Multiparametric fluorescence in situ hybridization

• US 5,759,781 A
• Filed: 05/01/1996
• Issued: 06/02/1998
• Est. Priority Date: 12/22/1995
• Status: Expired due to Term

• Current Assignee: Qiagen GmbH (Qiagen NV), Yale University
• Original Assignee: Yale University
• Inventors: Speicher, Michael, Ballard, Stephen Gwynn, Ward, David C.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Brusca, John S.
• Application Number:
  US08/640,657
• Time in Patent Office: 762 Days
• Field of Search: 435/6, 536/24.31, 536/25.3, 536/25.32
• 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

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 one predetermined autosomal or sex chromosome of a human karyotype, and to the use of such molecules to identify the individual autosomal and sex chromosomes of a human karyotype.

Claims

1. A set of combinatorially labeled oligonucleotide probes, each member thereof comprising a plurality of an oligonucleotide, said oligonucleotide:

(i) being linked or coupled to a predetermined label distinguishable from the label of any other member of said set, and (ii) being capable of specifically hybridizing with one predetermined autosomal or sex chromosome of a human karyotype;

said set having sufficient members to be capable of specifically hybridizing each autosomal or sex chromosome of said human karyotype to at least one member.

2. The set of combinatorially labeled oligonucleotide probes of claim 1, wherein said set contains at least one member probe specific for a sub-chromosomal fragment of an autosomal or sex chromosome of said human karyotype.

3. The set of combinatorially labeled oligonucleotide probes of claim 1, wherein said probes contain nucleotide residues that are labeled with a biotin moiety.

4. The set of combinatorially labeled oligonucleotide probes of claim 3, wherein said probes are additionally labeled with a labeled biotin-binding ligand.

5. The set of combinatorially labeled oligonucleotide probes of claim 4, wherein at least one of said probes is labeled with a biotin-binding ligand that comprises one or more fluorophores.

6. The set of combinatorially labeled oligonucleotide probes of claim 5, wherein at least one of said fluorophores is selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.

7. The set of combinatorially labeled oligonucleotide probes of claim 5, wherein at least one of said probes is labeled with a biotin-binding ligand that comprises more than one fluorophore.

8. The set of combinatorially labeled oligonucleotide probes of claim 7, wherein one of the fluorophores of said biotin-binding ligand that comprises more than one fluorophore is selected from the group consisting of fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.

9. The set of combinatorially labeled oligonucleotide probes of claim 8, wherein all of the fluorophores of said biotin-binding ligand that comprises more than one fluorophores are selected from the group consisting of fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.

10. The set of combinatorially labeled oligonucleotide probes of claim 5, wherein at least one member of said set is labeled with more than one fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy 3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled with at least one fluorophore selected from said fluorophore group.

11. The set of combinatorially labeled oligonucleotide probes of claim 10, wherein at least one member of said set is labeled with two fluorophores, each fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled with at least one fluorophore selected from said fluorophore group.

12. The set of combinatorially labeled oligonucleotide probes of claim 10, wherein at least one member of said set is labeled with three fluorophores, each fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled with at least one fluorophore selected from said fluorophore group.

13. A method of simultaneously identifying and distinguishing the individual autosomal and sex chromosomes of a human karyotype which comprises the steps:

(a) contacting a preparation of said chromosomes, in single-stranded form, under conditions sufficient to permit nucleic acid oligonucleotide probes, each member thereof comprising a plurality of an oligonucleotide, said oligonucleotide;

(i) being linked or coupled to a predetermined label distinguishable from the label of any other member of said set, and (ii) being capable of specifically hybridizing with one predetermined autosomal or sex chromosome of a human karyotype;

said set having sufficient members to be capable of specifically hybridizing each autosomal or sex chromosome of said human karyotype to at least one member;

wherein said contacting thereby causes at least one of each autosomal or sex chromosome of said preparation to become hybridized to at least one member of said set of probes;

(b) for each chromosome of said preparation hybridized to a member of said set probes, detecting and identifying the predetermined label of said hybridized probe member and correlating the identity of the label of said hybridized probe member with the identity of the autosomal or sex chromosome of said human karyotype with which said hybridized probe member specifically hybridizes, to thereby identify the chromosome hybridized to said member; and

(c) repeating step (b) until each autosomal and sex chromosome of said human karyotype has been identified in said preparation.

14. The method of claim 13, wherein said set of oligonucleotide probes contains at least one member probe specific for a sub-chromosomal fragment of an autosomal or sex chromosome of said human karyotype.

15. The method of claim 13, wherein said probes contain nucleotide residues that are labeled with a biotin moiety.

16. The method of claim 15, wherein said probes are additionally labeled with a labeled biotin-binding ligand.

17. The method of claim 16, wherein at least one of said probes is labeled with a biotin-binding ligand that comprises one or more fluorophores.

18. The method of claim 17, wherein at least one of said fluorophores is selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.

19. The method of claim 17, wherein at least one of said probes is labeled with a biotin-binding ligand that comprises more than one fluorophore.

20. The method of claim 19, wherein one of the fluorophores of said biotin-binding ligand that comprises more than one fluorophore is selected from the group consisting of fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7.

21. The method of claim 20, wherein all of the fluorophores of said biotin-binding ligand that comprises more than one fluorophore are selected from the group consisting of fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7.

22. The method of claim 17, wherein at least one member of said set is labeled with more than one fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled with at least one fluorophore selected from said fluorophore group.

23. The method of claim 22, wherein at least one member of said set is labeled with two fluorophores, each fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled with at least one fluorophore selected from said fluorophore group.

24. The method of claim 22, wherein at least one member of said set is labeled with three fluorophores, each fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled with at least one fluorophore selected from said fluorophore group.

25. The method of claim 13, wherein an optical comb is employed in step (b) to detect the predetermined label of said hybridized probe member.

26. The method of claim 13, wherein an optical filter is employed in step (b) to detect the predetermined label of said hybridized probe member.

27. The method of claim 26, wherein a plurality of optical filters is sequentially employed in step (b) to detect the predetermined label of said hybridized probe member.

28. The method of claim 26, wherein at least one member of said set is labeled with more than one fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled with at least one fluorophore selected from said fluorophore group.

29. The method of claim 26, wherein at least one member of said set is labeled with two fluorophores, each fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled with at least one fluorophore selected from said fluorophore group.

30. The method of claim 26, wherein at least one member of said set is labeled with three fluorophores, each fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled with at least one fluorophore selected from said fluorophore group.