Multiparametric fluorescence in situ hybridization

• US 20030235840A1
• Filed: 01/24/2003
• Published: 12/25/2003
• Est. Priority Date: 12/22/1995
• Status: Abandoned Application

• Current Assignee: David C. Ward, Michael Speicher, Stephen Gwyn Ballard
• Original Assignee: David C. Ward, Michael Speicher, Stephen Gwyn Ballard
• Inventors: Ward, David C., Speicher, Michael, Ballard, Stephen Gwyn
• Application Number:
  US10/350,042
• Publication Number:
  US 20030235840A1
• Time in Patent Office: Days
• US Class Current: 435/6.14
• 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 set of combinatorially labeled oligonucleotide probes comprised of a first and a second subset of probes, wherein:

(A) each member of said first subset of probes comprises a plurality of an oligonucleotide;

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

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

(B) each member of said second subset of probes comprises a plurality of an oligonucleotide;

(i) being linked or coupled to a predetermined label distinguishable from the label of any other member of said first or second subset, and (ii) being capable of specifically hybridizing with one extra-chromosomal polynucleotide copy of a predetermined region of an autosomal or sex chromosome of said human karyotype.

2. The set of combinatorially labeled oligonucleotide probes of claim 1, wherein said extra-chromosomal polynucleotide copy of a predetermined region of an autosomal or sex chromosome of said human karyotype is an RNA molecule.

3. The set of combinatorially labeled oligonucleotide probes of claim 1, wherein said extra-chromosomal polynucleotide copy of a predetermined region of an autosomal or sex chromosome of said human karyotype is a DNA molecule.

4. 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.

12. The set of combinatorially labeled oligonucleotide probes of any of claims 3 and 7, wherein said DNA molecule is produced through in situ nucleic acid amplification.

13. The set of combinatorially labeled oligonucleotide probes of any of claims 3 and 7, wherein said in situ nucleic acid amplification is an in situ polymerase chain reaction.

14. The set of combinatorially labeled oligonucleotide probes of any of claims 3 and 7, wherein said in situ nucleic acid amplification is an in situ rolling circle amplification reaction. ,

15. The set of combinatorially labeled oligonucleotide probes of any of claims 3 and 7, wherein said probes contain nucleotide residues that are labeled with a biotin moiety.

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

17. The set of combinatorially labeled oligonucleotide probes 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 set of combinatorially labeled oligonucleotide probes 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 set of combinatorially labeled oligonucleotide probes 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 set of combinatorially labeled oligonucleotide probes of claim 19, wherein one of the fluorophores of said biotin-binding ligand that comprises more than one fluorophore is selected from said group of fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.

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

22. The set of combinatorially labeled oligonucleotide probes 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 set of combinatorially labeled oligonucleotide probes of claim 22, wherein at least one member of said set is labeled with two fluorophores, each 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 set of combinatorially labeled oligonucleotide probes of claim 22, wherein at least one member of said set is labeled with three fluorophores, each 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.

5. A set of combinatorially labeled oligonucleotide probes comprised of a first subset of genotypic probes and a second subset of phenotypic probes, wherein:

(A) each member of said first subset of genotypic probes comprises a plurality of an oligonucleotide;

(i) being linked or coupled to a predetermined label distinguishable from the label of any other member of said first or second subsets of probes, and (ii) being capable of specifically hybridizing with a region of a nucleic acid of a preselected bacterium, virus or lower eukaryote;

said first subset of probes set having sufficient members to be capable of distinguishing said preselected bacterium, virus, or lower eukaryote from other bacteria, viruses, or lower eukaryotes; and

(B) each member of said second subset of phenotypic probes comprises a plurality of an oligonucleotide;

(i) being linked or coupled to a predetermined label distinguishable from the label of any other member of said first or second subset, and (ii) being capable of specifically hybridizing with a predetermined polynucleotide region of said chromosome of said preselected bacterium, virus, or lower eukaryote, or an extra-chromosomal copy thereof so as to permit the determination of whether said preselected bacterium, virus, or lower eukaryote exhibits a preselected phenotype.

6. The set of combinatorially labeled oligonucleotide probes of claim 5, wherein said extra-chromosomal polynucleotide copy of a predetermined region of said chromosome is an RNA molecule.

7. The set of combinatorially labeled oligonucleotide probes of claim 5, wherein said extra-chromosomal polynucleotide copy of a predetermined region of said chromosome is a DNA molecule.

8. The set of combinatorially labeled oligonucleotide probes of claim 5, wherein said predetermined region of said chromosome of said bacterium, virus or lower eukaryote encodes an antibiotic resistance deterninant.

9. The set of combinatorially labeled oligonucleotide probes of claim 5, wherein said predetermined region of said chromosome of said bacterium, virus or lower eukaryote encodes a toxin.

10. The set of combinatorially labeled oligonucleotide probes of claim 5, wherein said predetermined region of said chromosome of said bacterium, virus or lower eukaryote encodes a protein that is determinative of the species of said bacterium, virus or lower eukaryote.

11. The set of combinatorially labeled oligonucleotide probes of claim 10, wherein said predetermined region of said chromosome of said bacterium, virus or lower eukaryote encodes a protein that is determinative of the subspecies of said species of bacterium, virus or lower eukaryote.

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

(I) contacting a preparation of said chromosomes, in single-stranded form, under conditions sufficient to permit nucleic acid hybridization to occur with a set of combinatorially labeled oligonucleotide probes comprised of a first and a second subset of probes, wherein;

(A) each member of said first subset of probes comprises a plurality of an oligonucleotide;

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

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

(B) each member of said second subset of probes comprises a plurality of an oligonucleotide;

(i) being linked or coupled to a predetermined label distinguishable from the label of any other member of said first or second subset, and (ii) being capable of specifically hybridizing with an a predetermined extra-chromosomal polynucleotide copy of a region of an autosomal or sex chromosome of said human karyotype.

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

(III) repeating step (II) until each autosomal and sex chromosome of said human karyotype has been identified in said preparation

(IV) for each member of said second subset of probes hybridized to a predetermined extra-chromosomal polynucleotide copy of a region of an autosomal or sex chromosome detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the region of the autosomal or sex chromosome of said human karyotype with which that member specifically hybridizes, to thereby identify the region of said autosomal or sex chromosome hybridized to said member;

(V) repeating step (IV) for each member of said second subset of probes.

26. The method of claim 25, wherein said extra-chromosomal polynucleotide copy of said predetermined region of an autosomal or sex chromosome of said human karyotype is an RNA molecule.

27. The method of claim 25, wherein said extra-chromosomal polynucleotide copy of said predetermined region of an autosomal or sex chromosome of said human karyotype is a DNA molecule.

28. The method of claim 25, 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.

36. The method of any of claims 27 and 31, wherein said in situ nucleic acid amplification is an in situ polymerase chain reaction.

37. The method of claim 36, wherein said in situ nucleic acid amplification is an in situ rolling circle amplification reaction.

38. The method of claim 36, wherein said probes contain nucleotide residues that are labeled with a biotin moiety.

39. The method of claim 36, wherein said probes are additionally labeled with a labeled biotin-binding ligand.

40. The method of claim 39, wherein at least one of said probes is labeled with a biotin-binding ligand that comprises one or more fluorophores.

41. The method of claim 40, 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.

42. The method of claim 40, wherein at least one of said probes is labeled with a biotin-binding ligand that comprises more than one fluorophore.

43. The method of claim 42, wherein one of the fluorophores of said biotin-binding ligand that comprises more than one fluorophore is selected from said group of fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.

44. The method of claim 43, wherein all of the fluorophores of said biotin-binding ligand that comprises more than one fluorophore are selected from said group of fluorophores FlTC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.

45. The method of claim 40, 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.

46. The method of claim 45, wherein at least one member of said set is labeled with two fluorophores, each 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.

47. The method of claim 45, wherein at least one member of said set is labeled with three fluorophores, each 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.

48. The method of any of claims 25 and 29, wherein an optical optical comb is employed in step (b) to detect the predetermined label of said hybridized probe member.

49. The method of claims 25 and 29, wherein an optical filter is employed in step (b) to detect the predetermined label of said hybridized probe member.

50. The method of claim 49, wherein a plurality of optical filters is sequentially employed in step (b) to detect the predetermined label of said hybridized probe member.

51. The method of claim 48, wherein at least one member of said set is labeled with more than one fluorophore selected from the group consisting of the fluorophores FlTC, 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.

52. The method of claim 49, wherein at least one member of said set is labeled with two fluorophores, each 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.

53. The method of claim 49, wherein at least one member of said set is labeled with three fluorophores, each 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. A method of simultaneously identifying and distinguishing a preselected bacterium, virus, or lower eukaryote from other bacteria, viruses or lower eukaryotes that may be present in a sample which comprises the steps:

(I) contacting a preparation suspected to contain said preselected bacterium, virus, or lower eukaryote, under conditions sufficient to permit in situ nucleic acid hybridization to occur, with a set of combinatorially labeled oligonucleotide probes comprised of a first subset of genotypic probes and a second subset of phenotypic probes, wherein;

(A) each member of said first subset of genotypic probes comprises a plurality of an oligonucleotide;

(i) being linked or coupled to a predetermined label distinguishable from the label of any other member of said first or second subsets of probes, and (ii) being capable of specifically hybridizing with a region of a nucleic acid of said preselected bacterium, virus or lower eukaryote;

said first subset of probes set having sufficient members to be capable of distinguishing said preselected bacterium, virus, or lower eukaryote from other bacteria, viruses, or lower eukaryotes present in said preparation; and

(B) each member of said second subset of probes comprises a plurality of an oligonucleotide;

(i) being linked or coupled -to a predetermined label distinguishable from the label of any other member of said first or second subset, and (ii) being capable of specifically hybridizing with a predetermined polynucleotide region of said nucleic acid of said preselected bacterium, virus, or lower eukaryote, or an extra-chromosomal copy thereof so as to permit the determination of whether said preselected bacterium, virus, or lower eukaryote exhibits a preselected phenotype.

(II) for each member of said first subset of probes hybridized to a region of a chromosome of a preselected bacterium, virus or lower eukaryote, detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the bacterium, virus or lower eukaryote with which that member specifically hybridizes, to thereby identify said bacterium, virus or lower eukaryote hybridized to said member;

(III) repeating step (II) for each member of said first subset of probes;

(IV) for each member of said second subset of probes hybridized to a predetermined polynucleotide region of said chromosome of said preselected bacterium, virus, or lower eukaryote, or an extra-chromosomal copy thereof, detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of said predetermined region, to thereby identify the presence of said predetermined region on a chromosome of said preselected bacteria, virus or lower eukaryote;

(V) repeating step (IV) for each member of said second subset of probes.

30. The method of claim 29, wherein said extra-chromosomal polynucleotide copy of said predetermined region of said chromosome of said bacterium, virus or lower eukaryote is an RNA molecule.

31. The method of claim 29, wherein said extra-chromosomal polynucleotide copy of said predetermined region of said chromosome of said bacterium, virus or lower eukaryote is a DNA molecule.

32. The method of claim 29, wherein said predetermined region of said chromosome of said bacterium, virus or lower eukaryote encodes an antibiotic resistance determinant.

33. The method of claim 29, wherein said predetermined region of said chromosome of said bacterium, virus or lower eukaryote encodes a toxin.

34. The method of claim 29, wherein said predetermined region of said chromosome of said bacterium, virus or lower eukaryote encodes a protein that is determinative of the species of said bacterium, virus or lower eukaryote.

35. The method of claim 34, wherein said predetermined region of said chromosome of said bacterium, virus or lower eukaryote encodes a protein that is determinative of the subspecies of said species of bacterium, virus or lower eukaryote.

54. 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 telomeric region of 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.

55. The set of combinatorially labeled oligonucleotide probes of claim 54, wherein at least one of said labels is a fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.

56. 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 hybridization to occur with 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 telomeric region of 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 of probes, detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the autosomal or sex chromosome of said human karyotype with which that 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.

57. The method of claim 56, wherein at least one of said labels is a fluorophore selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.