Multiparametric fluorescence in situ hybridization

• US 6,548,259 B2
• Filed: 11/20/2001
• Issued: 04/15/2003
• Est. Priority Date: 12/22/1995
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Wilson, John T., Speicher, Michael, Ward, David C., Ballard, Stephen Gwyn
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/988,584
• Publication Number:
  US 20030027159A1
• Time in Patent Office: 511 Days
• Field of Search: 382/129, 382/133, 382/162, 382/165, 435/6, 435/91, 435/94, 435/504, 436/813, 536/27, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation
• 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 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]

Abstract

The invention relates to a set of combinatorially labeled oligonucleotide probes each member thereof: (i) having a predetermined label distinguishable from the label of any other member of said set, and (ii) being capable of specifically hybridizing with a predetermined chromosome or nucleic acid molecule, and to the use of such molecules, alone or in concert with nucleic acid amplification methods.

Claims

1. A method of achieving simultaneous visualization of at least 24 different genetic targets by multiparametric fluorescence in situ hybridization comprising:

(A) contacting a preparation of chromosomes that has been previously treated to render it in single-stranded form with a set of combinatorially and distinguishably labeled probes, wherein said set of combinatorially and distinguishably labeled probes are combinatorially linked or coupled to 5 fluorophores;

(B) hybridizing said set of combinatorially and distinguishably labeled probes with said preparation of chromosomes; and

(C) simultaneously visualizing said at least 24 different genetic targets.

2. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes are oligonucleotide probes.

3. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes are microdissected probes.

4. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes are probes for a sub-region of a target chromosome.

5. The method of claim 1, wherein said set of combinatorially and distinguishably labeled oligonucleotide probes is capable of detecting a telomere.

6. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes is capable of detecting a centromere.

7. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes is DNA probes.

8. The method of claim 1, wherein at least one member of the set is combinatorially labeled with a fluorophore selected from the group consisting of the fluorophores DAPI, FITC, Cy3, Cy3.5, Cy5, and Cy7.

9. The method of claim 8, wherein at least two members of the set are combinatorially labeled with a fluorophore selected from the group consisting of the fluorophores DAPI, FITC, Cy3, Cy3.5, Cy5, and Cy7.

10. The method of claim 9, wherein at least three members of the set are combinatorially labeled with a fluorophore selected from the group consisting of the fluorophores DAPI, FITC, Cy3, Cy3.5, Cy5, and Cy7.

11. The method of claim 10, wherein at least four members of the set are combinatorially labeled with a fluorophore selected from the group consisting of the fluorophores DAPI, FITC, Cy3, Cy3.5, Cy5, and Cy7.

12. The method of claim 8, wherein the fluorophore is conjugated to a ligand capable of binding to a modified nucleotide residue.

13. The method of claim 12, wherein said ligand is selected from the group consisting of avidin, streptavidin, biotin-binding antibodies and digoxigenin-binding antibodies.