) normalizing the ratios calculated in claim 15 so that the ratio for the target nucleic acid containing the calibration sequence is equal to the ratio of the average copy numbers determined in (d), the normalized ratio at any other target nucleic acid thereby giving the ratio of the copy numbers of the labeled nucleic acids that hybridize to that target nucleic acid.

18. The method of claim 1, wherein said target nucleic acids comprise nucleic acids from an antenna cell line.

19. The method of claim 1, wherein said labeled nucleic acids comprise cDNA molecules.